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WO2018101950A1 - Méthodes et compositions pour le diagnostic d'une leucémie aiguë myéloïde - Google Patents

Méthodes et compositions pour le diagnostic d'une leucémie aiguë myéloïde Download PDF

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Publication number
WO2018101950A1
WO2018101950A1 PCT/US2016/064442 US2016064442W WO2018101950A1 WO 2018101950 A1 WO2018101950 A1 WO 2018101950A1 US 2016064442 W US2016064442 W US 2016064442W WO 2018101950 A1 WO2018101950 A1 WO 2018101950A1
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Prior art keywords
son
polypeptide
nucleic acid
level
acid encoding
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Eun-Young Erin AHN
Jung-Hyun Kim
Ssang-Taek LIM
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University of South Alabama
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University of South Alabama
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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.
  • 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).
  • MLL mixed lineage leukemia
  • 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.
  • 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.
  • cellular factors that regulate MLL-menin interaction and MLL complex assembly are largely unknown.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10.
  • the short polypeptide comprises SEQ ID NOs:88 or 90.
  • the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.
  • 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.
  • the long polypeptide comprises SEQ ID NO:86.
  • the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.
  • the SON gene is a human SON gene.
  • Some embodiments also include obtaining a sample from the test subject.
  • 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.
  • the agent is a primer or a hybridization probe.
  • the primer or the hybridization probe comprises an oligonucleotide comprising SEQ ID NOs:06, 10 or 13-18
  • 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.
  • the agent is attached to a solid support.
  • the sample comprises nucleic acids or polypeptides.
  • 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.
  • the SON target gene is selected from the group consisting of FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFI1, EGR1, SRC, and GFI1B.
  • 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.
  • the multiprotein complex comprises a protein selected from menin, ASH2L, LEDGF, and WDR5.
  • Some embodiments also include determining an increased replating activity of a hematopoietic progenitor cell of the sample from the test subject.
  • the hematopoietic progenitor cell is a bone marrow cell.
  • 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.
  • the protein is selected from the group consisting of MLL1, MLL-N, MLL-C, MLL2, WDR5, ASH2L, menin, SET1A, SET1B, ASC2, and SUZ12.
  • the SON target gene is selected from the group consisting of CDKN1A, GADD45A, NOTCH2NL, ATF3, GFI1, EGR1, and SRC.
  • 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.
  • the sample comprises bone marrow mononuclear cells (BM-MNCs), or peripheral blood mononuclear cells (PB-MNCs).
  • BM-MNCs bone marrow mononuclear cells
  • PB-MNCs peripheral blood mononuclear cells
  • the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).
  • test subject is mammalian. In some embodiments, the test subject is human.
  • 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.
  • 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.
  • the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10.
  • the short polypeptide comprises SEQ ID NOs:88 or 90.
  • the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.
  • the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).
  • the subject is mammalian. In some embodiments, subject is human.
  • 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.
  • 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.
  • the nucleic acid comprises a sequence encoding a full length SON polypeptide.
  • the SON polypeptide is a human SON polypeptide.
  • the nucleic acid comprises SEQ ID NO.:85.
  • the polypeptide comprises SEQ ID NO.:86.
  • the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).
  • the subject is mammalian. In some embodiments, the subject is human.
  • kits 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the long polypeptide comprises SEQ ID NO:86.
  • the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.
  • the SON gene is a human SON gene.
  • Some embodiments also include an agent that specifically binds to a SON target gene, wherein the SON target gene has a SON binding site.
  • the SON target gene is selected from the group consisting of FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFI1, EGR1, SRC, and GFI1B.
  • 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.
  • the agent is a primer or a hybridization probe.
  • the primer or the hybridization probe comprises an oligonucleotide comprising SEQ ID NOs:06, 10, or 13-18.
  • the agent is an antibody or antigen-bind fragment thereof which specifically binds to the short polypeptide.
  • the agent is attached to a solid support.
  • 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.
  • 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).
  • 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).
  • FIG. ID depicts a heat map showing the overlap peak signal of SON ChIP around the transcription start site of genes.
  • 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.
  • 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.
  • 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.
  • FIG. 1H depicts a Venn diagram showing number of SON peaks determined by ChlP-seq using SON-N Ab and SON-C Ab.
  • FIG. II depicts top five motifs enriched in SON-binding sites analyzed by HOMER.
  • 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.
  • FIG. 2B depicts average signal profiles of indicated histone modifications and CpG islands around the SON-binding sites.
  • 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.
