WO2018101950A1

WO2018101950A1 - Methods and compositions for the diagnosis of acute myeloid leukemia

Info

Publication number: WO2018101950A1
Application number: PCT/US2016/064442
Authority: WO
Inventors: Eun-Young Erin AHN; Jung-Hyun Kim; Ssang-Taek LIM
Original assignee: University Of South Alabama
Priority date: 2016-12-01
Filing date: 2016-12-01
Publication date: 2018-06-07

Abstract

Embodiments of the present invention relate to the diagnosis of hematopoietic malignancies. Some embodiments include methods and kits for the diagnosis of hematopoietic malignancies, such as acute myeloid leukemia. Some embodiments relate to the presence of alternatively-spliced isoforms of a SON gene.

Description

METHODS AND COMPOSITIONS FOR THE DIAGNOSIS

OF ACUTE MYELOID LEUKEMIA

STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0001] This invention was made with government support under CA 190688 and CA185818 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled USA.018WO_SEQLIST, created November 30, 2016, which is approximately 183 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] Embodiments of the present invention relate to the diagnosis of hematopoietic malignancies. Some embodiments include methods and kits for the diagnosis of hematopoietic malignancies, such as acute myeloid leukemia. Some embodiments relate to the presence of alternatively-spliced isoforms of a SON gene.

BACKGROUND OF THE INVENTION

[0004] Methyl ation of lysine residues of histone H3 is an event dictating active or repressed status of chromatin. Tri-methylation of histone 3 lysine 4 (H3K4me3) near transcription start sites is associated with active transcription (Barski et al., 2007; Guenther et al., 2007), and in mammals, this modification is mediated by the SET1 and mixed lineage leukemia (MLL) family methyltransferases, SET1A, SET1B, and MLLl^t (Miller et al., 2001 ; Shilatifard, 2012). The SET1/MLL proteins are associated with multiple subunit proteins, such as WDR5, ASH2L and RBBP5, to acquire a maximum activity in methylation of H3K4 (Cao et al., 2010; Dou et al., 2006; Ernst and Vakoc, 2012). The N-terminal portion of the MLL1/2 protein interacts with the scaffold protein menin, facilitating LEDGF interaction and chromatin binding of the MLL complex (Yokoyama and Cleary, 2008). The MLL-menin interaction is required for leukemia-associated target gene expression (Yokoyama et al., 2005), suggesting that this interaction is involved in activating oncogenic MLL-target genes. Pharmacological inhibition of the MLL-menin interaction has been shown to effectively block leukemia progression (Borkin et al., 2015) and prostate cancer growth (Malik et al., 2015), indicating that MLL-menin interaction could be a promising target for cancer therapy. However, cellular factors that regulate MLL-menin interaction and MLL complex assembly are largely unknown.

SUMMARY OF THE INVENTION

[0005] Some embodiments of the methods and compositions provided herein include a method of diagnosing a hematopoietic malignancy or a likelihood of developing a hematopoietic malignancy in a test subject comprising: determining a ratio for the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject, wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene, and the long polypeptide is encoded by a full length transcript of the SON gene.

[0006] Some embodiments of the methods and compositions provided herein include a method of detecting a ratio for the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in a test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject comprising: contacting a nucleic acid or polypeptide sample from the test subject with an agent which indicates the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject; contacting the nucleic acid or polypeptide sample with an agent which indicates the level of the long polypeptide or a nucleic acid encoding the long polypeptide in the test subject; and determining the ratio for the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject to the level of the long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject, wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene, and the long polypeptide is encoded by a full length transcript of the SON gene. In some embodiments, the ratio is greater than about 30%, 40%, or 50%.

[0007] Some embodiments of the methods and compositions provided herein include a method of determining the level of a short polypeptide or a nucleic acid encoding the polypeptide in a nucleic acid or polypeptide sample from a test subject comprising: contacting the nucleic acid or polypeptide sample from the test subject with an agent which indicates the level of the short polypeptide or which indicates the level of a nucleic acid encoding the short polypeptide in the sample; and determining the level of the short polypeptide or a nucleic acid encoding the short polypeptide in the sample, wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.

[0008] Some embodiments of the methods and compositions provided herein include a method of diagnosing a hematopoietic malignancy or a likelihood of developing a hematopoietic malignancy in a test subject comprising: determining the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in a sample obtained from a test subject, wherein the short polypeptide is encoded by a truncated alternatively- spliced transcript of a SON gene.

[0009] Some embodiments also include comparing the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in a sample obtained from the test subject with the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in a subject not having a hematopoietic malignancy.

[0010] Some embodiments also include determining the level of a long polypeptide or a nucleic acid encoding the long polypeptide in the test subject, wherein the long polypeptide is encoded by a full length transcript of the SON gene.

[0011] Some embodiments also include determining a ratio for the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the sample from the test subject. In some embodiments, the ratio is greater than about 30%, 40%, or 50%. [0012] Some embodiments also include determining the level of an additional polypeptide in addition to the level of the short form of the SON polypeptide or the level of a nucleic acid encoding the additional polypeptide in addition to the level of a nucleic acid encoding the short form of the SON polypeptide in a test subject, wherein the additional polypeptide is encoded by a gene selected from CDKN1A, GFI1 and ATF3.

[0013] Some embodiments also include comparing the level of an additional polypeptide or the level of a nucleic acid encoding the additional polypeptide in the test subject with the level of an additional polypeptide or the level of a nucleic acid encoding the additional polypeptide in a subject not having a hematopoietic malignancy.

[0014] In some embodiments, the truncated alternatively-spliced transcript of a SON gene includes a SON E variant of a SON gene, and/or a SON B variant of a SON gene. In some embodiments, the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10. In some embodiments, the short polypeptide comprises SEQ ID NOs:88 or 90. In some embodiments, the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.

[0015] In some embodiments, the full length transcript of the SON gene comprises a sequence which specifically binds to an oligonucleotide comprising a sequence selected from SEQ ID NOs: 13-18. In some embodiments, the long polypeptide comprises SEQ ID NO:86. In some embodiments, the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.

[0016] In some embodiments, the SON gene is a human SON gene.

[0017] Some embodiments also include obtaining a sample from the test subject.

[0018] Some embodiments also include contacting the sample with an agent which specifically binds to the short polypeptide or a nucleic acid encoding the short polypeptide.

[0019] Some embodiments also include contacting the sample with an agent which specifically binds to the long polypeptide or a nucleic acid encoding the long polypeptide. [0020] In some embodiments, the agent is a primer or a hybridization probe. In some embodiments, the primer or the hybridization probe comprises an oligonucleotide comprising SEQ ID NOs:06, 10 or 13-18

[0021] In some embodiments, the agent is an antibody or antigen-binding fragment thereof which specifically binds to the short polypeptide. In some embodiments, the agent is an antibody or antigen-binding fragment thereof which specifically binds to the long polypeptide.

[0022] In some embodiments, the agent is attached to a solid support.

[0023] In some embodiments, the sample comprises nucleic acids or polypeptides.

[0024] Some embodiments also include determining an increased level of a polypeptide encoded by a SON target gene or a nucleic acid encoded by a SON target gene in the sample from the test subject, wherein the SON target gene has a SON binding site. In some embodiments, the SON target gene is selected from the group consisting of FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFI1, EGR1, SRC, and GFI1B.

[0025] Some embodiments also include determining an increased level of a MLL multi-protein complex in the sample from the test subject, wherein the MLL is selected from the group consisting of MLL1, MLL-N, MLL-C, and MLL2. In some embodiments, the multiprotein complex comprises a protein selected from menin, ASH2L, LEDGF, and WDR5.

[0026] Some embodiments also include determining an increased replating activity of a hematopoietic progenitor cell of the sample from the test subject.

[0027] In some embodiments, the hematopoietic progenitor cell is a bone marrow cell.

[0028] Some embodiments also include determining an increased binding of a protein at the or adjacent to the transcription start site of a SON target gene in the sample from the test subject. In some embodiments, the protein is selected from the group consisting of MLL1, MLL-N, MLL-C, MLL2, WDR5, ASH2L, menin, SET1A, SET1B, ASC2, and SUZ12. In some embodiments, the SON target gene is selected from the group consisting of CDKN1A, GADD45A, NOTCH2NL, ATF3, GFI1, EGR1, and SRC. [0029] Some embodiments also include determining an increased level in H3K4me3 in the sample from the test subject. In some embodiments, the level in H3K4me3 is increased at the or adjacent to a SON target gene selected from the group consisting of CDKN1A, GADD45A, NOTCH2NL, ATF3, GFI1, EGR1, and SRC.

[0030] In some embodiments, the sample comprises bone marrow mononuclear cells (BM-MNCs), or peripheral blood mononuclear cells (PB-MNCs).

[0031] In some embodiments, the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In some embodiments, the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).

[0032] In some embodiments, the test subject is mammalian. In some embodiments, the test subject is human.

[0033] Some embodiments of the methods and compositions provided herein include a method of diagnosing a hematopoietic malignancy or a likelihood of developing a hematopoietic malignancy in a test subject comprising: detecting increased levels of binding of a short polypeptide with a nucleic acid having a SON binding site in a sample from a test subject, wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.

[0034] In some embodiments, the truncated alternatively-spliced transcript of a SON gene includes a SON E variant of a SON gene, and/or a SON B variant of a SON gene. In some embodiments, the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10. In some embodiments, the short polypeptide comprises SEQ ID NOs:88 or 90. In some embodiments, the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.

[0035] In some embodiments, the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In some embodiments, the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).

[0036] In some embodiments, the subject is mammalian. In some embodiments, subject is human. [0037] Some embodiments of the methods and compositions provided herein include a method of ameliorating a hematopoietic malignancy comprising: increasing the level of a full length SON polypeptide or a nucleic acid encoding a full length SON polypeptide in a cell of a subject.

[0038] In some embodiments, the expression level of a nucleic acid encoding said full length SON polypeptide or the expression level of said full length SON polypeptide is increased by administering a composition comprising a nucleic acid to the subject.

[0039] In some embodiments, the nucleic acid comprises a sequence encoding a full length SON polypeptide. In some embodiments, the SON polypeptide is a human SON polypeptide. In some embodiments, the nucleic acid comprises SEQ ID NO.:85. In some embodiments, the polypeptide comprises SEQ ID NO.:86.

[0040] In some embodiments, the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In some embodiments, the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).

[0041] In some embodiments, the subject is mammalian. In some embodiments, the subject is human.

[0042] Some embodiments of the methods and compositions provided herein include a kit for the diagnosis of a hematopoietic malignancy in a test subject comprising: an agent that specifically binds to a short polypeptide or a nucleic acid encoding the short polypeptide, wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.

[0043] Some embodiments also include an agent that specifically binds to a long polypeptide or a nucleic acid encoding the long polypeptide, wherein the long polypeptide is encoded by a full length transcript of the SON gene.

[0044] Some embodiments also include an agent that specifically binds to an additional polypeptide in addition to the short polypeptide or a nucleic acid encoding the additional polypeptide in addition to the nucleic acid encoding the short polypeptide in the test subject, wherein the additional polypeptide is encoded by a gene selected from CDKN1A, GFI1 and ATF3. [0045] In some embodiments, the truncated alternatively-spliced transcript of a SON gene includes a SON E variant of a SON gene, and/or a SON B variant of a SON gene.

[0046] In some embodiments, the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10. In some embodiments, the short polypeptide comprises SEQ ID NOs:88 or 90. In some embodiments, the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.

[0047] In some embodiments, the full length transcript of the SON gene comprises a sequence which specifically binds to an oligonucleotide comprising a sequence selected from SEQ ID NOs: 13-18. In some embodiments, the long polypeptide comprises SEQ ID NO:86. In some embodiments, the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.

[0048] In some embodiments, the SON gene is a human SON gene.

[0049] Some embodiments also include an agent that specifically binds to a SON target gene, wherein the SON target gene has a SON binding site. In some embodiments, the SON target gene is selected from the group consisting of FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFI1, EGR1, SRC, and GFI1B.

[0050] Some embodiments also include an agent that specifically binds to a protein selected from MLL1, MLL-N, MLL-C, MLL2, menin, ASH2L, LEDGF, WDR5, SET1A, SET1B, ASC2, SUZ12, and H3K4me3.

[0051] In some embodiments, the agent is a primer or a hybridization probe. In some embodiments, the primer or the hybridization probe comprises an oligonucleotide comprising SEQ ID NOs:06, 10, or 13-18. In some embodiments, the agent is an antibody or antigen-bind fragment thereof which specifically binds to the short polypeptide. In some embodiments, the agent is attached to a solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1A depicts a schematic summarizing chromatin-immunoprecipitation (ChIP) using two different SON antibodies (Abs) and DN A- sequencing to determine overlap peaks. SON-N and SON-C Abs specifically bind to the N- and the C-terminus of SON, respectively.

[0053] FIG. IB depicts genomic distribution of SON-binding sites determined by SON-N and SON-C ChIP overlap peaks. The pie graphs show the percentage of peaks located at specific genomic regions indicated (transcription start site, transcription start site).

[0054] FIG. 1C depicts a Venn diagram showing the number of SON ChIP peaks (SON-N, SON-C and overlap) within ±5 kb from the transcription start site (Top). Pie graph illustrating genomic locations of overlap peaks within ±5kb from the transcription start site (Bottom).

[0055] FIG. ID depicts a heat map showing the overlap peak signal of SON ChIP around the transcription start site of genes.

