SUMMARY Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ~11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently-mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in MDS patients.
Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations—drug and mutation—compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins.
Mutations in spliceosome genes are detectable in ~50% of patients with myelodysplastic syndromes (MDS), making this cellular pathway the most commonly mutated in MDS and providing a novel target for therapeutic intervention. Spliceosome gene mutations are mutually exclusive, implying they are either redundant in pathogenic function or are not tolerated in a cell when they co-occur. Our group and others identified recurrent heterozygous missense mutations in the splicing factor gene U2AF1 in 11% of MDS patient samples. The most common U2AF1 mutation results in a conversion of serine to phenylalanine at position 34 (S34F) of the U2AF1 protein. We previously reported that expression of mutant U2AF1(S34F) in vivo using doxycycline-inducible U2AF1(S34F) transgenic mice revealed an expansion of hematopoietic bone marrow progenitor cells and leukopenia following transgene induction; both phenotypes are seen in patients with MDS. We also identified mutant U2AF1-specific alterations in pre-mRNA splicing in transgenic mouse bone marrow progenitor cells, primary AML patient samples, and CD34+ cells by RNA sequencing. We hypothesize that cells harboring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome by splicing modulator drugs, providing a new treatment approach for patients with U2AF1 mutations. For our studies, we utilize sudemycins, which are compounds that bind the SF3B1 spliceosome protein and modulate pre-mRNA splicing in non-hematopoietic tissues. We examined the effects of sudemycin treatment on pre-mRNA splicing in primary hematopoietic cells by treating CD34+ cells isolated from human umbilical cord blood with sudemycin in vitro. We performed whole transcriptome (RNA-seq) analysis following 6 hours of sudemycin treatment (1µM) of CD34+ cells and identified robustly altered pre-mRNA splicing patterns that are sudemycin-specific (26,120 splice junctions by DEXSeq, FDR<0.05, n=3), thereby validating that splicing is altered in hematopoietic cells treated with sudemycin. We confirmed a subset of these altered pre-mRNA splicing changes by RT-PCR and gel electrophoresis, as well as by Nanostring assay of RNA. We performed in vitro studies to examine the sensitivity of cells expressing U2AF1(S34F) to sudemycin treatment. Primary human MDS/AML cells with U2AF1(S34F) mutations display increased sensitivity to sudemycin, compared to non-mutant controls in a cell cycle (EdU incorporation) assay (n=3), while treatment with daunorubicin showed no specificity for mutant U2AF1(S34F) samples compared to non-mutant controls. Primary mouse c-Kit+ bone marrow cells transduced with a retrovirus expressing U2AF1(S34F) display a marked increase in apoptosis (by flow cytometry for Annexin V+ staining) in response to increasing concentrations of sudemycin, compared to controls (p<0.001, n=3-5). In addition, in vivo treatment of U2AF1(S34F) transgenic mice with sudemycin resulted in attenuation of hematopoietic progenitor cell expansion by colony forming unit (CFU-C) assay (p<0.01, n=6-11) and by flow cytometry for lineage-, c-Kit+, Sca-1+ (KLS) cells (p<0.001, n=6-11). Ongoing studies are examining the splicing alterations in U2AF1 mutant and wild-type transgenic mouse bone marrow cells treated with vehicle versus sudemycin. Taken together, these data suggest that we may be able to specifically treat hematological cancers with U2AF1 mutations using small molecule pre-mRNA splicing modulators such as sudemycin. Disclosures No relevant conflicts of interest to declare.
Our group and others discovered recurrent heterozygous missense mutations in U2AF1 in 11% of patients with myelodysplastic syndromes (MDS). The U2AF1 gene encodes a splicing factor involved in intronic 3’-splice site recognition, which suggests that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. To study the effects of the most common U2AF1 mutation, U2AF1(S34F), on hematopoiesis and pre-mRNA splicing in vivo, we created site-specific, single-copy, doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type recipient mice prior to induction of transgene expression. Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased Annexin V+ (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+/Lineage- cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for bone marrow KLS (c-Kit+/Lineage-/Sca-1+) cells (p≤0.05). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice is seen as early as 5 days and is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to U2AF1(WT) controls (p<0.05, n=8-13). Competitive repopulation studies show a disadvantage for bone marrow cells expressing mutant U2AF1(S34F) compared to U2AF1(WT) at ≥4 months post-transplant in both primary and secondary transplant recipient mice (p≤0.05, n=3-12), suggesting that the increase in KLS cell cycling following U2AF1(S34F) expression may lead to stem cell exhaustion. Collectively, these data indicate U2AF1(S34F) expression alters hematopoiesis in vivo. Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days of transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice (n=3 each). We identified 460 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) controls (FDR <5%). We observed a preference of the mutant U2AF1(S34F) to skip exons (p=1.3e-05, n=72) and alternative splice sites (p=0.014, n=45) with a T in the -3 position relative to the AG splice acceptor site of differentially-spliced genes; this effect has been previously reported in AML patient samples with U2AF1 mutations. To prioritize altered junctions for further analysis, we intersected mouse CMP junction results with RNA sequencing data from AML patient samples with and without U2AF1 mutations and primary human CD34+ cells over-expressing U2AF1(S34F) or U2AF1(WT). Across species and present in all 3 datasets, we identified homologous dysregulated junctions in 2 genes known to be involved in cancer and stem cell biology: H2AFY and MED24. We validated concordant changes in both H2AFY and MED24 isoform expression by RT-PCR using MDS patient bone marrow samples that have mutant U2AF1(S34F) versus U2AF1(WT) (p<0.001, n=5-6). We are currently testing these isoform changes for their functional contribution to mutant U2AF1-associated phenotypes. Together, these results suggest that mutant U2AF1 expression contributes to the altered hematopoiesis and pre-mRNA splicing observed in patients with U2AF1 mutations. This study also identifies changes in gene isoform expression unique to U2AF1 mutations that may have functional significance for MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.