  • IGF Integrative Genomics Viewer
  • FIG. 2D depicts close-up images of ChlP-seq peaks of SON-N, SON-C, H3K4me3, and H3K27ac in representative SON target genes.
  • 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.
  • 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 ⁇ , 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.
  • FIG. 2G depicts a Western blot that confirmed knockdown efficiency of two different siRNAs (#1 and #2) against SON in K562 cells.
  • FIG. 2H depicts average signal profiles of H3K4mel and H3K27ac around the intergenic SON-binding sites.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 4A depicts a Western blot verified SON knockdown by SON siRNA transfection in K562 cells.
  • FIG. 4B depicts a co-immunoprecipitation experiment with MLL-N antibodies.
  • FIG. 4C depicts a co-immunoprecipitation experiment with WDR5 antibodies.
  • FIG. 4D depicts a co-immunoprecipitation experiment with LEGF antibodies.
  • FIG. 4E depicts a co-immunoprecipitation experiment with SET1A antibodies.
  • 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.
  • 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.
  • 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.
  • MLL-N MLL
  • plasm id transfection two different amounts of SON-V5 construct, 5 ⁇ g (+) or 10 ⁇ g (++), were used.
  • 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).
  • FIG. 4J depicts a Western blot confirmed knockdown efficiency of SON siRNA in MV4; 11 cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • FIG. 5J depicts sequencing result of the 3' RACE PCR product from human SON E transcripts.
  • FIG. 5K depicts a schematic strategy for measurement of relative ratio of SON isoforms presented in FIG. 5D, FIG. 5E and FIG. 5G.
  • each qPCR analysis was performed using same forward primer and two different reverse primers.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • siRNA and SON constructs siRNA-resistant form
  • 3 ⁇ g (+) of SON F constructs plus increasing amounts of SON E construct, 0 (-), 3 (+) or 6 ⁇ g (++), or 3 ⁇ g 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.
  • FIG. 6F depicts TUBG1 exon 7-8 minigene splicing assay demonstrating that SON E does not interfere with SON F-mediated RNA splicing.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 ⁇ g (+) of SON F- V5 constructs plus increasing amounts of SON E-Flag, 5 (+) or 10 ⁇ g (++) 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.
  • 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.
  • IP co-immunoprecipitation
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 71 depicts models for SON and its alternative spliced isoform function in transcription.
  • 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.
  • MLL complex formation is facilitated, resulting in enhanced recruitment of the MLL complex and subsequent increase of H3K4me3 and promoter activity.
  • SON B and SON E 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 10 4 BM cells were cultured in MethoCult (M3434).
  • 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 ⁇ . DETAILED DESCRIPTION
  • 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.
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • a truncated alternatively-spliced transcript of a SON gene can indicate a subject having a hematopoietic malignancy, or the likelihood of the subject developing a hematopoietic malignancy.
  • 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.
  • 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.
  • the hematopoietic malignancy can include acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • the hematopoietic malignancy is an AML subtype including M2 without t(8;21), and t(8;21)(q22;q22).
  • the short polypeptide is a truncated alternatively-spliced transcript of a SON gene. See for example, TABLE 1.
  • 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.
  • the nucleic acid encoding the short polypeptide comprises a sequence which specifically binds to an oligonucleotide comprising SEQ ID NOs:06 or 10.
  • the short polypeptide comprises SEQ ID NOs:88 or 90.
  • the nucleic acid encoding the short polypeptide comprises SEQ ID NOs:87 or 89.
  • the long polypeptide is encoded by a full length transcript of the SON gene. See e.g., TABLE 1.
  • 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.
  • the long polypeptide comprises SEQ ID NO: 86.
  • the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.
  • the SON gene is a human SON gene.
  • 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.
  • the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene
  • the long polypeptide is encoded by a full length transcript of the SON gene.
  • 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.
  • the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene
  • the long polypeptide is encoded by a full length transcript of the SON gene.
  • 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.
  • the ratio is greater than about 30%, 40%, 50%, 60%, 70%, 80%, and 90%, or any range between any two of the foregoing numbers.
  • 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.
  • the short polypeptide is encoded by a truncated alternatively-spliced transcript of a SON gene.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the SON target gene has a SON binding site.
  • 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.
  • the SON target gene is selected from the group consisting of FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFT1 , EGR1 , SRC, and GFT1 B.
  • 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.
  • the MLL is selected from the group consisting of MLLl, MLL-N, MLL-C, and MLL2.
  • the multiprotein complex includes a protein selected from menin, ASH2L, LEDGF, and WDR5.