[0056] FIG. IE depicts the top five DNA sequence motifs identified in the SON- N and SON-C overlap peaks within ± 5 kb of the transcription start site.

[0057] FIG. IF depicts gene ontology (GO) term enrichment analysis using DAVID for the genes in which SON-N and SON-C overlap peaks were found within ±5 kb from the transcription start site.

[0058] FIG. 1G depicts the result of a pilot ChlP-seq study to verify specificity of two SON antibodies, SON-N Ab and SON-C Ab. The numbers of ChlP-seq peaks are summarized.

[0059] FIG. 1H depicts a Venn diagram showing number of SON peaks determined by ChlP-seq using SON-N Ab and SON-C Ab.

[0060] FIG. II depicts top five motifs enriched in SON-binding sites analyzed by HOMER.

[0061] FIG. 2A depicts qPCR analyses of SON ChlP-seq target genes in K562 cells transfected with control siRNA and two different SON siRNAs. GFI1B served as a negative control which does not have SON binding sites near the transcription start site. Values represent mean ± SD of four independent experiments. *p < 0.01.

[0062] FIG. 2B depicts average signal profiles of indicated histone modifications and CpG islands around the SON-binding sites. [0063] FIG. 2C depicts Integrative Genomics Viewer (IGV) images representing SON (SON-N), H3K4me3, and H3K27ac ChlP-seq read counts at the target gene locus in K562 cells.

[0064] FIG. 2D depicts close-up images of ChlP-seq peaks of SON-N, SON-C, H3K4me3, and H3K27ac in representative SON target genes.

[0065] FIG. 2E depicts SON ChlP-qPCR analysis (with SON-N Ab) confirming SON enrichment near the transcription start site of indicated genes in K562 cells. The number in the parenthesis indicates the base-pair counts from the transcription start site of each gene, and the primers for qPCR (TABLE 3) were designed to detect SON enrichment around these positions. *p-values <0.01.

[0066] FIG. 2F depicts HA ChlP-qPCR analysis measuring DNA-binding ability of FLA-tagged full-length SON and several deletion mutants in K562 cells; Full-length SON (SON F), potential DNA-binding region-deleted SON (SON ΑΌΒ, amino acids 1 ,263 - 1,818 deleted), G-patch-deleted SON (SON AG-patch), and double-stranded RNA binding motif- deleted SON (SON ADSRM). Potential DNA-binding region that has been shown to interact with human hepatitis B virus genome (Sun et al., 201 1) is indicated. Empty vector transfected sample was used as a control. *p-values <0.01.

[0067] FIG. 2G depicts a Western blot that confirmed knockdown efficiency of two different siRNAs (#1 and #2) against SON in K562 cells.

[0068] FIG. 2H depicts average signal profiles of H3K4mel and H3K27ac around the intergenic SON-binding sites.

[0069] FIG. 21 depicts an IGV browser image of density profiles of SON (SON-N Ab ChIP), CpG islands, H3K4me3 and H3K27ac at several examples of SON target genes. Scale bar lOkb.

[0070] FIG. 2J depicts close-up images of ChlP-seq peaks of SON-N, SON-C, H3K4me3, H3K27ac, H2A.Z and MNase-seq (nucleosome) peaks in representative SON target genes. Scale bar,lkb.

[0071] FIG. 3A depicts ChlP-qPCR analyses of various histone modification levels at the SON-binding regions near the transcription start sites of the indicated genes upon SON knockdown in K562 cells. Histone H3 ChlP-qPCR was used as a control. [0072] FIG. 3B depicts ChlP-qPCR analyses MLL and SET1 complex protein recruitment (MLL-N; MLL1 N-terminus region, MLL-C; MLL1 C-terminus region, MLL2, WDR5, ASH2L, menin, SET1A, SET1B and ASC2) to the regions near the transcription start sites of the indicated genes upon SON knockdown. Recruitment of SUZ12, a polycomb complex protein, was also examined, and H3 ChIP was done as a control. Depletion of SON at the transcription start site of indicated target regions upon SON siRNA-transfection was verified by SON ChIP. Signals for each experiment were represented as percentage of input chromatin. Results are expressed as mean ± SD of three biological replicates. * p < 0.01.

[0073] FIG. 3C depicts SON Depletion Leads to H3K4me3 Modification and MLL Complex Recruitment. ChlP-qPCR analysis of two SON target genes (CDKN1A and GADD45A), were conducted using indicated antibodies in K562 cells transfected with control or SON siRNA-#2. ChlP-qPCR results are plotted as percentage of input DNA. *p- values <0.05.

[0074] FIG. 3D depicts SON Depletion Leads to H3K4me3 Modification and MLL Complex Recruitment. ChlP-qPCR analysis of two non-targets (GFI1B and ATF4; B), were conducted using indicated antibodies in K562 cells transfected with control or SON siRNA-#l . ChlP-qPCR results are plotted as percentage of input DNA. *p-values <0.05.

[0075] FIG. 4A depicts a Western blot verified SON knockdown by SON siRNA transfection in K562 cells.

[0076] FIG. 4B depicts a co-immunoprecipitation experiment with MLL-N antibodies.

[0077] FIG. 4C depicts a co-immunoprecipitation experiment with WDR5 antibodies.

[0078] FIG. 4D depicts a co-immunoprecipitation experiment with LEGF antibodies.

[0079] FIG. 4E depicts a co-immunoprecipitation experiment with SET1A antibodies.

[0080] FIG. 4F depicts interaction of SON with menin. K562 nuclear extracts were immunoprecipitated with control IgG or SON antibody (SON-N Ab) and several components of the MLL complex were examined by Western blot. [0081] FIG. 4G depicts verification of the SON-menin interaction. HEK 293 cells transfected with HA-SON, Flag-menin or pcDNA3-control as indicated were used for co- immunoprecipitation with HA antibody followed by Western blotting with HA or Flag antibodies.

[0082] FIG. 4H depicts an immunoprecipitation experiment in K562 cells transfected with V5-tagged SON indicates that SON outcompetes MLL (MLL-N) for menin interaction in a dose-dependent manner. For plasm id transfection, two different amounts of SON-V5 construct, 5 μg (+) or 10 μg (++), were used.

[0083] FIG. 41 depicts an experiment in which nuclear extracts from control and SON knockdown-K562 cells were size- fractionated on FPLC and analyzed for MLL1 N- terminus (MLL-N) and menin distribution by Western blotting using indicated antibodies (bottom panels). Top panels are elution profiles: fraction numbers on elution profile (top) indicate MLL-N-enriched fractions (left panel of WB, fractions 12-19) and trace on elution profile (top) indicates further downstream fractions (right panel of WB, fractions 20-41).

[0084] FIG. 4J depicts a Western blot confirmed knockdown efficiency of SON siRNA in MV4; 11 cells.

[0085] FIG. 4K depicts nuclear extracts from MV4; 11 transfected by control and SON siRNA were used for immunoprecipitation with MLL-N antibody and Western blotted with indicated antibodies. The N-terminus of wild-type MLL1 is marked by an asterisk and the MLL-AF4 fusion protein is marked by a red arrow head.

[0086] FIG. 4L depicts 293T cells transiently co-expressing Flag-MLL-ENL and Myc-menin were transfected with control vector or SON-V5 construct and subjected to co- immunoprecipitation assays using a Flag antibody. Immunoprecipitated Flag-MLL-ENL and Myc-menin were analyzed by Western blotting.

[0087] FIG. 4M depicts expression of target genes of MLL fusion proteins (HOXA9 and MEIS1) or SON protein (CDKN1A, ATF3, and GFI1) measured by real-time qPCR in the control and SON siRNA-transfected MLL-rearranged leukemic cell lines, MV4;11 and ML-2. Note that the genes identified by our SON ChlP-seq in K562 (e.g. CDKN1A, ATF3 and GFI1) were also upregulated upon SON knockdown in MV4;1 1 cells (heterozygous for MLL-AF4) which retain one copy of wild-type MLL. However, these genes were not changed upon SON knockdown in ML2 cells which do not have wild-type MLL (homozygous for MLL-AF6), suggesting that the negative effect of SON on those target genes is exerted through inhibition of the MLL wild-type complex. Data represent three replicates from three independent experiments. *p values < 0.01.

[0088] FIG. 5A depicts a schematic representation of the SON gene and the SON proteins. Full-length SON (SON F) is generated by alignment of 12 constitutive exons. Inclusion of alternative exons (exon 7a, and exon 5a) produces two different alternatively spliced isoforms, SON B and E. Horizontal arrows indicate the specific position of the primers used in qPCR. Arrowheads, polyadenylation signal sequences; Hatched boxes, untranslated regions.

[0089] FIG. 5B depicts qPCR analysis of specific exon regions of SON in BM- MNCs of AML patients (P, red dots; n = 10) and healthy normal donors (N, black dots; n = 4). GAPDH was used for normalization. Horizontal bars indicating the median expression level of specific exon regions of SON are presented with the error bars indicating ±SD. *p < 0.05, **p < 0.01.

[0090] FIG. 5C depicts qPCR analysis of specific exon regions of SON in PB- MNCs of AML (n = 8) and MDS (n = 2) patients (P) and healthy normal donors (N; n = 7). GAPDH was used for normalization. Horizontal bars indicating the median expression level of specific exon regions of SON are presented with the error bars indicating ±SD. *p < 0.05, **p < 0.01.

[0091] FIG. 5D depicts relative ratio of SON F, SON B and SON E in the BM- MNCs from AML patients (PI - P10) and healthy normal donors (Nl - N4).

[0092] FIG. 5E depicts relative ratio of SON F, SON B and SON E in the PB- MNCs from AML patients (PI, P5, P9-14), MDS patients (P15 - 16) and healthy normal donors (N5 - Ni l).

[0093] FIG. 5F depicts analyses of SON isoform expression in normal mouse Lin- /c-Kit+ bone marrow (BM) cells and leukemic blasts from mice with AMLl-ET09a- and MLL-AF9-induced leukemia. Specific exon regions indicated in each graph were determined by qPCR with the primer sets indicated in FIG. 5H. Data are represented as mean ± SD of three independent experiments. *p < 0.01. [0094] FIG. 5G depicts relative ratios of three forms of Son in leukemic blasts (freshly isolated from the animals and also collected after methylcellulose culture) and normal Lin-/c-Kit+ mouse BM cells were determined.

[0095] FIG. 5H depicts a schematic representation of the mouse Son genes and the Son proteins. Similar to human SON, full-length mouse Son (Son f; mouse Son isoform 1) is generated by alignment of 12 constitutive exons. Inclusion of alternative exons (exon 7a; exons 5a) produces two different alternatively spliced isoforms, Son b (a predicted isoform) and Son e (mouse Son isoform 2). The primer sets used for mouse Son qPCR (presented in FIG. 5F) are indicated with horizontal arrows.

[0096] FIG. 51 depicts a structure of the SON E mRNA and position of PCR primers for 3' RACE PCR to confirm the 3' UTR sequence of SON E. Arrows indicate primers for 1st RACE PCR and arrows indicate primers for 2nd RACE PCR. EtBr-stained agarose gel showed 3' RACE-amplified PCR products (asterisk) that were used for sequencing. Decreased amount of the 3' RACE PCR product in total SON siRNA-transfected K562 cells, compared to control K562 cells, indicates the specificity of 3' RACE PCR.

[0097] FIG. 5J depicts sequencing result of the 3' RACE PCR product from human SON E transcripts.

[0098] FIG. 5K depicts a schematic strategy for measurement of relative ratio of SON isoforms presented in FIG. 5D, FIG. 5E and FIG. 5G. To calculate relative expression level of SON isoforms, each qPCR analysis was performed using same forward primer and two different reverse primers. At the 1st qPCR, the ratio of exon 5a-included transcripts (SON E) and exon 5-included transcripts (total of SON F and SON B) was determined using a common forward primer (F3) and exon-specific reverse primers (R5 or R5a). At the 2nd qPCR, the ratio of exon 7a-included transcripts (SON B) and exon 7-included transcript (SON F) was determined using a common forward primer (F5) and exon-specific reverse primers (R7 or R7a). Based on two qPCR analyses, relative levels of SON F, SON B and SON E were determined.

[0099] FIG. 5L depicts a schematic of genomic structure of full-length SON and SON B. Each bar with the probe set number indicates the specific position of DNA probes used in microarray analysis (Affymetrix U133A microarrays). [0100] FIG. 5M depicts relative expression levels of SON detected by three different probe sets. The microarray data were from Stegmaier Leukemia Dataset (Stegmaier et al., 2004) from Oncomine database. The values of log2 median-centered intensity detected by indicated probe sets were displayed as a boxplot according to Oncomine output.

[0101] FIG. 5N depicts relative expression levels of SON detected by three different probe sets. The microarray data were from Maia Leukemia Dataset (Maia et al., 2005) from Oncomine database. The values of log2 median-centered intensity detected by indicated probe sets were displayed as a boxplot according to Oncomine output.

[0102] FIG. 6A depicts a qPCR analysis of leukemia-associated SON target genes, CDKN1A, ATF3 and GFI1, in BM-MNCs from healthy normal donors (Nl - N4) and AML patients (P2 - P10). Black Broken lines indicate the average level of each gene in normal donor samples.

[0103] FIG. 6B depicts the effects of total SON knockdown (with SON siRNA-1) and SON E-specific knockdown (with SON E siRNA) on expression of leukemia-associated, ChlP-seq target genes in K562 cells. In addition, previously identified SON target genes regulated by RNA splicing function of SON (TUBG1, HDAC6 and AKT1) were also examined, revealing the effect of SON E on regulation of ChlP-seq target genes, but not RNA splicing target genes. *p < 0.05, **p < 0.01.

