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Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations.Significance:This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations.

Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations. Significance: This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations.

Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained wild-type allele is expressed, suggesting that mutant hematopoietic cells may require the residual wild-type allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knock-out mice was similar to control mice, but that deletion of the wild-type allele in U2AF1(S34F) heterozygous mutant expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of wild-type U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1 (S34F) expressing cells were also more sensitive to reduced expression of wild-type U2AF1 than non-mutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the wild-type U2af1 allele was deleted compared to when it was not deleted. These results suggest that selectively targeting the wild-type U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.

Background: Missense mutations of the TP53 gene frequently occur in patients with myelodysplastic syndrome and acute myeloid leukemia. The functional consequences of mutant p53 expression on hematopoiesis remain incompletely studied. Whether TP53 mutations confer a loss-of-function, gain-of-function, or dominant-negative effect in response to chemotherapies has not been fully explored. One of these hotspot mutations is a G-to-A substitution at nucleotide 525 of TP53, which codes for an Arg175His (R175H) substitution. Thus, we characterized hematopoiesis in Trp53R172H constitutive knock-in mice, corresponding to R175H TP53 mutation in patients, and compared the results to Trp53 knock-out and control mice. Methods: Five genotypes of mice were analyzed (Trp53+/+ [WT], Trp53R172H/+, Trp53R172H/R172H, Trp53+/-, and Trp53-/-). Frequencies of peripheral blood (PB) and bone marrow (BM) cells were measured by flow cytometry and fluorouracil (5-FU) and N-ethyl-N-nitrosourea (ENU) were used as hematopoietic stressors. Results: There was no change in the PB counts or lineage distribution of mature PB and BM cells in Trp53R172H/+ and Trp53R172H/R172H mice compared to WT or Trp53 knock-out mice at 8-15 weeks of age. We found that expression of Trp53R172H mutation in mice does not cause hematopoietic stem cell (HSC) expansion under basal conditions, in contrast to heterozygous and homozygous deletion of Trp53, but does confer a functional advantage to HSCs in a competitive repopulation assay. Trp53R172H HSCs expand further following ENU exposure, similar to Trp53-/- cells and consistent with a dominant-negative effect of Trp53R172H. Mechanistically, mutant p53R172H has a dominant-negative effect on p21-induced expression following ENU treatment. Repeated 5-FU injections drive proliferation of progenitors. To test the hematopoietic cell-intrinsic effects of mutant p53R172H on white blood cell count recovery, we exposed mice reconstituted with Trp53+/+, Trp53+/-, Trp53-/- or Trp53R172H/+ BM cells to a single dose of 5-FU and followed mice weekly for a month. Trp53R172H/+ mice were least affected by the 5-FU challenge and showed the fastest and largest recovery of WBC count compared to Trp53+/+, Trp53+/- and Trp53-/- mice. These results demonstrate that mutant p53R172H confers a gain-of-function property following 5-FU exposure that was not seen with deletion of Trp53. Conclusion: Our data suggest that in vivo expression of mutant Trp53R172H has minimal effects on basal hematopoiesis. However, mutant Trp53R172H induces either a gain-of-function or a dominant-negative effect depending on the type of hematopoietic stress and hematopoietic phenotype being observed. Overall, our results indicate that the functional consequences induced by various Trp53 mutations (e.g., deletion and point mutation) are not identical, but are influenced by the type of hematopoietic stress. Citation Format: Tuoen Liu, Tanzir Ahmed, Michael O. Alberti, Brian A. Wadugu, Jin Shao, Sarah Grieb, Grace Przybyl, Matthew Ndonwi, Matthew J. Walter. Mutant Trp53 R172H has gain-of-function or dominant-negative effects in response to hematopoietic stressors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 841.

Nonsense-mediated RNA decay (NMD) is well recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTCs) for degradation; however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system that can accurately measure NMD activity in individual cells. By carrying out a genome-wide CRISPR/Cas9 knockout screen using this reporter system, we identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, we also found that cells with mutations in the U2 spliceosome genes SF3B1 and U2AF1—which are commonly found in myelodysplastic syndrome (MDS) and cancers—have overall attenuated NMD activity. Furthermore, we found that compared to wild type cells, SF3B1 and U2AF1 mutant cells are more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition could be rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, our findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel strategy for the treatment of MDS and cancers with spliceosome mutations.

Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation; however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes SF3B1 and U2AF1, which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared to wild type cells, SF3B1 and U2AF1 mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations.

Somatic mutations in the spliceosome geneU2AF1are common in patients with myelodysplastic syndromes.U2AF1mutations that code for the most common amino acid substitutions are always heterozygous, and the retained wild-type allele is expressed, suggesting that mutant hematopoietic cells may require the residual wild-type allele to be viable and cause disease. We show that hematopoiesis and RNA splicing inU2af1heterozygous knock-out mice was similar to control mice, but that deletion of the wild-type allele in U2AF1(S34F) heterozygous mutant expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of wild-type U2AF1 for survivalin vivoand thatU2AF1is a haplo-essential cancer gene. Mutant U2AF1 (S34F) expressing cells were also more sensitive to reduced, but not absent, expression of wild-type U2AF1 than non-mutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the wild-typeU2af1allele was deleted compared to when it was not deleted. These results suggest that selectively targeting the wild-typeU2AF1allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboringU2AF1mutations.

