Splicing Factor Mutations Research Articles

Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function invivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and+1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

In This Issue

In This Issue

Translating recent advances in the pathogenesis of acute myeloid leukemia to the clinic.

Despite FDA approval of nine new drugs for patients with acute myeloid leukemia (AML) in the United States over the last 4 years, AML remains a major area of unmet medical need among hematologic malignancies. In this review, we discuss the development of promising new molecular targeted approaches for AML, including menin inhibition, novel IDH1/2 inhibitors, and preclinical means to target TET2, ASXL1, and RNA splicing factor mutations. In addition, we review progress in immune targeting of AML through anti-CD47, anti-SIRPα, and anti-TIM-3 antibodies; bispecific and trispecific antibodies; and new cellular therapies in development for AML.

Representative cancer-associated U2AF2 mutations occurring at the inter-RRM interface alter RNA interactions and splicing

Recurrent missense mutations of pre-mRNA splicing factors involved in the early stages of splicing are associated with hematologic malignancies. Most mutational hotspots recur at the protein–RNA interfaces of SF3B1, SRSF2 and U2AF1. At lower frequencies, we identified cancer-associated mutations of an essential pre-mRNA splicing factor, U2AF2. These mutations either lie at the inter-RRM interface or the RNA interacting interface of U2AF2. Normally, U2AF2 recognizes the polypyrimidine (Py) tract preceding the 3′ splice site and initiates spliceosome assembly.

Coordinated missplicing of TMEM14C and ABCB7 causes ring sideroblast formation in SF3B1-mutant myelodysplastic syndrome

AbstractSF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5′ UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non–rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation.

Roles of RNA alternative splicing during the occurrence and development of lung cancer

<p indent="0mm">In the human genome, more than 90% of the human genes undergo alternative splicing, which participates in the entire life activity. Abnormal RNA splicing is closely associated with human diseases, including cancer. Cancer is still a serious public health concern as the second leading cause of death. Every year, lung cancer ranks first in the number of new cancer cases and deaths. Abnormal alternative splicing occurs extensively in lung cancer, regulating tumorigenesis and lung cancer progression. Splicing factor mutations and aberrant expression are well-known drivers of lung cancer progression. Furthermore, alternative splicing plays a critical role in drug resistance, radiotherapy resistance, angiogenesis, and tumor metabolism.

Clonal architecture predicts clinical outcomes and drug sensitivity in acute myeloid leukemia

The impact of clonal heterogeneity on disease behavior or drug response in acute myeloid leukemia remains poorly understood. Using a cohort of 2,829 patients, we identify features of clonality associated with clinical features and drug sensitivities. High variant allele frequency for 7 mutations (including NRAS and TET2) associate with dismal prognosis; elevated GATA2 variant allele frequency correlates with better outcomes. Clinical features such as white blood cell count and blast percentage correlate with the subclonal abundance of mutations such as TP53 and IDH1. Furthermore, patients with cohesin mutations occurring before NPM1, or transcription factor mutations occurring before splicing factor mutations, show shorter survival. Surprisingly, a branched pattern of clonal evolution is associated with superior clinical outcomes. Finally, several mutations (including NRAS and IDH1) predict drug sensitivity based on their subclonal abundance. Together, these results demonstrate the importance of assessing clonal heterogeneity with implications for prognosis and actionable biomarkers for therapy.

Abstract P242: Preclinical efficacy landscape of the pan-CLK/DYRK inhibitor Cirtuvivint (SM08502)