[0104] FIG. 6C depicts the effects of total SON knockdown (with SON siRNA-1) and SON E-specific knockdown (with SON E siRNA) on expression of leukemia-associated, ChlP-seq target genes in primary human CD34+ BM cells.

[0105] FIG. 6D depicts effects of SON F and SON E overexpression on SON target gene expression (ChlP-seq target genes and RNA splicing target genes) in human CD34+ BM cells. Error bars represent SD from three independent experiments. *p < 0.05, **p < 0.01.

[0106] FIG. 6E depicts a ChlP-qPCR assay to analyze the H3K4me3 levels at SON-binding sites near the promoter of the CDKN1A and GFI1 genes upon SON F and SON E expression. K562 cells were transfected with indicated siRNA and SON constructs (siRNA-resistant form). For plasmid transfection, 3 μg (+) of SON F constructs plus increasing amounts of SON E construct, 0 (-), 3 (+) or 6 μg (++), or 3 μg of SON E alone were used as indicated. H3K4me3 antibody or H3 antibody were used for ChlP. Asterisks indicate statistical significances of H3K4me3 reduction, as compared to the sample transfected with SON siRNA alone (the second lane). Inter-peak asterisks indicate statistical significance of the difference between two indicated samples. *p < 0.01.

[0107] FIG. 6F depicts TUBG1 exon 7-8 minigene splicing assay demonstrating that SON E does not interfere with SON F-mediated RNA splicing.

[0108] FIG. 6G depicts the location of the target regions of total SON siRNAs (siRNAs #1 and #2, targeting exon 3) and SON E siRNA (targeting exon 5a). SON siRNA sequences are described in herein.

[0109] FIG. 6H depicts verification of SON F and SON E overexpression in primary human CD34+ bone marrow cells after nucleofection of the expression constructs. Exogenous expression of SON was determined by qPCR using the forward primers (F- hSON-Exonl2 for SON F, F-hSON-Exon4 for SON E) together with a reverse primer targeting the V5 sequence (R-V5-exo) in the plasmid. **p-values <0.01.

[0110] FIG. 61 depicts the expression level of SON F-V5 and SON E-Flag in the K562 cells used for V5-CMP and Flag-ChIP presented in FIG. 6E.

[0111] FIG. 6 J depicts strategies of minigene assay with the TUBG1 exon7- intron 7 - exon8 minigene model (containing SON-dependent splicing sites; Ahn et al., 201 1; Lu et al., 2013). Splicing efficiencies of transfected SON F and SON E (expressed by the siRNA-resistant form of cDNA; Ahn et al., 201 1) were accessed by RT-PCR using a forward primer (F) and a reverse primer (R) to detect unspliced and spliced RNA.

[0112] FIG. 6K depicts RT-PCR results demonstrating minigene splicing efficiencies. The numbers above the each lane indicates the relative amount of exogenous SON F and/or SON E transfected together with the minigene.

[0113] FIG. 7A depicts a schematic diagram of expression constructs used for the experiments presented in panels B— E; V5-tagged SON F (full-length), Flag-tagged SON E, V5-tagged SON E, and the HA-tagged SON C-terminal fragment containing the RS domain and RNA-binding motifs (SR+RB).

[0114] FIG. 7B depicts ChlP-qPCR analysis of SON F-V5 or SON E-Flag binding on target regions near the transcription start site of CDKN1 A. Five μg (+) of SON F- V5 constructs plus increasing amounts of SON E-Flag, 5 (+) or 10 μg (++) constructs were used for transfection into K562 cells. Shown are representative results of three independent experiments (mean ± S.D. from triplicate). Asterisks indicate statistical significances of SON F-V5 or SON E-Flag enrichment, as compared to the background signal from negative control samples (SON E Flag-only sample in V5-ChlP and SON F-V5-only sample in Flag ChIP). Inter-peak asterisks indicate statistical significance of the difference between two indicated samples. *p < 0.05, **p < 0.01.

[0115] FIG. 7C depicts co-immunoprecipitation (IP) experiments demonstrating the lack of menin-binding ability of SON E. Lysates of HEK 293 cells co-transfected with Flag-menin and SON F-V5 or SON E-V5 were subjected to IP with anti-V5, followed by Western blotting with indicated antibodies.

[0116] FIG. 7D depicts the C-terminus of SON interacts with menin. The HA- tagged SR+RB fragment was expressed in HEK 293 cells with or without Flag-menin, and subjected to HA-IP followed by Western blotting with HA or Flag antibodies.

[0117] FIG. 7E depicts SON E overexpression enhances the interactions between MLL complex components, while SON F overexpression shows inhibitory effects. Empty vector (pcDNA3), SON F-V5, or SON E-Flag were transfected into K562 cells (without depletion of endogenous SON) and nuclear extract was used for immunoprecipitation with MLL-N antibody followed by Western blotting with indicated antibodies.

[0118] FIG. 7F depicts a Western blot verifying overexpression of SON E in primary mouse bone marrow cells infected with lentivirus carrying flag-tagged SON E.

[0119] FIG. 7G depicts number of colony-forming units in serial replating assay using primary mouse bone marrow cells infected with lentivirus carrying the control vector or SON E. The results represent 3 independent experiments. *p < 0.05, **p < 0.01.

[0120] FIG. 7H depicts representative images of control cells and the colony formed by SON E-overexpressing cells in the third round of the replating assay.

[0121] FIG. 71 depicts models for SON and its alternative spliced isoform function in transcription. Under normal conditions, SON binds G/C-rich sequence near the transcription start site to inhibit transcriptional activity. Interaction of the SON C-terminal region with menin diminishes MLL complex assembly and its recruitment to target DNA, leading to the low level of H3K4me3 and transcriptional repression. Upon SON depletion, MLL complex formation is facilitated, resulting in enhanced recruitment of the MLL complex and subsequent increase of H3K4me3 and promoter activity. In leukemic condition, a high level of short SON isoforms (SON B and SON E) interrupts DNA-binding of full- length SON, but cannot interfere with MLL-menin interaction, resulting in abrogation of the full-length SON function and de-repression/activation of multiple leukemia-associated genes.

[0122] FIG. 7J depicts co-immunoprecipitation of HA-tagged SON SR+RB with Myc-tagged full-length/wild type (WT) or several partial fragments of menin after transient transfections of the constructs into 293T cells as indicated. Immunoprecipitations were performed using an HA antibody and analyzed by Western blotting using a Myc antibody. Wild type and several partial fragments of menin were detected in the precipitates (upper panel, lane 3, 5, 6, and 8; arrowheads). Asterisks indicate IgG.

[0123] FIG. 7K depicts a schematic diagram shows the structure of the menin and its deletion constructs used in FIG. 7J. The region previously shown to interact with MLL is indicated with a bar.

[0124] FIG. 7L depicts a Western blot verified expression of V5-tagged SON F (SON F-V5) and Flag-tagged SON E (SON E-Flag) in K562 cells used for the experiment presented in FIG. 7E.

[0125] FIG. 7M depicts schematic structures of the lentiviral construct for SON E overexpression and the illustration of the experimental design. Bone marrow (BM) cells were isolated from mice, infected with empty lentivirus (Control) or Flag-tagged SON E— expressing lentivirus (SON E) and subjected to the colony forming and serial replating assays. For each round of plating, a total of 2 ^χ 10⁴ BM cells were cultured in MethoCult (M3434).

[0126] FIG. 7N depicts representative photomicrographs of colonies from control and SON E lentivirus-infected BM cells from the first plating, demonstrating the high efficiency of infection (GFP-positive cells). Scale bars on pictures represent 100 μιη. DETAILED DESCRIPTION

[0127] Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. As described herein, SON, previously known as an RNA splicing factor, has been found to also control MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased histone 3 lysine 4 (H3K4me3) and transcriptional repression. Alternatively-spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full- length SON for chromatin occupancy, but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full- length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. These findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia.

[0128] SON is a ubiquitously expressed nuclear protein recently identified as an SR-like splicing co-factor. SON is required for proper RNA splicing of selective genes (Ahn et al., 201 1; Hickey et al., 2014; Lu et al., 2013; Lu et al., 2014; Martello, 2013; Sharma et al., 2011). Knockdown of SON leads to splicing defects in transcripts of other genes containing weak splice sites, and many of the affected genes are necessary for cell cycle progression and epigenetic modification (Ahn et al., 201 1; Sharma et al., 201 1). Interestingly, SON is highly expressed in human embryonic stem cells and is an essential factor in stem cell pluripotency (Chia et al., 2010; Lu et al., 2013). Further RNA-seq analyses revealed that SON knockdown causes intron retention and exon skipping at several pluripotency genes, such as OCT4, PRDM14 and E4F 1 (Lu et al., 2013).

[0129] While the role of SON in RNA-binding and splicing has been highlighted, a few studies have also suggested that SON may function in transcriptional regulation. SON has been implicated in DNA-binding (Mattioni et al., 1992; Sun et al., 2001), and SON suppresses the promoter activity of the miR-23a~27a~24-2 cluster (Ahn et al., 2013). In addition, microarray and RNA-seq experiments have shown that SON knockdown not only leads to gene downregulation which is mainly due to splicing defects, but also upregulates a substantial number of genes (Ahn et al., 2011; Lu et al., 2013; Sharma et al., 2011), strongly suggesting that SON has a repressive function in gene expression. However, whether SON is directly associated with chromosomal loci in the mammalian genome and how SON regulates transcription is completely unknown. Interestingly, in addition to full-length SON, various splice isoforms of SON have been predicted based on analyses of expressed sequence tags (ESTs) and genomic DNA sequence. Nevertheless, the functional significance of SON isoforms remains unexplored.

[0130] As described herein, SON has an unexpected role in interacting with menin and regulating MLL complex activity, H3K4me3, and transcriptional initiation of multiple leukemia-associated genes. Described herein are significant increases in acute myeloid leukemia in short splice variants of SON, which lack menin-interacting ability, and their functional significance in blocking full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing.

[0131] Applicant has discovered that an increase in the ratio of the level of a truncated alternatively-spliced transcript of a SON gene, such as SON E and/or SON B transcripts of the SON gene, to the level of a full length transcript of the SON gene, such as a SON F transcript can indicate a subject having a hematopoietic malignancy, or the likelihood of the subject developing a hematopoietic malignancy. Applicant has also discovered that increases in the level of a truncated alternatively-spliced transcript of a SON gene, such as SON E and/or SON B transcripts of the SON gene, can indicate a subject having a hematopoietic malignancy, or the likelihood of the subject developing a hematopoietic malignancy. Applicant has also discovered that an increase in the levels of additional markers, such CDKN1A, GFI1 and ATF3 can also indicate a subject having a hematopoietic malignancy, or the likelihood of the subject developing a hematopoietic malignancy.

Methods for diagnosis

[0132] Some embodiments of the methods and compositions provided herein include methods for the diagnosis of a hematopoietic malignancy, or a likelihood of developing a hematopoietic malignancy in a test subject. In some embodiments the hematopoietic malignancy can include acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In some embodiments, the hematopoietic malignancy is an AML subtype including M2 without t(8;21), and t(8;21)(q22;q22).

[0133] Some embodiments of the methods and compositions provided herein relate to a short polypeptide or a nucleic acid encoding the short polypeptide. In some embodiments, the short polypeptide is a truncated alternatively-spliced transcript of a SON gene. See for example, TABLE 1. In some embodiments, the truncated alternatively-spliced transcript of a SON gene comprises a SON E variant of a SON gene, or a SON B variant of a SON gene. In some embodiments, the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10. In some embodiments, the short polypeptide comprises SEQ ID NOs:88 or 90. In some embodiments, the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.

[0134] Some embodiments of the methods and compositions provided herein relate to a long polypeptide or a nucleic acid encoding the long polypeptide. In some embodiments, the long polypeptide is encoded by a full length transcript of the SON gene. See e.g., TABLE 1. In some embodiments, the full length transcript of the SON gene comprises a sequence which specifically binds to an oligonucleotide comprising a sequence selected from SEQ ID NOs:13-18. In some embodiments, the long polypeptide comprises SEQ ID NO: 86. In some embodiments, the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.

[0135] In some embodiments, the SON gene is a human SON gene.

[0136] Some embodiments include determining a ratio for the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject. In some such embodiments, the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene, and the long polypeptide is encoded by a full length transcript of the SON gene.

[0137] Some embodiments include methods of detecting a ratio for the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in a test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject. Such methods can include obtaining a nucleic acid or polypeptide sample from the test subject; contacting the nucleic acid or polypeptide sample with an agent which indicates the level of the short polypeptide or a nucleic acid encoding the short polypeptide in the test subject; contacting the nucleic acid or polypeptide sample with an agent which indicates the level of the long polypeptide or a nucleic acid encoding the long polypeptide in the test subject; and determining the ratio for the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject to the level of the long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject. In such embodiments, the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene, and the long polypeptide is encoded by a full length transcript of the SON gene.

[0138] In the foregoing methods, a ratio of the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in a test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject can be indicative of a test subject having a diagnosis for a hematopoietic malignancy, or a likelihood of developing a hematopoietic malignancy. In some embodiments, the ratio is greater than about 30%, 40%, 50%, 60%, 70%, 80%, and 90%, or any range between any two of the foregoing numbers.