  • 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.
  • 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.
  • the protein can include MLL1, MLL-N, MLL-C, MLL2, WDR5, ASH2L, menin, SET1A, SET1B, ASC2, and SUZ12.
  • the SON target gene can include CDKN1 A, GADD45A, NOTCH2NL, ATF3, GFI1, EGR1, and SRC.
  • 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.
  • a SON target gene including CDKN1A, GADD45A, NOTCH2NL, ATF3, GFI1, EGR1, and SRC.
  • Some embodiments also include a sample from the test subject.
  • the sample can include nucleic acids or polypeptides.
  • the sample comprises bone marrow mononuclear cells (BM-MNCs), or peripheral blood mononuclear cells (PB-MNCs).
  • BM-MNCs bone marrow mononuclear cells
  • PB-MNCs peripheral blood mononuclear cells
  • 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.
  • 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.
  • 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.
  • a SON target gene such as a SON target gene having a SON binding site.
  • the SON target includes FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFI1, EGR1, SRC, and GFI1B.
  • 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.
  • 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.
  • the agent is an antibody or antigen-binding fragment thereof which specifically binds to the short polypeptide.
  • the agent can be immobilized on a solid support.
  • 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.
  • 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.
  • 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
  • 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
  • the hybridization probe or primer can include an oligonucleotide comprising any one of SEQ ID NOs:06, 10, 13-18.
  • 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.
  • the agent comprises an antibody or antigen- binding fragment thereof.
  • 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.
  • 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.
  • the SON polypeptide is a human SON polypeptide.
  • the nucleic acid comprises SEQ ID NO.:85.
  • the polypeptide comprises SEQ ID NO.: 86.
  • the hematopoietic malignancy is selected from acute myeloid leukemia (AML) and myelodysplasia syndrome (MDS).
  • the hematopoietic malignancy is an acute myeloid leukemia (AML) subtype selected from M2 without t(8;21), and t(8;21)(q22;q22).
  • the subject is mammalian. In some embodiments, the subject is human.
  • 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.
  • 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.
  • 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).
  • compositions can be administered by various means known in the art.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024.
  • 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.
  • 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.
  • 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.
  • Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
  • donor construct such as expression vectors comprising a nucleic acid encoding a full length SON polypeptide
  • naked DNA complexed/formulated with a delivery vehicle e.g. liposome or poloxamer
  • 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.
  • 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.
  • 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 for the diagnosis of a hematopoietic malignancy, or likelihood of developing a hematopoietic malignancy in a test subject can include an agent that specifically binds to a short polypeptide or a nucleic acid encoding the short polypeptide.
  • 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.
  • the long polypeptide is encoded by a full length transcript of the SON gene.
  • 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.
  • 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.
  • 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.
  • 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.
  • the long polypeptide comprises SEQ ID NO:86.
  • the nucleic acid encoding the long polypeptide comprises SEQ ID NO:85.
  • the SON gene is a human SON gene.
  • Some embodiments also include an agent that specifically binds to a SON target gene, wherein the SON target gene has a SON binding site.
  • the SON target gene can include FOX03, NOTCH2NL, SRC, GADD45A, CDKN1A, ATF3, GFI1, EGR1, SRC, and GFI1B.
  • 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.
  • the agent is a primer or a hybridization probe.
  • the primer or the hybridization probe comprises an oligonucleotide comprising SEQ ID NOs:06, 10, or 13-18.
  • the agent is an antibody or antigen-binding fragment thereof which specifically binds to the short polypeptide.
  • the agent is attached to a solid support.
  • 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.
  • FIG. 2E 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.
  • 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).
  • SON depletion leads to increased recruitment of MLL complex components to the SON target genes and enhanced MLL complex formation
  • 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).
  • IP immunoprecipitation
  • MLL-N MLL1 N-terminus
  • SON interacts with menin, an MLL1/2 complex component, and SON-menin interaction diminishes MLL-menin interaction
  • menin-MLL-N interaction was assessed as well as menin-SON interaction by IP with menin antibody.
  • 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).
  • BM-MNCs Bone marrow mononuclear cells
  • FAB subtype M2 Bone marrow mononuclear cells
  • TABLE 2 healthy donors
  • FIG. 5A Similar to previous results (Ahn et al., 2013), most patient samples showed high levels of total SON (exon 1—3 region).
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • FIG. 6G 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.
  • BM bone marrow
  • SON E overexpression resultsed 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.
  • 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).
  • expression of SON E alone failed to reduce the H3K4me3 level, indicating its lack of ability to suppress H3K4me3.