Introduction. Mutations in TP53 are common (~18%) in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia and often undergo loss of heterozygosity. Our understanding of the hematopoietic consequences of expressing mutant TP53-R175H, one of the most common mutations in MDS, is incomplete. In addition, whether TP53-R175H confers a loss-of-function, gain-of-function, or dominant-negative effect in response to chemotherapy has not been fully explored. Methods. We used a constitutive knock-in mouse model expressing TRP53-R172H (G-to-A substitution at nucleotide 515), corresponding to human mutant TP53-R175H. We generated Trp53R172H/+ and Trp53R172H/R172H mice and compared them to wild type (WT), Trp53+/-, and Trp53-/- mice. Peripheral blood (PB) and bone marrow (BM) was analyzed in non-BM transplant conditions, following a non-competitive BM transplant, and following a competitive BM transplant with or without exposure to N-ethyl-N-nitrosourea (ENU) and 5-fluorouracil (5FU). Results. BM hematopoietic stem and progenitor cells (HSPC), including LSK-SLAM cells, were increased in Trp53+/- and Trp53-/-mice (n=4-8, 8-15 weeks old, P TP53 mutant cells can clonally expand in patients following cytotoxic chemotherapy. Therefore, we investigated the response of TRP53R172H/+ mutant cells to alkylator (ENU) exposure. We created mixed BM chimeric mice by transplanting test (WT, Trp53+/-, Trp53R172H/+ and Trp53-/-) and WT competitor BM in a 1:3 ratio, respectively. Following engraftment, chimeric mice received vehicle or ENU (2 doses of 100 mg/kg, 9 days apart). ENU-exposed Trp53R172H/+ cells have a robust PB multilineage competitive advantage relative to placebo (Fig. 1A, n = 4-5, 2-fold increase at 10 weeks post-ENU, P Next, we asked whether mutant TRP53-R172H has similar or different effects as Trp53 deletion following exposure to an alternative chemotherapy (5FU). We first treated mice with a single dose of 5FU (200 mg/kg) to deplete cells and monitored WBC count recovery for 4 weeks. Trp53R172H/+ mice had significantly higher recovery WBC counts compared to WT, Trp53+/-, andTrp53-/- mice (n = 5-15, P Collectively, the results indicate that mutant TRP53-R172H may induce a gain-of-function or a dominant-negative effect depending on the exposure to specific hematopoietic stresses. Disclosures No relevant conflicts of interest to declare.