Abstract A monumental challenge facing cancer therapy is the vast mechanistic diversity of tumors among and even within cancer patients. This severely limits the number of patients that respond to any given therapy and the durability of responses that do occur. Deep phenotypic profiling of almost 9000 patient samples indicates perturbation of alternative pre-mRNA splicing (AS) is often a root cause of cancer. This offers an extraordinary opportunity to restore health through normalization of AS in diseased tissue. The bottleneck to progress has been finding and drugging the right targets. The CLK/DYRK kinases modulate AS efficiency and specificity, and therefore, targeting them offers the potential to address this bottleneck. The isoquinoline cirtuvivint is a potent ATP-competitive inhibitor of the Cdc2-like kinases (CLK1-4) and the dual-specificity tyrosine phosphorylation-regulated kinases (DYRK1-4), with activity against only a minimal number of the remaining members of the CMGC-family kinases or the kinome as a whole. Cellular target engagement assays indicated biological IC50s below 0.06 μM across the CLK/DYRK target class. Thus, cirtuvivint has utility for robust chemical evaluation of the overarching contribution of CLK/DYRK family activity to tumor biology in general and context- dependent AS in particular. To help assess the breadth and depth of CLK/DYRK dependencies in human cancers, the consequences of pan-CLK/DYRK inhibition on growth and survival of 154 cancer cell line models, 46 PDX models, and 43 CDX models were tested. EC50s in cell viability assays ranged from 0.014 to 0.73 μM in culture with strong effects observed in subsets of cell lines across all lineages tested. Molecular profiles associated with high sensitivity to cirtuvivint included somatic mutations in the RBM10 splicing factor. Tumor growth inhibition assays with daily dosing at exposures about 2X below the MTD resulted in significant disease control (≥75% TGI) in 15/46 PDX and 18/43 CDX models. Together, these results indicate broad cancer relevance, at least in the preclinical setting, and are consistent with a common reliance on CLK/DYRK-dependent alternative splicing among otherwise highly mechanistically heterogeneous disease. Citation Format: Carine Bossard, Emily Creger, Elizabeth A. McMillan, Carsten Merkwirth, Maureen Ibanez, Deepti Bhat, Josh Stewart, Brian Eastman, Chi-Ching Mak, Vishal Deshmukh, Michael A. White. Preclinical efficacy landscape of the pan-CLK/DYRK inhibitor Cirtuvivint (SM08502) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P242.

Donor Clonal Hematopoiesis and Recipient Outcomes After Transplantation.

Clonal hematopoiesis (CH) can be transmitted from a donor to a recipient during allogeneic hematopoietic cell transplantation. Exclusion of candidate donors with CH is controversial since its impact on recipient outcomes and graft alloimmune function is uncertain. We performed targeted error-corrected sequencing on samples from 1,727 donors age 40 years or older and assessed the effect of donor CH on recipient clinical outcomes. We measured long-term engraftment of 102 donor clones and cytokine levels in 256 recipients at 3 and 12 months after transplant. CH was present in 22.5% of donors, with DNMT3A (14.6%) and TET2 (5.2%) mutations being most common; 85% of donor clones showed long-term engraftment in recipients after transplantation, including clones with a variant allele fraction < 0.01. DNMT3A-CH with a variant allele fraction ≥ 0.01, but not smaller clones, was associated with improved recipient overall (hazard ratio [HR], 0.79; P = .042) and progression-free survival (HR, 0.72; P = .003) after adjustment for significant clinical variables. In patients who received calcineurin-based graft-versus-host disease prophylaxis, donor DNMT3A-CH was associated with reduced relapse (subdistribution HR, 0.59; P = .014), increased chronic graft-versus-host disease (subdistribution HR, 1.36; P = .042), and higher interleukin-12p70 levels in recipients. No recipient of sole DNMT3A or TET2-CH developed donor cell leukemia (DCL). In seven of eight cases, DCL evolved from donor CH with rare TP53 or splicing factor mutations or from donors carrying germline DDX41 mutations. Donor CH is closely associated with clinical outcomes in transplant recipients, with differential impact on graft alloimmune function and potential for leukemic transformation related to mutated gene and somatic clonal abundance. Donor DNMT3A-CH is associated with improved recipient survival because of reduced relapse risk and with an augmented network of inflammatory cytokines in recipients. Risk of DCL in allogeneic hematopoietic cell transplantation is driven by somatic myelodysplastic syndrome-associated mutations or germline predisposition in donors.

Predictive Biomarkers of Response to the Polo-like Kinase 1 (PLK1) Inhibitor, Onvansertib, in Combination with Decitabine in Relapsed or Refractory Acute Myeloid Leukemia (R/R AML)

Predictive Biomarkers of Response to the Polo-like Kinase 1 (PLK1) Inhibitor, Onvansertib, in Combination with Decitabine in Relapsed or Refractory Acute Myeloid Leukemia (R/R AML)

Single-Cell Multi-Omics Defines the Cell-Type Specific Impact of SF3B1 Splicing Factor Mutations on Hematopoietic Differentiation in Human Clonal Hematopoiesis and Myelodysplastic Syndromes

Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the importance of alternative splicing regulation in hematopoiesis. Specifically, mutations in splicing factor 3B subunit 1 (SF3B1) are implicated in the pathogenesis of myelodysplastic syndromes (MDS) and linked to a high-risk of leukemic transformation in clonal hematopoiesis (CH). SF3B1 mutations are associated with aberrant RNA splicing, leading to increased cryptic 3' splice site (ss) usage and MDS with ring sideroblasts phenotype.The study of mutant SF3B1-driven splicing aberrations in humans has been hampered by the inability to distinguish mutant and wildtype single cells in patient samples and the inadequate coverage of short-read sequencing over splice junctions. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT; Nam et al. 2019) with Nanopore long-read single-cell transcriptome profiling and CITE-seq (Fig. A). This allowed for the simultaneous single-cell profiling of protein and gene expression, somatic mutation status, and alternative splicing. Our method selectively enriched full-length sequencing reads with the accurate structure, enabling the capture of higher number of junctions per cell and greater coverage uniformity vs. short-read sequencing (10x Genomics; Fig. B, C).We applied GoT-Splice to CD34+ bone marrow progenitor cells from MDS (n = 15,436 cells across 3 patients; VAF: [0.38-0.4]) to study how SF3B1 mutations corrupt human hematopoiesis (Fig. D). High-resolution mapping of SF3B1 mut vs. SF3B1 wt hematopoietic progenitors revealed an increasing fitness advantage of SF3B1 mut cells towards the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1 muterythroid progenitor (EP) cells (Fig. E, F). Accordingly, SF3B1 mutEP cells displayed higher protein expression of erythroid lineage markers, CD71 and CD36, vs. SF3B1 wt cells (Fig. G). In these SF3B1 mutEP cells, we identified up-regulation of genes involved in regulation of cell cycle and checkpoint controls (e.g., CCNE1, TP53), and mRNA translation (eIFs gene family; Fig. H).Next, while SF3B1 mut cells showed the expected increase of cryptic 3' splicing vs. SF3B1 wt cells (Fig. I), they exhibited distinct cryptic 3' ss usage as a function of hematopoietic progenitor cell identity, displaying stage-specific aberrant splicing during erythroid maturation (Fig. J). In less differentiated EP cells, we observed mis-splicing of genes involved in iron homeostasis, such as the hypoxia-inducible factor HIF1A, and key regulators of erythroid cell growth, such as SEPT2. At later stages, we observed mis-splicing of BAX, a pro-apoptotic member of the Bcl-2 gene family and transcriptional target of p53, and erythroid-specific genes (e.g., PPOX). We further predicted 54% of the aberrantly spliced mRNAs to introduce premature stop codons, promoting RNA degradation through nonsense-mediated decay (NMD). In line with this notion, we observed a significant decrease in expression of NMD-inducing genes in SF3B1 mut vs . SF3B1 wt EP cells (Fig. K).Lastly, splicing factor mutations observed in CH subjects provide an opportunity to interrogate the downstream impact of SF3B1 mutations prior to development of disease. Like MDS, by applying GoT-splice to CD34+ progenitor cells from SF3B1 mut CH subjects (n = 9,007 cells across 2 subjects; VAF: [0.15-0.22]; Fig. L), we revealed increased mutant cell frequency in EP cells (Fig. M) with concomitant increased expression of genes involved in mRNA translation (Fig. N), consistent with SF3B1 mutation causing mis-splicing injury to translational machinery and ineffective erythropoiesis. Notably, CH patients already exhibited cell-type specific cryptic 3' ss usage in SF3B1 mut cells (Fig. O).In summary, we developed a novel multi-omics single-cell toolkit to examine the impact of splicing factor mutations on cellular fitness directly in human samples. With this approach, we showed that, while SF3B1 mutations arise in uncommitted HSCs, their effect on fitness increases with differentiation into committed EPs, in line with the mutant SF3B1-driven dyserythropoiesis phenotype. We revealed that SF3B1 mutations exert cell-type specific mis-splicing that leads to abnormal erythropoiesis. Finally, we demonstrated that the impact of SF3B1 mutations on EP cells begins before disease onset, as observed in CH subjects. [Display omitted] DisclosuresDai: Oxford Nanopore Technologies: Current Employment. Beaulaurier: Oxford Nanopore Technologies: Current Employment. Drong: Oxford Nanopore Technologies: Current Employment. Hickey: Oxford Nanopore Technologies: Current Employment. Juul: Oxford Nanopore Technologies: Current Employment. Wiseman: Astex: Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy; StemLine: Consultancy. Harrington: Oxford Nanopore Technologies: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Characterization of Somatic Mosaicism and Mutational Profiling of Clonal Hematopoiesis Compared to MDS and sAML Depicts Diversities of Clonal Evolution