  • 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).
  • 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).
  • 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
  • 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).
  • 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.
  • 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).
  • MLL-menin interaction is also involved in promoting hepatocellular carcinoma development (Xu et al., 2013).
  • MLL-menin interaction 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.
  • menin 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
  • 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.
  • overexpression of SON E specifically weakened the full-length SON function in transcriptional initiation, but does not impair full-length SON- mediated RNA splicing.
  • 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.
  • ChlP-Sequencing ChlP-seq
  • ChlP-qPCR ChlP-qPCR
  • ChIP Chromatin Immunoprecpitation
  • 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).
  • 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.
  • 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.
  • GC AAGTGATGTTGGACGTGACAGATC-3 ' (SEQ ID NO:79) and V5-SON F Notl-R: 5'- TTTGCGGCCGCtacgtagaatcgagaccgaggagagggttagggataggcttaccTGGCCGGCCatacctatt caagaaaacatacaatt-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).
  • RT-qPCR Reverse transcription and Quantitative PCR
  • RT-qPCR Quantitative real time PCR
  • 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.
  • 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).
  • lysis buffer 5 mM PIPES pH 8.0 / 85 mM KC1 / 0.5% NP-40 / l
  • 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
  • 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).
  • 0.5 ⁇ 10 6 cells were nucleofected with 100 - 200 pmol of siRNA or 5 ⁇ g of plasmid using Cell Line Nucleofector Kit V (Lonza) according to the manufacturer's instructions.
  • 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.
  • human CD34+ bone marrow cells 0.4x 106 cells were nucleofected with 80 - 150 pmol of siRNA or 4 ⁇ g 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 ⁇ g of plasmids using PEL
  • 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.
  • FDR false discovery rate
  • TD tag density
  • the genomic distributions of binding sites 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.
  • FIG. 2F were generated using IGV tools and were viewed in Integrative Genomics Viewer (IGV) (http://www.broadinstitute.org/igv/).
  • IGV Integrative Genomics Viewer
  • 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.
  • 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 MgCl 2 / 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.
  • buffer A (10 mM HEPES pH 7.9 / 1.5 mM MgCl 2 / 10 mM KC1 / 1 mM DTT / 0.1% NP-40 / Protease Inhibitor Cocktail
  • 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 MgCl 2 / 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.
  • 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.
  • wash buffer (20 mM HEPES pH 7.9 / 150 mM NaCl / 0.05% (v/v) NP-40
  • 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.
  • 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)
  • 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.
  • BGH bovine growth hormone
  • the transcription terminating site of the primary transcript of SON E was determined by 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.
  • the bone marrow mononuclear cells and/or peripheral blood mononuclear cells from AML patients 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.
  • 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 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.
  • 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 10 4 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.
  • CFUs colony-forming units
  • Histone methylase MLLl has critical roles in tumor growth and angiogenesis and its knockdown suppresses tumor growth in vivo. Oncogene 32, 3359-3370.
  • MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer cell 20, 66-78.
  • RNAi screen reveals determinants of human embryonic stem cell identity. Nature 468, 316-320.
  • An M11-AF9 fusion gene made by homologous recombination causes acute leukemia in chimeric mice: a method to create fusion oncogenes. Cell 85, 853-861.
  • SON connects the splicing-regulatory network with pluripotency in human embryonic stem cells. Nature cell biology 15, 1 141-1 152.
  • Gene expression profiling identifies BAX-delta as a novel tumor antigen in acute lymphoblastic leukemia. Cancer Res 65, 10050- 10058.
  • COMPASS a complex of proteins associated with a trithorax-related SET domain protein. Proceedings of the National Academy of Sciences of the United States of America 98, 12902-12907.
  • the menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis. Cell 123, 207-218.

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Abstract

Certains modes de réalisation de la présente invention concernent le diagnostic d'affections malignes hématopoïétiques. Certains modes de réalisation comprennent des méthodes et des kits pour le diagnostic d'affections malignes hématopoïétiques, telles que la leucémie aiguë myéloïde. Certains modes de réalisation concernent la présence d'isoformes épissées en alternance d'un gène SON.
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Cited By (2)

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JPWO2020013233A1 (ja) * 2018-07-10 2021-08-02 国立大学法人 東京大学 Ａｌｓのバイオマーカーおよびａｌｓの診断方法
EP4232020A4 (fr) * 2020-10-21 2024-08-07 Kura Oncology, Inc. Traitement de malignités hématologiques par des inhibiteurs de ménine

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