[0139] Some embodiments include methods of detecting an increase in the level of a short polypeptide or a nucleic acid encoding the polypeptide in a test subject. Such methods can include obtaining a nucleic acid or polypeptide sample from the test subject; contacting the nucleic acid or polypeptide sample with an agent which indicates an increase in the level of the short polypeptide or a nucleic acid encoding the short polypeptide in the test subject; and determining the level the short polypeptide or a nucleic acid encoding the short polypeptide in the test subject. In such embodiments, the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.

[0140] Some embodiments include methods of diagnosing a hematopoietic malignancy, or the likelihood of developing a hematopoietic malignancy in a test subject. Such embodiments can include determining the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in a subject. In such embodiments, the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.

[0141] Some methods also include comparing the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in a test subject with the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in a subject not having a hematopoietic malignancy. In some embodiments, an increase in the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in a subject is indicative of a test subject having a diagnosis for a hematopoietic malignancy, or a likelihood of developing a hematopoietic malignancy. In some embodiments, the increase is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, and 200%, or any range between any two of the foregoing numbers, compared to the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in a subject not having a hematopoietic malignancy.

[0142] Some methods also include determining the level of a long polypeptide or a nucleic acid encoding the long polypeptide in the test subject, wherein the long polypeptide is encoded by a full length transcript of the SON gene. Some such methods also include determining a ratio for the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject. In the foregoing methods, a ratio can be indicative of a test subject having a diagnosis for a hematopoietic malignancy, or a likelihood of developing a hematopoietic malignancy. In some embodiments, the ratio is greater than about 30%, 40%, 50%, 60%, 70%, 80%, and 90%, or any range between any two of the foregoing numbers.

[0143] Some methods also include determining the level of a polypeptide in addition to the level of the short form of the SON polypeptide or the level of a nucleic acid encoding the additional polypeptide in addition to the level of a nucleic acid encoding the short form of the SON polypeptide, wherein the additional polypeptide is encoded by a gene selected from CDKN1A, GFI1 and ATF3. Some methods also include comparing the level of an additional polypeptide or the level of a nucleic acid encoding the additional polypeptide in the test subject with the level of an additional polypeptide or the level of a nucleic acid encoding the additional polypeptide in a subject not having a hematopoietic malignancy. In some embodiments, the increase is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, and 200%, or any range between any two of the foregoing numbers, compared to the level of an additional polypeptide or the level of a nucleic acid encoding the additional polypeptide in a subject not having a hematopoietic malignancy.

[0144] Some embodiments also include determining the level of a polypeptide encoded by a SON target gene or a nucleic acid encoded by a SON target gene in a sample from a test subject. In some embodiments, the SON target gene has a SON binding site. In some embodiments, an increased level of a polypeptide encoded by a SON target gene or a nucleic acid encoded by a SON target gene in the sample from the test subject compared to the level of a polypeptide encoded by a SON target gene or a nucleic acid encoded by a SON target gene in a sample from a subject not having a hematopoietic malignancy can be indicative of a diagnosis of a hematopoietic malignancy, or a likelihood of developing a hematopoietic malignancy in a test subject. In some embodiments, the SON target gene is selected from the group consisting of FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFT1 , EGR1 , SRC, and GFT1 B.

[0145] Some embodiments also include determining the level of a MLL multi- protein complex in a sample from a test subject. Some embodiments also include comparing the level of the MLL multi-protein complex in the sample from the test subject with the level of a MLL multi-protein complex in a sample from a sample from a subject not having a hematopoietic malignancy. In some embodiments, an increase in the level of a MLL multi- protein complex in the sample from a test subject can be indicative of a diagnosis of a hematopoietic malignancy, or a likelihood of developing a hematopoietic malignancy in a test subject. In some embodiments, the MLL is selected from the group consisting of MLLl, MLL-N, MLL-C, and MLL2. In some embodiments, the multiprotein complex includes a protein selected from menin, ASH2L, LEDGF, and WDR5.

[0146] Some embodiments also include determining the level of a replating activity of a hematopoietic progenitor cell of a sample from a test subject. Some embodiments also include comparing the level of replating activity of a hematopoietic progenitor cell of the sample from the test subject with the level of replating activity of a hematopoietic progenitor cell of the sample from the test subject from a subject not having a hematopoietic malignancy. In some embodiments, an increase in the level of replating activity of a hematopoietic progenitor cell of the sample from the test subject can be indicative of a hematopoietic malignancy. In some embodiments, the hematopoietic progenitor cell is a bone marrow cell.

[0147] Some embodiments also include determining the level of binding of a protein at the or adjacent to the transcription start site of a SON target gene in a sample from a test subject. Some embodiments also include comparing the level the level of binding of a protein at the or adjacent to the transcription start site of a SON target gene in a sample from a test subject with the level of binding of a protein at the or adjacent to the transcription start site of a SON target gene in a sample from a subject not having a hematopoietic malignancy. In some embodiments, an increased binding of a protein at the or adjacent to the transcription start site of a SON target gene in the sample from the test subject can be indicative of a hematopoietic malignancy. In some embodiments, the protein can include MLL1, MLL-N, MLL-C, MLL2, WDR5, ASH2L, menin, SET1A, SET1B, ASC2, and SUZ12. In some embodiments, the SON target gene can include CDKN1 A, GADD45A, NOTCH2NL, ATF3, GFI1, EGR1, and SRC.

[0148] Some embodiments also include determining the level in H3K4me3 in the sample from the test subject. Some embodiments also include comparing the level in H3K4me3 in the sample from the test subject with the level in H3K4me3 in the sample from a subject not having a hematopoietic malignancy. In some embodiments an increased level in H3K4me3 in a sample from a test subject can be indicative of a hematopoietic malignancy. In some embodiments, the level in H3K4me3 is increased at the or adjacent to a SON target gene including CDKN1A, GADD45A, NOTCH2NL, ATF3, GFI1, EGR1, and SRC.

[0149] Some embodiments also include a sample from the test subject. In some embodiments, the sample can include nucleic acids or polypeptides. In some embodiments, the sample comprises bone marrow mononuclear cells (BM-MNCs), or peripheral blood mononuclear cells (PB-MNCs).

[0150] Some embodiments also include contacting the sample with an agent which specifically binds to the short polypeptide or a nucleic acid encoding the short polypeptide. Some embodiments also include contacting the sample with an agent which specifically binds to the long polypeptide or a nucleic acid encoding the long polypeptide. In some embodiments, the agent is a primer or a hybridization probe. In some embodiments, the primer or the hybridization probe comprises an oligonucleotide comprising SEQ ID NOs:06, 10 or 13-18.

[0151] Some embodiments also include contacting the sample with an agent which specifically binds to a SON target gene, such as a SON target gene having a SON binding site. In some embodiments, the SON target includes FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFI1, EGR1, SRC, and GFI1B.

[0152] Some embodiments also include contacting the sample with an agent which specifically binds to a protein selected from MLL1, MLL-N, MLL-C, MLL2, menin, ASH2L, LEDGF, WDR5, SET1A, SET1B, ASC2, SUZ12, and H3K4me3.

[0153] In some embodiments, the agent is an antibody or antigen-binding fragment thereof which specifically binds to the short polypeptide.

[0154] In some embodiments, the agent can be immobilized on a solid support. Examples of solid supports include a test well of a microtiter plate, a membrane, such as nitrocellulose membrane, a particle, such as a bead, comprising glass, fiberglass, latex, or a plastic material, such as polystyrene or polyvinylchloride. The solid support can include a magnetic particle. The agent can be immobilized on the solid support by a noncovalent association, such as adsorption, and/or a covalent attachment which can include a direct linkage between the agent and functional groups on the solid support, or may be a linkage by way of a cross-linking agent.

[0155] Methods to determine the level of a nucleic acid, such as a transcript of a SON gene, such as a truncated alternatively-spliced transcript of a SON gene, such as SON B variant, and SON E variant; a full length transcript of a SON gene, such as SON F; and nucleic acids encoded by CDKN1A, GFI1 and ATF3 are also well known in the art. Examples of the foregoing methods include hybridizing a hybridization probe or primer with a nucleic acid. Methods can include PCR, quantative methods of PCR, such as real time PCR, and Northern blot analysis. Techniques for both PCR based assays and hybridization assays are well known in the art {See e.g., Mullis et ah, Cold Spring Harbor Symp. Quant. Biol., 51 :263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989). In some embodiments, the hybridization probe or primer can include an oligonucleotide comprising any one of SEQ ID NOs:06, 10, 13-18.

[0156] Methods to determine the level of a polypeptide, such as a polypeptide encoded by a truncated alternatively-spliced transcript of a SON gene, such as SON B variant, and SON E variant; a full length transcript of a SON gene, such as SON F; and nucleic acids encoded by CDKN1A, GFI1 and ATF3 are well known in the art. Examples of the foregoing methods include contacting the polypeptide with an agent that that specifically binds to the polypeptide. In some embodiments, the agent comprises an antibody or antigen- binding fragment thereof.

[0157] Some embodiments of the methods and compositions provided herein also include a method of diagnosing a hematopoietic malignancy or a likelihood of developing a hematopoietic malignancy in a test subject that includes detecting increased levels of binding of a short polypeptide with a nucleic acid having a SON binding site in a sample from a test subject, wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.

Methods of treatment

[0158] Some embodiments of the methods and compositions provided herein also include methods of ameliorating a hematopoietic malignancy. Some such methods include treat, preventing, and/or reducing the symptoms of the hematopoietic malignancy. Some embodiments include increasing the level of a full length SON polypeptide or a nucleic acid encoding a full length SON polypeptide in a cell of a subject. In some embodiments, the expression level of a nucleic acid encoding SON or the expression level of SON protein is increased by administering an isolated nucleic acid to the subject. In some embodiments, the nucleic acid comprises a sequence encoding a full length SON polypeptide. In some embodiments, the SON polypeptide is a human SON polypeptide. In some embodiments, the nucleic acid comprises SEQ ID NO.:85. In some embodiments, the polypeptide comprises SEQ ID NO.: 86. In some embodiments, the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplasia syndrome (MDS). In some embodiments, the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22). In some embodiments, the subject is mammalian. In some embodiments, the subject is human.

[0159] Methods to increase the level of a polypeptide or a nucleic acid encoding a polypeptide, such as a full length SON polypeptide, in a cell of a subject are well known in the art. For example, some methods can include administering an expression vector to the subject. The expression vector can include the nucleic acid and a promoter. The promoter can be tissue-specific, constitutive, and/or inducible.

[0160] Some embodiments include pharmaceutical compositions that include the nucleic acid, such as an expression vector including the nucleic acid, and a pharmaceutically effective carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

[0161] The pharmaceutical compositions can be administered by various means known in the art. For example, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy), followed by re-implantation of the cells into a subject, usually after selection for cells which have incorporated the vector. Therapies where cells are genetically modified ex vivo, and then re-introduced into a subject can be referred to as cell-based therapies or cell therapies. In a preferred embodiment, cells are isolated from the subject organism, transduced with an exogenous gene (gene or cDNA) according to the present disclosure, and re-infused back into the subject (e.g., a patient).

[0162] Expression vectors can include plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, helper-dependent adenovirus, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,1 13; 6,979,539; 7,013,219; and 7,163,824, incorporated by reference herein in their entireties. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding a full length SON polynucleotide in cells (e.g., mammalian cells) and target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, T1BTECH 1 1:211-217 (1993); Mitani & Caskey, T1BTECH 1 1 : 162-166 (1993); Dillon, TIBTECH 1 1 :167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1 149-1 154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31- 44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1 :13-26 (1994). Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, dendrimers, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, agent-enhanced uptake of DNA or use of macromolecules such as dendrimers (see Wijagkanalen et al (201 1) Pharm Res 28(7) p. 1500-19). Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.

[0163] Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336). Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024.

[0164] In applications in which transient expression is preferred, adenoviral based systems can be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno -associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81 :6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0165] Gene therapy vectors, such as expression vectors comprising a nucleic acid encoding a full length SON polypeptide, can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem/progenitor cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.

[0166] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing donor construct, such as expression vectors comprising a nucleic acid encoding a full length SON polypeptide, can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA complexed/formulated with a delivery vehicle (e.g. liposome or poloxamer) can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0167] Formulations for both ex vivo and in vivo administrations include suspensions in liquid or emulsified liquids. The active ingredients often are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances, such as, wetting or emulsifying agents, pH buffering agents, stabilizing agents or other reagents that enhance the effectiveness of the pharmaceutical composition.

Kits

[0168] Some embodiments of the methods and compositions provided herein include kits for the diagnosis of a hematopoietic malignancy, or likelihood of developing a hematopoietic malignancy in a test subject. Some such embodiments can include an agent that specifically binds to a short polypeptide or a nucleic acid encoding the short polypeptide. In such embodiments, the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene. Some such embodiments can include an agent that specifically binds to a long polypeptide or a nucleic acid encoding the long polypeptide. In such embodiments, the long polypeptide is encoded by a full length transcript of the SON gene.

[0169] Some embodiments also include an agent that specifically binds to an additional polypeptide or a nucleic acid encoding the additional polypeptide in the test subject, wherein the additional polypeptide is encoded by a gene selected from CDKN1A, GFI1 and ATF3.

[0170] In some embodiments, the truncated alternatively-spliced transcript of a SON gene comprises a SON E variant of a SON gene. In some embodiments, the truncated alternatively-spliced transcript of a SON gene comprises a SON B variant of a SON gene. In some embodiments, the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10. In some embodiments, the short polypeptide comprises SEQ ID NOs:88 or 90. In some embodiments, the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.