Background:Clonal hematopoiesis (CH) describes the presence of genetic alterations and expansion of clonal cell populations in the peripheral blood (PB) of individuals without clinical manifestation of a hematologic malignancy. CH is a common, age-related state. However, individuals carrying CH are at greater risk for hematologic cancer. It has been shown that presence of TP53- and other high-risk mutations, variant allele frequency (VAF) >10% or multiple mutations in CH carriers may confer a higher risk of preleukemic development and transformation to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Nevertheless, further insights into the role of CH in clonal evolution towards leukemia and elaborating features that are predictive of leukemic progression are needed.Aims:We have recently shown that CH is common in individuals undergoing hip replacement surgery (Hecker, Hartmann et al., Blood 2021). Here, we compared mutational spectrum in these CH individuals to MDS and secondary AML (sAML) cohorts. Additionally, we analyzed spatial and lineage distribution of CH and longitudinally monitored CH and MDS clones over time.Methods:Samples from individuals without known hematologic disease undergoing hip replacement surgery (n=288 samples from 261 individuals), patients with MDS (n=92) or sAML (n=123) were screened for variants in 68 leukemia-associated genes using a targeted sequencing approach (VAF cut off, 1%). Follow-up (FU) PB samples were available for 21 individuals with CH and 16 untreated low-risk MDS patients, 6-24 months after screening. n=5 CH bone marrow (BM) samples carrying six ASXL1-mutations were sorted for seven different cell fractions.Results:At screening, variants were detected in 127/261 (49%) healthy individuals, 84/92 (91%) MDS and 117/123 (95%) sAML patients, with median VAFs of 2.7% (ranging from 1-44%), 18.8% (1.1-87%) and 37.1% (1-99%), respectively. Individuals with CH had a median number of 1 variant per individual, whereas median detected variants per patient increased with clonal evolution with 3 variants in the MDS and 4 variants per patient in the sAML cohort.CH, MDS and sAML showed entity-specific mutation profiles (Figure 1A). Most variants in CH affected epigenetic modifiers, while mutations in splicing factors, signaling pathways and transcription factors increased with clonal progression.During FU, untreated low-risk MDS patients more frequently gained additional mutations compared to CH individuals (7/16 vs 2/21, respectively; p=0.024). However, we did not observe significant changes in clone sizes over time.In 17 hip replacement individuals both femoral heads were removed simultaneously, allowing paired analysis of two different hematopoietic sites. CH prevalence in this subgroup was 70.6% (12/17). Ten individuals showed identical mutation patterns in BM obtained from the right and left femoral head, with little differences in VAFs (Table 1). In contrast, two other individuals showed significant differences in variant detection comparing one to the other side: ASXL1-mutations were only present in one hip sample, whereas all the other variants were detectable in both sides (p<0.001), indicating possible spatial heterogeneity of CH clones in the BM compartment.To further characterize ASXL1 mosaicism across hematopoietic lineages and differentiation stages, we sorted and sequenced n=5 CH BM samples carrying six ASXL1-mutations for seven different hematopoietic cell fractions. In all cases ASXL1-mutations were identified in CD34 +CD38 -CD90 + hematopoietic stem cells (HSC). VAFs of ASXL1-mutations were differentially distributed (p=0.0049, Kruskal Wallis test): detected VAFs were significantly higher in HSC and common myeloid progenitors (CMP) compared to VAFs in T-cells (p=0.045 and p=0.011, respectively; Dunn´s multiple comparison test; Figure 1B).Conclusions:CH, MDS and sAML show characteristic mutation profiles that remained stable over a 6-24-month period. Gains of additional variants and clonal expansion associated with disease progression. Cellular distribution analysis of ASXL1 CH variants revealed characteristic repartition patterns within the hematopoietic differentiation tree. Additionally, differences in variant detection between cellular BM compartments indicates spatial heterogeneity of CH clones warranting further investigation.J.S.H. and L.H. contributed equally. [Display omitted] DisclosuresPlatzbecker: Geron: Honoraria; Takeda: Honoraria; AbbVie: Honoraria; Celgene/BMS: Honoraria; Janssen: Honoraria; Novartis: Honoraria. Goetze: BMS/Celgene: Other: Advisory Board, Research Funding; Abbvie: Other: Advisory Board.

Phase 1 Study of JNJ-64619178, a Protein Arginine Methyltransferase 5 Inhibitor, in Patients with Lower-Risk Myelodysplastic Syndromes

Phase 1 Study of JNJ-64619178, a Protein Arginine Methyltransferase 5 Inhibitor, in Patients with Lower-Risk Myelodysplastic Syndromes

Molecular Derivation of Extramedullary Myeloid Sarcomas Based on Machine Learning Analysis of Genomic Clusters in AML

Molecular Derivation of Extramedullary Myeloid Sarcomas Based on Machine Learning Analysis of Genomic Clusters in AML

Bone marrow ribonucleotide reductase mRNA levels and methylation status as prognostic factors in patients with myelodysplastic syndrome treated with 5-Azacytidine