Mutations in 8 spliceosome genes have been identified in up to half of patients with myelodysplastic syndromes (MDS), suggesting that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. Our group and others discovered recurrent heterozygous mutations in U2AF1, a gene encoding a splicing factor involved in intronic 3’-splice site recognition, in 11% of MDS patients. The most common U2AF1 mutation causes a serine to phenylalanine substitution at amino acid 34 (S34F). To study the effects of U2AF1(S34F) expression on hematopoiesis, we created site-specific, single-copy, doxycycline-inducible (controlled by the reverse tetracycline-transactivator [rtTA] transgene) U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type mouse recipients prior to doxycycline induction of transgene expression. We observed a doxycycline dose-dependent increase in expression of U2AF1(S34F) and U2AF1(WT) transgenes by RT-PCR and pyrosequencing analysis, and we subsequently chose a dose that induces equivalent levels of exogenous U2AF1 transgene to endogenous mouse U2af1 transcripts. Following 4 weeks of doxycycline induction of transgene expression in vivo, U2AF1(S34F)-recipient mouse bone marrow cells showed increased alternative splicing of endogenous Fmr1 pre-mRNA, a transcript previously shown to be alternatively-spliced in U2AF1 mutant MDS patient samples, when compared to U2AF1(WT)- and rtTA only-recipient mouse bone marrow cells (p<0.02, n=9-11). This effect was seen as early as 5 days (p<0.001, n=3-7). Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice display a decrease in WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (average 4.5 vs 6.7 and 7.1 K/µl, respectively; p≤0.01). However, there are no significant differences in bone marrow cellularity or spleen size in these mice (n=9-11). U2AF1(S34F)-recipient mice have reduced frequency of peripheral blood monocytes (average 1.2 vs 3.2 and 2.6%, p<0.05), as well as bone marrow monocytes (3.9 vs 7 and 6.1%, p<0.001) and B cells (average 10.1 vs 20.7 and 20.6%, p<0.001) when compared to U2AF1(WT)- and rtTA only-recipient mice, respectively (n=9-11). Moreover, bone marrow monocytes in U2AF1(S34F)-recipient mice have an increased frequency of Annexin V+ staining compared to controls (average 11.2 vs 6.9 and 7.5%, p<0.05, n=5-6), indicating that loss of these cells may be due to apoptosis. U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow neutrophils compared to U2AF1(WT)- and rtTA only-recipient mice (average 65 vs 53 and 50%, respectively, p<0.001, n=9-11). Taken together, these data indicate U2AF1(S34F) expression alters hematopoietic lineage distribution in vivo. In addition, 4 weeks of U2AF1(S34F) expression results in alterations of progenitor and stem cell frequency. U2AF1(S34F)-recipient mice have approximately a 1.5-fold increase in the frequency of progenitor cells in their bone marrow measured by flow cytometry (linlow/c-kit+/Sca-1-) (average 4.1 vs 2.6 and 2.9%; p<0.001, n=5-6) and methylcellulose progenitor colony forming assays (average 85 vs 55 and 59 colonies/plate; p<0.02, n=9-11) when compared to U2AF1(WT)- and rtTA only-recipient mice, respectively. U2AF1(S34F)-recipient mice also display a 1.5-fold increase in the frequency of common myeloid progenitors compared to U2AF1(WT) and rtTA only controls in both bone marrow and spleen (p≤0.02), along with a 2-fold increase of the granulocyte-macrophage progenitors in the spleen (p≤0.03) (n=5-6). In addition to changes in progenitors, U2AF1(S34F)-recipient mice have a 1.4-fold increase in the frequency of bone marrow KLS cells, short-term HSCs, and dormant long-term HSCs (p≤0.05), as well as a 2-fold increase in spleen dormant long-term HSCs (p≤0.03) compared to U2AF1(WT)- and rtTA only-recipient controls (n=5-6). Competitive repopulation studies are ongoing. Collectively, these data indicate that mutant U2AF1(S34F) expression in mouse bone marrow cells results in altered pre-mRNA splicing and changes in hematopoietic cell lineage distribution, as well as increased bone marrow and spleen progenitor and stem cell populations. Furthermore, these results suggest that mutant U2AF1 may contribute to altered hematopoiesis in patients. Disclosures: No relevant conflicts of interest to declare.
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