[0171] In some embodiments, the full length transcript of the SON gene comprises a sequence which specifically binds to an oligonucleotide comprising a sequence selected from SEQ ID NOs: 13-18. In some embodiments, the long polypeptide comprises SEQ ID NO:86. In some embodiments, the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.

[0172] In some embodiments, the SON gene is a human SON gene. [0173] Some embodiments also include an agent that specifically binds to a SON target gene, wherein the SON target gene has a SON binding site. In some embodiments, the SON target gene can include FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFI1, EGR1, SRC, and GFI1B.

[0174] Some embodiments also include an agent that specifically binds to a includingMLLl, MLL-N, MLL-C, MLL2, menin, ASH2L, LEDGF, WDR5, SET1A, SET IB, ASC2, SUZ12, and H3K4me3.

[0175] In some embodiments, the agent is a primer or a hybridization probe. In some embodiments, the primer or the hybridization probe comprises an oligonucleotide comprising SEQ ID NOs:06, 10, or 13-18. In some embodiments, the agent is an antibody or antigen-binding fragment thereof which specifically binds to the short polypeptide. In some embodiments, the agent is attached to a solid support.

EXAMPLES

Genome-wide analyses of SON binding sites revealed SON interaction with G/C-rich sequences near transcription start sites and SON depletion caused activation of SON target genes

[0176] To explore SON function in genome-wide DNA-binding and gene regulation, chromatin immunoprecipitation and sequencing (ChlP-seq) in K562 leukemic cells was performed with two different SON antibodies (SON-N and SON-C Abs) (FIG. 1 A). Through pilot experiments, these antibodies were validated to be suitable for ChIP experiments based on enrichment and reproducibility of peaks (FIG. 1G, FIG. 1H). The results from ChlP-seq with SON-N and SON-C Abs identified the genomic distribution of SON binding sites at both intergenic and intragenic regions, with a significant portion of the intragenic peaks located at promoters and introns (FIG. IB). To focus on SON function near transcription start sites, the ChlP-seq peaks located 5 kb upstream and downstream (± 5kb) from the transcription start site were analyzed, which were mainly localized in the promoter, the 5' UTR, and the first exon or intron of the target genes (FIG. 1C). The heat map and motif analysis confirmed that SON binding sites are indeed enriched near transcription start sites (FIG. ID) and contain repetitive G or C tracts as well as GC dinucleotide repeats (FIG. IE and FIG. II). The genes bearing SON peaks at their transcription start site have functions in DNA-binding / transcription (e.g. ATF3, GFI1, EGR1, FOX03A), receptor signal transduction (NOTCH2NL, SRC) and cell cycle regulation (CDKN1A, GADD45A) (FIG. IF). Altered expression of these genes has been implicated in perturbed hematopoiesis, leukemia and other cancers (Janz et al., 2006; Joslin et al., 2007; Khandanpour et al., 2013; Liebermann et al., 201 1; Phelan et al., 2010; Viale et al., 2009). Enrichment of SON near transcription start sites of these genes was further confirmed by ChlP-qPCR (FIG. 2E) and deletion of the potential DNA-binding region (Sun et al., 2001) reduced SON interaction with target DNA (FIG. 2F). Interestingly, knockdown of SON by siRNA (FIG. 2G) caused upregulation of SON ChIP target genes (FIG. 2A), indicating that SON interaction with transcription start sites of target genes exerts inhibitory effects on transcription.

SON binding sites near the transcription start site overlap with the locations of the histone modification H3K4me3 and SON depletion leads to increased levels of H3K4me3

[0177] To elucidate the function of SON near the transcription start site, the genomic location of SON peaks were compared with the location of several histone modifications which are involved in regulation of transcriptional initiation. Interestingly, while the regions with intergenic SON peaks were enriched with mono-methylation of histone 3 lysine 4 (H3K4mel) (FIG. 2H), SON peaks near transcription start sites were closely associated with the locations of H3K4me3, a marker of active or potentially active promoters, and CpG islands (FIG. 2B). Concurrence of SON peaks with H3K4me3 peaks and CpG islands were further visualized and confirmed in several SON target genes (FIG. 2C, FIG. 21). The genomic regions bearing SON peaks near transcription start sites show a low level of conventional nucleosomes and the presence of variant histone, H2A.Z, indicating the active chromatin status (FIG. 2J). A close look at the peak regions revealed that the high peaks of H3K4me3 were precisely aligned with the valleys of SON peaks, and vice versa (FIG. 2D, FIG. 2J). These observations suggest that SON may bind to the region between nucleosomes and modify histones within adjacent nucleosomes.

[0178] Extensive ChlP-qPCR analyses revealed that the level of H3K4me3 at SON target sites near the transcription start site was significantly increased (FIG. 3A, FIG. 3C, FIG. 3D), while H3K4me3 at the transcription start site of unrelated genes (non-targets) did not show any significant changes upon SON knockdown (FIG. 3D). The levels of H3K4mel and H3K27ac also showed an increase in a few sites, while H3K27me3 did not show any changes (FIG. 3A, FIG. 3C). Taken together, these data demonstrate that SON functions to lower the level of H3K4me3 near transcription start sites.

SON depletion leads to increased recruitment of MLL complex components to the SON target genes and enhanced MLL complex formation

[0179] To understand the mechanism of the increased H3K4me3 upon SON knockdown, the occupancy of MLL and SET1 complex components at SON target sites near the transcription start site was measured. Interestingly, significant increases in occupancies of MLL (MLL1 N-terminus and C-terminus), MLL2, WDR5, ASH2L and menin at SON target genes were detected in SON-depleted cells (FIG. 3B, FIG. 3C, FIG. 3D). In contrast, recruitment of SET1A, SET1B and ASC2/NCOA6 (a component of MLL3/4 complex) to the SON target sites was not increased upon SON knockdown (FIG. 3B). These results indicate that SON inhibits recruitment of MLL 1, MLL2 and their associated components to the target chromatin region.

[0180] While MLL itself is a weak H3K4 methyltransferase, its interaction with the multiple subunit proteins greatly increases the ability to induce H3K4me3 (Dou et al., 2006; Steward et al., 2006). Since increases in both chromatin occupancy of the MLL complex and H3K4me3 in SON-depleted cells were detected, it was hypothesized that the protein interactions between the MLL complex subunits may be altered by SON knockdown. To this end, immunoprecipitation (IP) with an MLL1 N-terminus (MLL-N) antibody was performed and the presence of other MLL complex components by Western blotting was examined. Surprisingly, depletion of SON (FIG. 4A) significantly facilitated MLL-N interaction with MLL1 C-terminus (MLL-C), WDR5, ASH2L, menin and LEDGF (FIG. 4B). Immunoprecipitation with a WDR5 antibody further confirmed the enhanced interaction of WDR5 with MLL-C, MLL2, and menin upon SON depletion (FIG. 4C). Increased LEDGF interaction with MLL-N and menin in SON siRNA-transfected cells was also confirmed by LEDGF IP (FIG. 4D). In contrast, both WDR5 IP and SET1A IP experiments demonstrated that the protein interactions within the SET1A complex and the MLL3/4 complex were not affected upon SON knockdown (FIG. 4C and FIG. 4E). These findings revealed that SON exerts its inhibitory effect specifically on MLL1/2 complex assembly.

SON interacts with menin, an MLL1/2 complex component, and SON-menin interaction diminishes MLL-menin interaction

[0181] The findings on the inhibitory effect of SON on MIX 1/2 complex formation prompted the examination of the possibility of physical association of SON with MLL1/2 complex subunits. Immunoprecipitation with SON antibody to detect SON- associated proteins revealed that SON interacts with menin, an MLL1/2 complex component involved in MLL function in oncogenesis (FIG. 4F). SON interaction with menin was further confirmed by overexpression and IP experiments (FIG. 4G). However, none of other components of the MLL complex, such as MLL-N, MLL-C, ASH2L, WDR5 and LEDGF, were detected in SON co-IP (FIG. 4F), suggesting that the SON-menin complex is not incorporated into a complete MLL complex. To examine the effect of SON on the interaction of menin with its direct binding partner MLL-N, menin-MLL-N interaction was assessed as well as menin-SON interaction by IP with menin antibody. Surprisingly, SON overexpression increased the menin-SON complex formation and at the same time, menin interaction with MLL-N was significantly decreased (FIG. 4H). Size-exclusion chromatography further demonstrated that a higher amount of menin was detectable in the MLL 1 -containing fractions when SON was depleted by siRNA (FIG. 41). These findings demonstrate an inhibitory effect of menin-SON interaction on menin-MLL interaction.

[0182] An increased interaction between MLL and menin upon SON knockdown was also observed in MV4; 11 cells which express MLL-fusion protein (MLL-AF4) as well as wild-type MLL (FIG. 4J and FIG. 4K). SON having an inhibitory effect on the interaction between menin and the MLL-fusion protein MLL-ENL was demonstrated (FIG. 4L). HOXA9 and MEIS 1, well-known transcriptional targets of MLL-fusion proteins, were also upregulated upon SON knockdown in MLL-rearranged cell lines (FIG. 4M), indicating inhibitory effects of SON on expression of MLL-fusion protein target genes. Short isoforms of SON generated by alternative splicing are upregulated in acute myeloid leukemia

[0183] In addition to full-length SON (isoform F; SON F hereafter), several splice variants of SON have been predicted in genome databases (TABLE 1). An alternative exon is located between exon 6 and exon 7 of the human SON gene (labeled as exon 7a), and inclusion of exon 7a generates the isoform B (SON B). There is another alternative exon within intron 4 (labeled as exon 5a), and inclusion of this exon generates the isoform E (SON E). Both SON B and SON E are C-terminally truncated forms. Similar forms of full-length and the short splice variants (Son f, Son b and Son e) have been predicted in mice (FIG. 5A, FIG. 5H, and TABLE 1).

[0184] Interestingly, exon 5a is extremely well conserved between human and mouse (Wynn et al., 2000), suggesting functional significance of the isoforms made by inclusion of this alternative exon. Despite such information, no experimental data have proven the expression of these SON splice variants. 3' rapid amplification of cDNA ends (3' RACE) in K562 cells was performed and confirmed that mRNA of SON E with its own poly(A) site is indeed expressed (FIG. 51 and FIG. 5J).

[0185] To address the functional significance of SON splice variants, whether SON splice variants are differentially expressed in AML was examined. Bone marrow mononuclear cells (BM-MNCs) from AML patients (FAB subtype M2) and healthy donors (TABLE 2) were analyzed by qPCR using several primer sets (FIG. 5A). Similar to previous results (Ahn et al., 2013), most patient samples showed high levels of total SON (exon 1—3 region). Surprisingly, while expression levels of the exon 9— 12 region specific for SON F did not show significant upregulation, the expression level of alternative exon (exon 5a or 7a)-containing transcripts were significantly increased in AML patient BM-MNC samples (FIG. 5B). the SON isoform levels in peripheral blood mononuclear cells (PB-MNCs) isolated from AML and myelodysplastic syndrome (MDS) patients were measured (TABLE 2), and significant upregulation of exon 5 a- and exon 7a-containing transcripts were observed (FIG. 5C). These findings revealed that the SON upregulation in AML patients is largely attributable to upregulation of short isoforms. [0186] The relative proportions of full-length SON and the isoforms in normal donors and AML patients were measured (FIG. 5K). The results revealed that SON F is the major form of SON in normal human BM-MNCs, and the short isoforms (SON B and SON E) occupy -20% of total SON (FIG. 5D). In contrast, the portion of SON E was remarkably increased in AML patient BM-MNCs, resulting in SON E accounting for 30 - 70% of total SON (FIG. 5D). PB-MNCs were also analyzed for relative ratios of full-length and SON isoforms. While PB-MNCs from 7 normal healthy donors represent almost identical patterns showing that only -10% of total SON is taken by SON E and SON B, the percentage of SON E and especially SON B are markedly increased in AML and MDS patients (FIG. 5E). Further evidence of short isoform expression was demonstrated in leukemic blasts isolated from mouse models of AMLl-ET09a-mediated leukemia and MLL-AF9-induced leukemia (FIG. 5F and FIG. 5G). In addition, microarray data available from Oncomine revealed that expression of exon 7a detected by the exon 7a-specific probe sets (FIG. 5L) was significantly increased in leukemia and lymphoma samples (FIG. 5M and FIG. 5N). Collectively, all of these results demonstrated the C-terminally truncated short isoforms of SON are aberrantly upregulated in hematopoietic malignancies, particularly in AML.

SON E attenuates the full-length SON function in transcriptional repression, resulting in derepression of SON ChIP target genes, but does not impair full-length SON-mediated RNA splicing

[0187] Whether the increase of SON short isoforms has any effect on expression of SON target genes in AML patients was investigated. Among SON ChIP target genes, CDKN1A, GFI1 and ATF3 were selected (FIG. 6A) since the importance of tight regulation of these genes in leukemia and other cancers has been demonstrated (Abbas and Dutta, 2009; Janz et al., 2006; Khandanpour et al., 2013; Phelan et al., 2010; Viale et al., 2009). These target genes were indeed significantly upregulated in BM-MNCs from AML patients compared with healthy donors (FIG. 6A), indicating that increased expression of SON short isoforms is associated with de-repression of SON ChIP target genes in AML.