Myelodysplastic syndromes (MDS) are the most common myeloid malignancies among the elderly. Patients present with bone marrow (BM) failure manifested by low peripheral blood (PB) counts and are at increased risk of developing acute myeloid leukemia. Mutations of U2AF1, a gene that encodes a spliceosome protein, are identified in 11% of MDS patients. The two most common U2AF1 mutants, S34F and Q157P, alter the splicing of two distinct sets of pre-mRNA targets in vitro and each co-occur with unique gene mutations in MDS patients, suggesting these mutants may affect MDS pathogenesis differently. In mice, U2AF1S34F expression leads to altered splicing, reduced B-cell counts, and features of MDS. Similar studies have not been performed for U2AF1Q157P. To study the impact of U2AF1Q157P expression on splicing and hematopoiesis in vivo, we created a doxycycline (DOX)-inducible ("Tet-On") transgenic mouse that expresses mutant U2AF1Q157P and is isogenic to our previously reported U2AF1S34F and U2AF1WT transgenic mice. First, we confirmed DOX-inducible expression of the U2AF1Q157P transgene in BM by RT-PCR-seq. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P, we performed non-competitive BM transplants into lethally irradiated congenic recipient mice. Donor BM from U2AF1WT or U2AF1S34F mice was also transplanted for comparison. Six weeks after transplant, mice were maintained on DOX chow to induce U2AF1 transgene (U2AF1WT, U2AF1S34F, or U2AF1Q157P) expression (n = 10 mice per genotype). After six weeks on DOX, there were no significant changes in PB counts for U2AF1Q157P mice compared to U2AF1WT controls. In contrast, white blood cell (WBC) and B-cell counts were significantly reduced in U2AF1S34F mice, as reported previously. Assessment of the BM revealed increased numbers (per five leg bones) of hematopoietic stem and progenitor cells (LSK [Lin− Sca-1+ c-kit+] and LK [Lin− Sca-1− c-kit+]) in U2AF1S34F mice (1.33×105 LSK and 7.13×105 LK cells) compared to U2AF1WT (1.04×105 LSK and 5.69×105 LK cells; p < 0.05 for LSK and LK), as reported previously. In contrast, there was no change in LSK cells (1.03×105, p = 0.9668) and a non-significant increase in LK cells (6.84×105, p = 0.0547) in U2AF1Q157P mice compared to U2AF1WT. Both U2AF1S34F and U2AF1Q157P mice shared a significant increase in the number of common myeloid progenitors (CMP) compared to U2AF1WT (2.43×105 and 2.39×105 vs. 1.66×105 cells; p < 0.001 and p < 0.01, respectively), although CFU-C interrogated by methylcellulose assay were significantly increased only for U2AF1S34F mice. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P on stem cell function, we mixed equal numbers of whole BM test cells (CD45.2+; U2AF1Q157P or U2AF1WT) with congenic control wild-type BM competitor cells (CD45.1+/CD45.2+) and transplanted them into lethally irradiated congenic recipient mice (CD45.1+/CD45.2+ ; n = 6 per genotype). As in non-competitive transplants, DOX chow was administered six weeks after transplant. After six weeks on DOX, we observed a relative multi-lineage competitive disadvantage by analysis of peripheral blood chimerism (%CD45.2+ WBC) for U2AF1Q157P test compared to U2AF1WT test cells (49.5% vs. 71.7%, respectively, p < 0.001). In addition, stem and progenitor cells were all significantly reduced in the BM of U2AF1Q157P competitive transplant mice compared to U2AF1WT after 18 weeks of DOX (LSK, 36.1% vs. 92.2%, respectively, p < 0.001; LK, 53.1% vs. 92.0%, p < 0.001). Lastly, using a Nanostring array, we identified consensus 3' splice sites of cassette exons that were increased or decreased in RNA from c-kit enriched mutant (U2AF1S34F or U2AF1Q157P) BM cells relative to U2AF1WT (FDR < 0.1). As expected, we observed altered consensus 3' splice sites at the −3 position (for U2AF1S34F) and +1 position (for U2AF1Q157P) of differentially spliced exons, indicating altered but different pre-mRNA splicing induced by either U2AF1 mutant. In aggregate, hematopoietic expression of U2AF1Q157P causes a multi-lineage competitive disadvantage of BM stem cells and expanded myeloid progenitors in the non-competitive transplant setting, like U2AF1S34F. However, PB counts and lineage distribution are not affected, indicating that the two common U2AF1 mutants, Q157P and S34F, are associated with different hematopoietic phenotypes and alterations to splicing, and may have different roles in MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Somatic mutations in genes encoding factors involved in pre-mRNA splicing (i.e., spliceosome genes) have been identified in more than half of myelodysplastic syndrome (MDS) patients. We are interested in the role of U2AF1, a spliceosome gene mutated in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1 have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type (WT) U2AF1 in normal hematopoiesis has not been studied. A complete understanding of the role of WT U2AF1 on hematopoiesis and RNA splicing is critical to our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of WT U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse. Homozygous embryonic deletion of U2af1 using Vav1-Cre was lethal, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wt or Mx1-Cre/U2af1wt/wt into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1 deletion, homozygous U2af1 KO mice, but not other genotypes, became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KO mice compared to all other genotypes (p ≤ 0.001, n=5). Deletion of U2af1 also led to rapid bone marrow failure and a reduction in bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (p ≤ 0.001, n=5). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and WT congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cells (HSCs). As early as 10 days, and up to 4 months following Mx1-Cre-induction, we observed a significant decrease in white blood cell count chimerism and complete loss of homozygous U2af1 KO HSCs, neutrophils and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). Collectively, the data indicate that normal hematopoiesis is dependent on U2af1 expression. In ongoing studies, we crossed the U2af1 KO mouse to existing transgenic mutant U2AF1-expressing mice to determine if mutant U2AF1 expression is sufficient to rescue cell survival and normal hematopoiesis when endogenous WT U2af1 is deleted. If U2AF1 mutant cells are vulnerable to loss of the WT U2AF1 allele, then selectively targeting the WT U2AF1 allele in heterozygous mutant cells could be a novel therapeutic strategy. Citation Format: Brian A. Wadugu, Amanda Heard, Joseph Bradley, Matthew Ndonwi, Matthew J. Walter. U2af1, a spliceosome gene commonly mutated in MDS, is required for hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5112.

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 patients with MDS.

With the goal of elucidating the molecular and cellular mechanisms of chloroprene toxicity, we examined the potential DNA cross-linking of the bifunctional chloroprene metabolite, (1-chloroethenyl)oxirane (CEO). We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of interstrand cross-links by CEO within synthetic DNA duplexes. Our data suggest interstrand cross-linking at deoxyguanosine residues within 5'-GC and 5'-GGC sites, with the rate of cross-linking depending on pH (pH 5.0 > pH 6.0 > pH 7.0). A comparison of the cross-linking efficiencies of CEO and the structurally similar cross-linkers diepoxybutane (DEB) and epichlorohydrin (ECH) revealed that DEB > CEO > or = ECH. Furthermore, we found that cytotoxicity correlates with cross-linking efficiency, supporting a role for interstrand cross-links in the genotoxicology of chloroprene.

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 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 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.