[0188] To examine the exact effect of SON short isoforms on SON target gene expression, a specific siRNA targeting the SON E-specific exon 5a was developed (FIG. 6G), and confirmed that this siRNA lowers the level of SON E, but not SON F (FIG. 6B and FIG. 6C). Interestingly, while the total SON siRNA that targets all forms of SON significantly upregulated SON ChIP target genes, transfection of SON E-specific siRNA led to downregulation of theses target genes in both K562 cells and human CD34+ bone marrow (BM) cells (FIG. 6B and FIG. 6C), indicating that a stronger repression of target genes occurred in the absence of SON E. Furthermore, unlike SON F overexpression which causes SON ChIP target gene repression, SON E overexpression (FIG. 6H) resulted in upregulation of those target genes in human CD34+ BM cells (FIG. 6D). These results strongly support the concept that SON E weakens the inhibitory effect of full-length SON on target gene transcription.

[0189] Since the critical role of SON in RNA splicing of a group of genes has been previously demonstrated (Ahn et al., 201 1; Lu et al., 2013; Sharma et al., 2011), how those genes are regulated upon SON E knockdown and overexpression was examined. While total SON siRNA significantly reduced the level of SON's splicing target genes, such as TUBG1, HDAC6 and A T1, knockdown of SON E caused no change or a marginal decrease in the expression of these splicing target genes (FIG. 6B). Furthermore, SON E overexpression in human CD34+ BM cells did not affect the level of SON's RNA splicing target genes (FIG. 6D), indicating that short SON isoforms do not exert an inhibitory effect on the expression of the target genes undergoing SON-mediated RNA splicing.

[0190] Whether SON E expression affects SON F function in repressing H3K4me3 was examined. SON F (siRNA-resistant form) expression could lower the H3K4me3 level, which was increased by SON siRNA, near the transcription start sites of the CDKN1A and GFI1 genes (FIG. 6E). In contrast, expression of SON E alone (siRNA- resistant form) failed to reduce the H3K4me3 level, indicating its lack of ability to suppress H3K4me3. Interestingly, when SON E was co-expressed with SON F (FIG. 61), SON E could attenuate the repressive effect of SON F on H3K4me3 in a dose-dependent manner (FIG. 6E).

[0191] A minigene splicing assay was performed using the TUBG1 exon 7-8 minigene model (Ahn et al., 201 1) and various ratios of SON F and SON E transfection to assess the effect of SON E expression on SON F-mediated RNA splicing (FIG. 6J). SON E expression did not attenuate SON F-mediated RNA splicing (FIG. 6F and FIG. 6K). Taken together, these results demonstrate that SON E overexpression causes dysregulation of SON- mediated transcriptional repression, but not SON-mediated RNA splicing.

SON E competes with full-length SON for DNA-binding, but lacks the menin-binding ability, thus attenuating the inhibitory effect of full-length SON on MLL complex assembly

[0192] To gain further mechanistic insights, the DNA-binding ability of SON E was investigated. ChIP was performed to pull down V5-tagged SON F and Flag-tagged SON E (FIG. 7A) after expression of different amounts of SON E together with SON F. ChlP- qPCR revealed the enrichment of transfected SON E at SON target sites near transcription start sites of the CDKN1A and ATF3 genes (FIG. 7B), demonstrating that SON E indeed retains its ability to associate with target DNA. Increased SON E binding to the target sites concomitantly led to decreased SON F binding to the same sites (FIG. 7B). These data demonstrate that SON F and SON E share the common target DNA for binding, and they compete with each other to occupy the same chromatin region. Therefore, overexpression of SON E results in displacement of full-length SON from the target DNA.

[0193] Since the data demonstrated that SON F interaction with menin abrogates MLL-menin interaction (FIG. 4F), whether SON E retains the menin-binding ability was investigated. Surprisingly, unlike SON F, SON E does not interact with menin (FIG. 7C). It was demonstrated that the C-terminus of SON containing the RS domain and RNA-binding motifs (SR+RB in FIG. 7A) indeed interacts with menin (FIG. 7D) through the central region of menin (FIG. 7J and FIG. 7K). This region has been known to be important for menin interaction with MLL (Murai et al., 2011), suggesting that the C-terminal region of SON potentially occupies the MLL-binding site within menin.

[0194] The effects of SON F and SON E overexpression (FIG. 7L) on MLL interaction with menin and MLL complex formation were examined. While SON F overexpression inhibits MLL-N interaction with menin, SON E overexpression further enhanced the interactions between MLL-N with menin. SON E overexpression also strengthened MLL-N interaction with MLL-C and WDR5, indicating that the whole MLL complex assembly is enhanced upon increased SON E expression (FIG. 7E). SON E overexpression enhances in vitro replating capacity of primary mouse bone marrow cells

[0195] To further assess the functional significance of SON E in hematopoietic cells, SON E was overexpressed in mouse primary bone marrow cells through lenviral transduction (FIG. 7F, FIG. 7M, and FIG. 7N), and measured the colony forming ability in methylcellulose replating assays (FIG. 7M). Surprisingly, SON E-overexpressing cells were able to produce significantly increased numbers of colonies and were able to maintain colony forming cells even in the 5th round of plating (FIG. 7G and FIG. 7H), indicating higher clonogenic potential. The findings demonstrate that increased expression of SON short isoforms enhances the preservation of sternness in normal hematopoietic progenitors, suggesting its potential contribution to stem cell self-renewal and development and/or maintenance of leukemic stem cells.

Discussion

[0196] SON was previously known as an RNA splicing co-factor. This study demonstrated that SON and its splice variants regulate MLL complex assembly and H3K4me3, affect gene expression of multiple leukemia-associated genes, and affect replating potential of hematopoietic progenitors. The data suggest that although the short splice variants, such as SON E, interact with target DNA, they cannot exert an inhibitory effect on MLL complex assembly due to the lack of menin-binding ability. Therefore, overexpression of "short SON" in pathological conditions, such as AML, attenuates the inhibitory effects of full-length SON on MLL complex assembly, resulting in activation of multiple target gene transcription (modeled in FIG. 71). These current findings suggest that there are two arms for the biological function of SON: one supporting efficient RNA splicing and the other antagonizing MLL complex-mediated transcriptional initiation.

SON as a negative regulator of MLL-menin interaction and MLL complex assembly

[0197] It has been demonstrated that the direct interaction between MLL and menin is required for MLL target gene expression and oncogenic property of the MLL fusion protein. The importance of the MLL-menin interaction was highlighted by a recent study showing that pharmacologic inhibition of this interaction is able to block progression of MLL-rearranged leukemia (Borkin et al., 2015; Grembecka et al., 2012). In addition, blocking MLL-menin interaction with a small molecule inhibitor could effectively block growth of hormone-refractory prostate cancers (Malik et al., 2015). MLL-menin interaction is also involved in promoting hepatocellular carcinoma development (Xu et al., 2013). Given the significance of MLL-menin interaction in disease-associated gene expression, identification of endogenous regulatory factors affecting this interaction would generate valuable information for understanding and targeting the MLL complex. Our discoveries of SON as a menin-binding protein and a negative regulator of MLL-menin interaction support a potential clinical impact of SON. In addition to MLL, menin also interacts with other transcription factors and hormone receptors, such as JUND, estrogen receptor and androgen receptor (Agarwal et al., 1999; Dreijerink et al., 2006; Malik et al., 2015).

Short splice variants of SON: their effects on two arms of SON function and clinical significance

[0198] A finding in this study is identification of functional significance of SON isoforms generated by alternative splicing. This data demonstrated that a short isoform of SON (SON E) which is expressed in both human and mouse has a functional defect in menin interaction and transcriptional repression but retains the ability to interact with target DNA sequences. Although this study did not examine the function of SON B, another short isoform of SON, SON B may have similar functions as SON E based on the role of the C-terminus of SON in menin interaction (FIG. 7D and FIG. 7E). SON ChIP target genes identified include critical factors for cell-cycle and hematopoietic differentiation, such as CDKN1A, GFI1 and ATF3, which are aberrantly upregulated in AML patients with elevated levels of short SON isoforms (FIG. 5 and FIG. 6). Therefore, increased SON isoforms in hematopoietic stem cells or progenitors could potentially lead to failures in dosage controls of multiple leukemia- associated target genes. Interestingly, overexpression of SON E specifically weakened the full-length SON function in transcriptional initiation, but does not impair full-length SON- mediated RNA splicing. These results suggest that overexpression of short SON observed in leukemia patients would selectively affect SON's transcription target genes, leading to de- repression, while not blocking full-length SON-mediated RNA splicing which is involved in cell proliferation and survival.

[0199] So far, the biological significance of the MLL complex in leukemia has been studied mainly in MLL-rearranged leukemia (Bernt et al., 2011; Dorrance et al., 2006; Popovic and Licht, 2012). Dysregulation of wild-type MLL function has not been clearly linked to diseases, although wild-type MLL function was recently shown to be necessary for growth and survival of MLL-fusion leukemia (Thiel et al., 2010) and other solid tumors (Ansari et al., 2013). This work suggests that MLL functions could be altered in cancer patients without MLL-rearrangement when "short SON" is overexpressed. In addition, the increased level of short SON isoforms could serve as a biomarker indicating dysregulation of MLL-mediated H3K4me3.

[0200] SON E overexpression markedly enhances replating capacity of primary hematopoietic progenitors, shedding light on functional significance of aberrant upregulation of "short SON" in AML. This study suggests that overexpression of short SON alone may not be sufficient to drive oncogenic transformation of hematopoietic cells, but implies potential roles of short SON in self-renewal and clonogenic abilities of leukemic cells.

Experimental Procedures

ChlP-Sequencing (ChlP-seq) and ChlP-qPCR

[0201] Chromatin Immunoprecpitation (ChIP) was performed with the methods adapted from the protocol of the laboratory of Richard M. Myers which has been used in the part of the ENCODE project (http://research.hudsonalpha.org/Myers/?page_id=142). For ChlP-qPCR, all ChIP signals were normalized to total input and each experiment was performed 3 times independently. The primer sets for qPCR on the specific region of the target genes are listed in TABLE 3.

TABLE 3

[0202] ChlP-seq libraries were generated using Gnomegen Library Preparation Kits (Gnomegen), and ChlP-seq libraries were cluster-amplified and sequenced with the Illumina HiSeq2000 sequencer (50-nucleotide pair-ended read).

Primary Patient Samples

[0203] Cells were purified by Ficoll-Paque (GE Healthcare) density-gradient centrifugation and frozen as viable cells. Details of the patient samples are listed in TABLE 2.

Cell Lines and Cell Culture

[0204] K562, MV4; 1 1 and ML-2 cell lines were cultured in RPMI 1640 medium with 10% fetal bovine serum. Primary human CD34+ bone marrow cells were purchased from Lonza and were cultured in STEMSPAN SFEM (StemCell Technologies) supplemented with 1% penicillin and streptomycin, 100 ng/ml of recombinant human SCF, 100 ng/ml of recombinant human FLT3L and 100 ng/ml of recombinant human TPO for 1 to 2 days before transfection. Primary mouse bone marrow cells were cultured in STEMSPAN supplemented with 15% FBS, 1% penicillin and streptomycin, 1% Glutamate, 10 ng/ml recombinant mouse IL-3 50ng/ml recombinant mouse SCF and 50 ng/ml recombinant mouse FLT3L for 1 day after lentivirus infection. All the cytokines were purchased from PeproTech.

Plasmid Construction

[0205] Plasmids containing siRNA-resistant full-length SON cDNA (siRR-SON F) and SON SR+RB (serine/arginine-rich region and RNA-binding domain) were created as previously described (Ahn et al., 201 1). The plasmid containing SON E was constructed as follows. The region from internal Hpal site to the 3' end of SON E cDNA was amplified using the forward primer (SON Hpal-F: 5'-GAATCTTCAATTACGTTAACA-3') (SEQ ID NO:77) and the reverse primer (Flag-SON E Notl-R: 5'- TTTGCGGCCGCTATTTGTCATCGTCATCCTTGTAGTCTGGCCGGCC AAACTCAGTTTAGTTCTTCTATAGT AGCTCCTCCTG-3 ' ) (SEQ ID NO:78). The sequence for the Flag tag was added as indicated in the reverse primer. Then, the C-terminus of SON F in pcDNA3 HA-siRR SON F- Flag was replaced by the amplified SON E C- terminal fragment at the Hpal and Notl sites to create pcDNA3 HA-siRR SON E-Flag construct. To construct V5-tagged plasmids, the C-terminus region was amplified from cDNA using specific primers (SON BmgBl-F: 5'-

GC AAGTGATGTTGGACGTGACAGATC-3 ' (SEQ ID NO:79) and V5-SON F Notl-R: 5'- TTTGCGGCCGCtacgtagaatcgagaccgaggagagggttagggataggcttaccTGGCCGGCCatacctatt caagaaaaacatacaatt-3' (SEQ ID NO:80)) and subcloned into plasmids. Full-length cDNA clones of human menin (pcDNA3 Flag-menin, #32079) and MLL-ENL (pMSCV Flag-MLL- pl-ENL, #20873) were purchased from Addgene).

Reverse transcription and Quantitative PCR (RT-qPCR)

[0206] Human leukemic cell lines from one 6-well, human CD34+ BM cells from one 24-well, and patient samples were lysed and total RNA was isolated using the RNeasy Mini Kit (Qiagen). Total RNA from each sample was treated with RNase-free DNase I and used as a template to produce cDNA with oligo-dT using the Superscript III First Strand Synthesis Kit (Life Technologies). Quantitative real time PCR (RT-qPCR) was performed in triplicate reactions on the iQ5 Real Time PCR Detection System (Bio-Rad) using the Fast Start Universal SYBR Green Master (Roche) and standard deviations were calculated. All PCR reactions were finished under the following program: initial denaturation step was 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 seconds, and annealing at 60°C for 60 seconds. PCR primers were listed in TABLE 3 in the supplemental material. Gene expression levels were normalized to GAPDH.

Antibodies

[0207] The antibodies used for ChIP, IP, and Western blotting were the following; SON antibody recognizing the N-terminus of SON (SON-N Ab) was generated against amino acids 74-88 of human SON. SON-N antibody was used for both ChIP and Western blot. Anti- SON (SON-C Ab, abl21759, for ChIP), anti-H3K27ac (ab4729), anti-H3K4me3 (ab8580), anti-SUZ12 (ab l2073), anti-WDR5 (ab56919), anti-H3 (abl791), and anti-H3K4mel (ab8895), anti-H3K79me2 (ab3594) were purchased from Abeam. Anti-TRX2/MLL2 (A300- 1 13A), anti-ASH2L (A300-489A), anti-menin (A300-105A), anti-MLL-C (MLL1, C- terminus, A300-374A), anti-LEDGF (A300-848A), anti-SETIA (A300-289A), anti-SETIB (A302-281A), anti-CFPl (A303-161A), and anti-MLL-N for ChIP (MLL1 , N-terminus, A300-086A) were purchased from Bethyl Laboratories. ASC2 antibody was a gift from Dr. Jae W Lee (Oregon Health & Science University). Anti-H3K27me3 (07-4490, Millipore), anti-MLL-N for IP and WB (MLL1 N-terminus, 39829, Active motif), anti-V5 (R960-25, Invitrogen), anti-HA (#2367, Cell Signaling Technology), anti-Flag M2 (F3165, Sigma), and anti-Actin (A5441 , Sigma) were purchased from the indicated companies.

Chromatin Immunoprecpitation (ChIP)

[0208] K562 cells were incubated with 1% formaldehyde in 5 ml growth medium for 10 min at room temperature and cross-linking reaction was terminated by incubation with 125 raM glycine for 10 min. Subsequently cells were incubated for 15 min at 4°C with lysis buffer (5 mM PIPES pH 8.0 / 85 mM KC1 / 0.5% NP-40 / lx Complete Protease Inhibitor Cocktail (Roche)), collected by centrifugation for 5 min at 3,000g and resuspended in RIPA buffer (150 mM NaCl / 50 mM Tris-HCl, pH 8.0/ 1 mM EDTA / 1% sodium deoxycholate / 0.1% SDS / 1% Triton X-100 / lx Complete Protease Inhibitor Cocktail). To shear chromatin to lengths ranging between 200—500 base pairs, crude nuclei were sonicated with the Ultrasonic disintegrator Sonicator S-4000 (Misonix). Sonicated DNA from each sample were incubated at 4°C overnight with 1—5 μg of specific antibodies or normal immunoglobulin G (IgG) as controls and magnetic bead protein A or G (Dynabeads Protein A or Protein G, Life Technologies). The magnetic beads were washed 5 times for lOmin at 4°C on a rotating platform with 1ml wash buffer (100 mM Tris pH 7.5 / 500 mM LiCl / 1% NP-40 / 1% Sodium deoxycholate) and washed once with TE (10 mM Tris pH 7.5 / 0.1 mM EDTA). After washing, the washed beads were eluted by heating for 2hr at 65 °C in elution buffer (1% SDS / 0.1 M NaHC03) with proteinase K. ChIP DNA were purified and concentrated using the QIAquick PCR Purification Kit (Qiagen). siRNA and Plasmid Transfection

[0209] Total SON siRNAs directed against human SON (siSON #1 : GCAUUUGGCCCAUCUGAGAtt, (SEQ ID NO:81) Ahn et al, 2011 ; siSON #2: UGAGCGCUCUAUGAUGUCAtt, (SEQ ID NO:82) Lu et al, 2013), human SON E-specific siRNA (siSON E: CACCGGAGCUUGGAAAUUAtt (SEQ ID NO:83)), and negative control siRNA (UAACGACGCGACGACGUAAtt (SEQ ID NO:84)) were custom synthesis products by Life Technologies (Silencer Select siRNA). For K562 and ML2 cells, 0.5^χ 10⁶ cells were nucleofected with 100 - 200 pmol of siRNA or 5 μg of plasmid using Cell Line Nucleofector Kit V (Lonza) according to the manufacturer's instructions. For MV4;11 cells, 0.5x 106 cells were nucleofected with 150 - 200 pmol of siRNA using Cell Line Nucleofector Kit L (Lonza) according to the manufacturer's instructions. For human CD34+ bone marrow cells, 0.4x 106 cells were nucleofected with 80 - 150 pmol of siRNA or 4 μg of plasmid using Human CD34+ Cell Nucleofector Kit (Lonza) according to the manufacturer's instructions. Human embryonic kidney (HEK) 293 cells were transiently transfected with 5 μg of plasmids using PEL

ChlP-Seq Analysis

[0210] ChlP-sequencing data files were aligned to the hgl9 human reference genome using Bowtie (version 0.12.9) and standard parameters. Peak calling was performed with MACS vl .4.2 software using default parameters. To identify high confidence SON binding peaks, the MACS peak calling output from two different experimental samples were used. BigWig files were generated by first extending the 5' ends of uniquely aligned, non- duplicate ChlP-seq reads by the average DNA fragment length (150 bp for histone marks, 250 bp for transcription factors) in the 3' direction using BEDtools. Identified peaks were then annotated to the nearest transcription start site. Peaks that were identified in both experimental samples (overlapping peaks) with a false discovery rate (FDR) of 0.001 and a tag density (TD) of 12 in at least one of the experimental samples were identified as significant high confidence peaks. When determining the peak overlaps from each analysis, an in-house script was used to determine the percentage of the region of the smaller peak that overlapped with the larger peak. Overlap cut-off threshold was set to 50%, such that 50% of the smaller peak in one replicate was required to overlap with the peak in the other replicate to be considered an overlapping peak.

[0211] The genomic distributions of binding sites (FIG. IB and FIG. 1C) were analyzed using the cis-regulatory element annotation system (CEAS v 1.0.2). The genes closest to the binding site on both strands were classified into functional categories such as promoter (from - lkb to +100bp), 5' UTR, first exon, first intron, exon, intron, 3' UTR, and intergenic region. The genes were also divided into defined groups according to the enrichment of the SON across transcription start site regions (5 kb window surrounding the transcription start site). Tag density heatmap (FIG. ID) was generated using the R package pheatmap vO.7.7 (peak extensions 5 kb upstream and 5 kb downstream of the peak summit and bin size 10 bp). Motif analyses (FIG. IE and FIG. II) were performed using HOMER, the methods of which are freely available at http://biowhat.ucsd.edu/homer/. Gene-associated region annotations (FIG. IF) were obtained with Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7. ChIP density plots (FIG. 2B and FIG. 2H) were made using HOMER by calculating average tag densities across ±5kb regions surrounding indicated peaks. ChTP-seq read density files (FTG. 2C, FIG. 2D, FIG. 2F) were generated using IGV tools and were viewed in Integrative Genomics Viewer (IGV) (http://www.broadinstitute.org/igv/). Previously published ChlP-seq data for H3K27ac, H3K4me3, and H2A.Z from K562 (downloaded from ENCODE/Broad Institute) and MNase- seq data for nucleosome position from K562 (downloaded from ENCODE/Stanford/BYU) were analyzed. Enriched ChlP-seq regions at promoters (5 kb window surrounding the transcription start site) for SON and histone marks were combined together to generate a unified track consisting of all merged enriched regions.

Preparation of Nuclear Extraction and Immunoprecipitation (IP)

[0212] Nuclear extracts were prepared from control, siRNA- or plasmid- transfected K562 or MV4; 1 1 cells using the Dignam protocol (Dignam et al., 1983). In brief, harvested cells were resuspended in three packed cell volumes of buffer A (10 mM HEPES pH 7.9 / 1.5 mM MgCl₂ / 10 mM KC1 / 1 mM DTT / 0.1% NP-40 / Protease Inhibitor Cocktail) and homogenized using needle and syringe with 25 to 30 gentle strokes. Lysed cells were centrifuged at 13,000xg for 10 min and the nuclei pellet was resuspended in two packed volumes of buffer C (20 mm HEPES pH 7.9 / 420 mM KC1 / 1.5 mM MgCl₂ / 1 mM DTT / 25% glycerol / Protease Inhibitor Cocktail). The nuclei suspension was gently stirred for 30 min at 4°C and centrifuged 15 min at 13,000 g to remove debris. Sufficient volume of buffer D (20 mM HEPES pH 7.9 / 0.5 mM DTT / 25% glycerol / Protease Inhibitor Cocktail) was added to the nuclei extract. For IP with K562 cells, nuclear extracts were pre-cleared with protein A-sepharose beads (Life Technologies) for 1 hour and incubated either with rabbit IgG or SON antibody at 4°C overnight on a rotator. Beads were washed four times with wash buffer (20 mM HEPES pH 7.9 / 150 mM NaCl / 0.05% (v/v) NP-40) and eluted by boiling in SDS buffer and analyzed by SDS-PAGE.

[0213] For Co-IP with exogenously expressed proteins, HEK293 cells were co- transfected with V5 and HA-tagged plasmids encoding wild type SON F or its alternative spliced variant SON E and either Flag-tagged menin plasmid or empty vector. Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 8.0 / 150 mM NaCl / 0.5% NP-40 / 10% glycerol / Protease Inhibitor Cocktail / 50 U/mL of Benzonase nuclease (Sigma)) for 1 hour. Whole cell extracts were pre-cleared and incubated with the V5 or HA antibody and protein G-sepharose beads (Life technologies) overnight. Co-IP complexes precipitated by bead were eluted by boiling in SDS buffer and subjected to Western blot analyses.

Size Fractionation and Analysis

[0214] Gel filtration chromatography was carried out at 4 °C using AKTA system (GE Healthcare). Nuclear extract from control or SON siRNA transfected K562 cells were prepared fresh, passed through a 0.22-μηι pore size MILLEX-GS filter (Millipore) and size- fractionated by fast-protein liquid chromatography (FPLC). 5mg of nuclear protein was applied to a Superose 6 10/300 GL column (GE Healthcare) equilibrated in FPLC buffer (20 mM Tris-HCl / 0.2 mM EDTA / 5 mM MgC12 / 0.1 M KC1 / 10% glycerol / 0.5 mM DTT / 1 mM benzamidine / 0.2 mM PMSF / pH 7.9) at 0.3 mL/min. 0.5 mL of elutes were collected and prepared for Western blot. Analysis of Minigene Splicing

[0215] The minigene construct containing TUBG1 exon 7- intron 7- exon 8 were used to examine SON-mediated splicing as described previously (Ahn et al., 201 1). Briefly, HeLa cells in 6 well plates were transfected with 100 pmol of control siRNA or SON siRNA. The next day, minigene alone or minigene plus various amounts of SON F or SON E constructs (siRNA-resistant form) were transfected as indicated in each experiment. RNAs were isolated 30h after minigene transfection, and RT-PCR was performed using forward primer targeting TUBG1 exon 7 and the reverse primer targeting the bovine growth hormone (BGH) terminator sequence present in the pcDNA vector.

3' RACE PCR

[0216] The transcription terminating site of the primary transcript of SON E was determined by 3' RACE PCR. For the 3' RACE PCR, K562 total RNA was reverse transcribed by Superscript III First Strand Synthesis Kit (Life Technologies) with the adapter oligo-dT primer. The first PCR was performed using the first adapter primer (Adapter Reverse Parti) and SON E primer (SON El), which specifically bind with SON exon 5a region. A second PCR was achieved using the second adapter primer (Adapter Reverse Part2) and SON E primer (SON E2). PCR products were visualized on a 1% agarose gel and cDNA fragments were cloned and sequenced. The primer sets for RACE PCR are listed in TABLE 3.

Primary Patient Samples

[0217] The bone marrow mononuclear cells and/or peripheral blood mononuclear cells from AML patients (FAB subtype M2, Pl - Pl l) as well as bone marrow mononuclear cells from healthy donors (Nl — N4) were obtained from the Stem Cell and Xenotransplantation Core Facility of the University of Pennsylvania. Peripheral blood samples from additional 5 patients diagnosed with AML or MDS (P12— PI 6) and healthy donors (N5— Nl 1) were obtained from the BioBank of Mitchell Cancer Institute, University of South Alabama. Cells were purified by Ficoll-Paque (GE Healthcare) density-gradient centrifugation and frozen as viable cells. Details of the patient samples were listed in TABLE 2. Preparation of Leukemic Blasts and Lin- / c-Kit+ Bone Marrow Cells from Mice

[0218] Mouse leukemic blasts were obtained from C57BL/6 mice with AML1- ET09a-induced leukemia generated by retroviral transduction-transplantation (Yan et al., 2006). MLL-AF9 leukemic blasts were obtained from the spleen and bone marrow of MLL- AF9a knock-in mice (Corral et al., 1996). Normal mouse Lin-/c-Kit+ bone marrow cells were prepared by Lineage Cell Depletion Kit and CD1 17 MicroBeads (Miltenyl Biotec).

Lentiviral Vector Construction and Lentivirus Production

[0219] Lentiviral vector for SON E overexpression was prepared by subcloning SON E cDNAs into the pCDH-MCS-T2A-copGFP-MSCV vector (System Bioscience). Lentivirus was produced by cotransfection of HEK 293T cells with expression plasmid, pMDLg/pRRE, pRSV-REV, and pVSVG. Viral supernatants were collected after 48 h and clarified by filtration before use. Ultracentrifugation was performed for lentivirus concentration with the Optima L-100 XP centrifuge (Beckman) using an SW55TI rotor (Beckman) at 19,400 rpm for 2 hr at 20°C. Supernatant was completely removed and virus pellets resuspended in PBS.

Colony Forming Unit Assay and Serial Replating

[0220] Mouse total bone marrow cells were transduced with recombinant lentivirus in the 4 ug/ml polybrene. Infected cells were incubated overnight in the above mouse BM culture media. After 3 days of culture, 2 x 10⁴ cells were plated in methylcellulose medium (Methocult GF M3434, StemCell Technologies). Colony number counting and re- plating were repeated every 7-10 days. The colony-forming units (CFUs) were quantified as the average and standard deviation of at least triplicate determinations.

Oncomine Database Analysis

[0221] Analysis of SON isoform expression in leukemia and normal cells were conducted using the Oncomine database (www.oncomine.org). The Oncomine database was used as previously described (Rhodes et al., 2004). Statistical Analysis

[0222] In all graphs data are expressed as mean ± SD of three independent experiments, except when otherwise indicated. Differences were analyzed by Student's t test. P-values < 0.05 were considered significant.

[0223] Each of the following references is incorporated herein by reference in its entirety.

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[0224] The term "comprising" as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0225] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

[0226] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing a hematopoietic malignancy or a likelihood of developing a hematopoietic malignancy in a test subject comprising:

determining a ratio for the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject,

wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene, and the long polypeptide is encoded by a full length transcript of the SON gene.

2. A method of detecting a ratio for the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in a test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject comprising:

contacting a nucleic acid or polypeptide sample from the test subject with an agent which indicates the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject;

contacting the nucleic acid or polypeptide sample with an agent which indicates the level of the long polypeptide or a nucleic acid encoding the long polypeptide in the test subject; and

determining the ratio for the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject to the level of the long polypeptide or the level of a nucleic acid encoding the long polypeptide in the test subject,

3. The method of claim 1 or 2, wherein the ratio is greater than about 30%.

4. The method of any one of claims 1-3, wherein the ratio is greater than about

40%.

5. The method of any one of claims 1-4, wherein the ratio is greater than about

50%.

6. A method of determining the level of a short polypeptide or a nucleic acid encoding the polypeptide in a nucleic acid or polypeptide sample from a test subject comprising:

contacting the nucleic acid or polypeptide sample from the test subject with an agent which indicates the level of the short polypeptide or which indicates the level of a nucleic acid encoding the short polypeptide in the sample; and

determining the level of the short polypeptide or a nucleic acid encoding the short polypeptide in the sample, wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.

7. A method of diagnosing a hematopoietic malignancy or a likelihood of developing a hematopoietic malignancy in a test subject comprising: determining the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in a sample obtained from a test subject, wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.

8. The method of any one of claims 1-7, further comprising comparing the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in a sample obtained from the test subject with the level of the short polypeptide or the level of a nucleic acid encoding the short polypeptide in a subject not having a hematopoietic malignancy.

9. The method of any one of claims 1-8, further comprising determining the level of a long polypeptide or a nucleic acid encoding the long polypeptide in the test subject, wherein the long polypeptide is encoded by a full length transcript of the SON gene.

10. The method of claim 9, further comprising determining a ratio for the level of a short polypeptide or the level of a nucleic acid encoding the short polypeptide in the test subject to the level of a long polypeptide or the level of a nucleic acid encoding the long polypeptide in the sample from the test subject

1 1. The method of claim 10, wherein the ratio is greater than about 30%.

12. The method of claim 10, wherein the ratio is greater than about 40%.

13. The method of claim 10, wherein the ratio is greater than about 50%.

14. The method of any one of claims 1-13, further comprising determining the level of an additional polypeptide in addition to the level of the short form of the SON polypeptide or the level of a nucleic acid encoding the additional polypeptide in addition to the level of a nucleic acid encoding the short form of the SON polypeptide in a test subject, wherein the additional polypeptide is encoded by a gene selected from CDK 1A, GF11 and ATF3.

15. The method of claim 14, further comprising comparing the level of an additional polypeptide or the level of a nucleic acid encoding the additional polypeptide in the test subject with the level of an additional polypeptide or the level of a nucleic acid encoding the additional polypeptide in a subject not having a hematopoietic malignancy.

16. The method of any one of claims 1-15, wherein the truncated alternatively- spliced transcript of a SON gene comprises a SON E variant of a SON gene.

17. The method of any one of claims 1-16, wherein the truncated alternatively- spliced transcript of a SON gene comprises a SON B variant of a SON gene.

18. The method of claim any one of claims 1-17, wherein the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10.

19. The method of any one of claims 1-18, wherein the short polypeptide comprises SEQ ID NOs:88 or 90.

20. The method of any one of claims 1-19, wherein the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.

21. The method of any one of claims 1 -20, wherein the full length transcript of the SON gene comprises a sequence which specifically binds to an oligonucleotide comprising a sequence selected from SEQ ID NOs: 13-18.

22. The method of any one of claims 1 -21 , wherein the long polypeptide comprises SEQ ID NO:86.

23. The method of any one of claims 1-22, wherein the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.

24. The method of any one of claims 1-23, wherein the SON gene is a human SON gene.

25. The method of any one of claims 1-24, further comprising obtaining a sample from the test subject.

26. The method of claim 25, further comprising contacting the sample with an agent which specifically binds to the short polypeptide or a nucleic acid encoding the short polypeptide.

27. The method of claim 25, further comprising contacting the sample with an agent which specifically binds to the long polypeptide or a nucleic acid encoding the long polypeptide.

28. The method of claim 26 or 27, wherein the agent is a primer or a hybridization probe.

29. The method of claim 28, wherein the primer or the hybridization probe comprises an oligonucleotide comprising SEQ ID NOs:06, 10 or 13-18

30. The method of claim 26 or 27, wherein the agent is an antibody or antigen- binding fragment thereof which specifically binds to the short polypeptide.

31. The method of claim 26 or 27, wherein the agent is an antibody or antigen- binding fragment thereof which specifically binds to the long polypeptide.

32. The method of any one of claims 26-31, wherein the agent is attached to a solid support.

33. The method of any one of claims 2-32, wherein the sample comprises nucleic acids or polypeptides.

34. The method of any one of claims 1 -33, further comprising determining an increased level of a polypeptide encoded by a SON target gene or a nucleic acid encoded by a SON target gene in the sample from the test subject, wherein the SON target gene has a SON binding site.

35. The method of claim 34, wherein the SON target gene is selected from the group consisting of FOX03, NOTCH2NL, SRC, GADD45A, CD N1A, ATF3, GFI1, EGR1, SRC, and GFI1B.

36. The method of any one of claims 1-35, further comprising determining an increased level of a MLL multi-protein complex in the sample from the test subject, wherein the MLL is selected from the group consisting of MLL1, MLL-N, MLL-C, and MLL2.

37. The method of claim 36, wherein the multiprotein complex comprises a protein selected from menin, ASH2L, LEDGF, and WDR5.

38. The method of any one of claims 1-37 further comprising determining an increased replating activity of a hematopoietic progenitor cell of the sample from the test subject.

39. The method of claim 38, wherein the hematopoietic progenitor cell is a bone marrow cell.

40. The method of any one of claims 1-39, further comprising determining an increased binding of a protein at the or adjacent to the transcription start site of a SON target gene in the sample from the test subject.

41. The methods of claim 40, wherein the protein is selected from the group consisting of MLL1, MLL-N, MLL-C, MLL2, WDR5, ASH2L, menin, SET1A, SET IB, ASC2, and SUZ12.

42. The method of claim 40 or 41, wherein the SON target gene is selected from the group consisting of CDKN1A, GADD45A, NOTCH2NL, ATF3, GFI1, EGR1, and SRC.

43. The method of any one of claims 1-42 further comprising determining an increased level in H3K4me3 in the sample from the test subject.

44. The method of claim 43, wherein the level in H3K4me3 is increased at the or adjacent to a SON target gene selected from the group consisting of CDKN1 A, GADD45A, NOTCH2NL, ATF3, GFI1, EGR1, and SRC.

45. The method of any one of claims 2-44, wherein the sample comprises bone marrow mononuclear cells (BM-MNCs), or peripheral blood mononuclear cells (PB-MNCs).

46. The method of any one of claims 1-45, wherein the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

47. The method of any one of claims 1-46, wherein the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).

48. The method of any one of claims 1-47, wherein the test subject is mammalian.

49. The method of any one of claims 1-48, wherein the test subject is human.

50. A method of diagnosing a hematopoietic malignancy or a likelihood of developing a hematopoietic malignancy in a test subject comprising: detecting increased levels of binding of a short polypeptide with a nucleic acid having a SON binding site in a sample from a test subject, wherein the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.

51. The method of claim 50, wherein the truncated alternatively-spliced transcript of a SON gene comprises a SON E variant of a SON gene.

52. The method of any one of claims 50-51, wherein the truncated alternatively- spliced transcript of a SON gene comprises a SON B variant of a SON gene.

53. The method of claim any one of claims 50-52, wherein the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10.

54. The method of any one of claims 50-53, wherein the short polypeptide comprises SEQ ID NOs:88 or 90.

55. The method of any one of claims 50-54, wherein the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.

56. The method of any one of claims 50-55, wherein the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

57. The method of any one of claims 50-56, wherein the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).

58. The method of any one of claims 50-57, wherein the subject is mammalian.

59. The method of any one of claims 50-58, wherein subject is human.

60. A method of ameliorating a hematopoietic malignancy comprising: increasing the level of a full length SON polypeptide or a nucleic acid encoding a full length SON polypeptide in a cell of a subject.

61. The method of claim 60, wherein the expression level of a nucleic acid encoding said full length SON polypeptide or the expression level of said full length SON polypeptide is increased by administering a composition comprising a nucleic acid to the subject.

62. The method of claim 60 or 61 , wherein the nucleic acid comprises a sequence encoding a full length SON polypeptide.

63. The method of any one of claims 60-62, wherein the SON polypeptide is a human SON polypeptide.

64. The method of any one of claims 60-63, wherein the nucleic acid comprises SEQ ID NO.:85.

65. The method of any one of claims 60-64, wherein the polypeptide comprises SEQ ID NO.:86.

66. The method of any one of claims 60-65, wherein the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

67. The method of any one of claims 60-66, wherein the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).

68. The method of any one of claims 60-67, wherein the subject is mammalian.

69. The method of any one of claims 60-68, wherein subject is human.

70. A kit for the diagnosis of a hematopoietic malignancy in a test subject comprising: an agent that specifically binds to a short polypeptide or a nucleic acid encoding the short polypeptide, wherein the short polypeptide is encoded by a truncated alternatively- spliced transcript of a SON gene.

71. The kit of claim 70, further comprising an agent that specifically binds to a long polypeptide or a nucleic acid encoding the long polypeptide, wherein the long polypeptide is encoded by a full length transcript of the SON gene.

72. The kit of any one of claims 70-71, further comprising an agent that specifically binds to an additional polypeptide in addition to the short polypeptide or a nucleic acid encoding the additional polypeptide in addition to the nucleic acid encoding the short polypeptide in the test subject, wherein the additional polypeptide is encoded by a gene selected from CDK 1A, GF11 and ATF3.

73. The kit of any one of claims 70-72, wherein the truncated alternatively-spliced transcript of a SON gene comprises a SON E variant of a SON gene.

74. The kit of any one of claims 70-73, wherein the truncated alternatively-spliced transcript of a SON gene comprises a SON B variant of a SON gene.

75. The kit of any one of claims 70-74, wherein the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10.

76. The kit of any one of claims 70-75, wherein the short polypeptide comprises SEQ ID NOs:88 or 90.

77. The kit of any one of claims 70-76, wherein the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.

78. The kit of any one of claims 70-77, wherein the full length transcript of the SON gene comprises a sequence which specifically binds to an oligonucleotide comprising a sequence selected from SEQ ID NOs: 13-18.

79. The kit of any one of claims 70-78, wherein the long polypeptide comprises SEQ ID NO:86.

80. The kit of any one of claims 70-79, wherein the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.

81. The kit of any one of claims 70-80, wherein the SON gene is a human SON gene.

82. The kit of any one of claims 70-81, further comprising an agent that specifically binds to a SON target gene, wherein the SON target gene has a SON binding site.

83. The kit of claim 82, wherein the SON target gene is selected from the group consisting of FOX03, NOTCH2NL, SRC, GADD45A, CDKN 1 A, ATF3, GFI1, EGR1, SRC, and GFI1B.

84. The kit of any one of claims 70-83, further comprising an agent that specifically binds to a protein selected from MLL1, MLL-N, MLL-C, MLL2, menin, ASH2L, LEDGF, WDR5, SET1A, SET IB, ASC2, SUZ12, and H3K4me3.

85. The kit of any one of claims 70-84, wherein the agent is a primer or a hybridization probe.

86. The kit of any one of claims 70-85, wherein the primer or the hybridization probe comprises an oligonucleotide comprising SEQ ID NOs:06, 10, or 13-18.

87. The kit of any one of claims 70-86, wherein the agent is an antibody or antigen-bind fragment thereof which specifically binds to the short polypeptide.

88. The kit of any one of claims 70-87, wherein the agent is attached to a solid support.