Ribonucleotide Reductase (RNR) is a two-subunit (RRM1, RRM2) enzyme, responsible for the conversion of ribonucleotides to deoxyribonucleotides required for DNA replication. To evaluate RNR as a biomarker of response to 5-azacytidine, we measured RNR mRNA levels by a quantitative real-time PCR in bone marrow samples of 98 patients with myelodysplastic syndrome (MDS) treated with 5-azacytidine with parallel quantification of the gene promoter’s methylation. Patients with low RRM1 levels had a high RRM1 methylation status (p = 0.005) and a better response to treatment with 5-azacytidine (p = 0.019). A next-generation sequencing for genes of interest in MDS was also carried out in a subset of 61 samples. Splicing factor mutations were correlated with lower RRM1 mRNA levels (p = 0.044). Our results suggest that the expression of RNR is correlated with clinical outcomes, thus its expression could be used as a prognostic factor for response to 5-azacytidine and a possible therapeutic target in MDS.

U2AF1 Mutations Enhance Stress Granule Response in Myeloid Malignancies

Somatic mutations in splicing factor genes are drivers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The splicing factors U2AF1 and U2AF2 form the U2AF heterodimer that is critical in the 3' splice site (3'SS) recognition and in the recruitment of U2 small nuclear ribonucleoproteins for the activation of the spliceosome complex. U2AF1 carries hotspot mutations in its two RNA binding motifs; yet the molecular mechanisms affecting the splicing process and promoting clonal advantage remain unclear, albeit necessary to develop effective targeted therapies.We applied a multi-omics approach comparing the activities of two U2AF1 mutants (S34F and Q157R) in MDS/AML cell lines and primary samples. Using a novel approach of fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA-sequencing (freCLIP-seq), we mapped transcriptome-wide binding at nucleotide resolution and we identified conformational changes in mutant vs wild-type U2AF1 binding. Specifically, we observed an emergent peak in position -3 of the 3'SS for the S34F mutant and in position +1 for the Q157R mutant, matching the critical positions observed by differential splicing analysis on RNA-seq data. Altered U2AF1-RNA binding compromised U2AF2-RNA interactions, resulting predominantly in exon exclusion and intron retention. Combined binding-splicing analysis showed that while the Q157R mutant mainly exhibits loss of binding, the S34F mutant follows a gain-of-binding pattern, where splicing progression appears impaired by increased mutant binding.Functional analysis of genes affected by both binding and splicing alterations revealed that U2AF1 mutants alter RNA granule biology, affecting in particular stress granule-enriched transcripts and proteins. Stress granules are membrane-less cytoplasmic assemblies of RNAs and RNA binding proteins that improve cellular adaptation in response to stress conditions. Increased stress granule formation has been linked to tumorigenesis as a strategy exploited by cancer cells to regulate gene expression and signal transduction, enhancing their fitness under stress. To probe how aberrant binding and splicing of stress granule components affected stress granule biology, we assessed stress granule formation in U2AF1 mutant vs wild-type cells at steady state and after stress induction with sodium arsenite treatment. Immunofluorescent staining followed by confocal imaging demonstrated that U2AF1 mutations enhance stress granule formation upon arsenite stress in both cell lines and primary samples. RNA turnover analysis by TimeLapse-seq confirmed that U2AF1 S34F and Q157R mutations promote stability/synthesis of transcripts that are enriched in stress granules and determine degradation/shutdown of transcripts that are depleted in stress granules, providing a molecular explanation for the increase in stress granules observed by imaging. Finally, we were able to corroborate our observations by single-cell RNA-seq in patient-derived U2AF1-mutant MDS blasts, establishing the causal link between U2AF1 mutations and upregulation of stress granule components.Collectively, this multi-omics analysis identified biological processes directly influenced by mutant U2AF1 binding and splicing, laying the foundation for a new paradigm where splicing factor mutations enhance stress granule formation by acting on the availability of their RNA and protein components. The enhanced formation of stress granules potentially fosters the stress adaptation of U2AF1-mutant cells, contributing to their clonal advantage in MDS/AML. Stress granule perturbations may therefore represent a novel therapeutic vulnerability in U2AF1-mutant MDS/AML patients and possibly in patients carrying other splicing factor mutations. DisclosuresHunck: Boehringer Ingelheim: Other: Fellowship.

Isogenic MDS-RS Patient-Derived iPSCs Define the Mis-Spliced Transcript Repertoire and Chromatin Landscape of SF3B1-Mutant Hematopoietic Stem/Progenitor Cells

▪The somatic hotspot mutation SF3B1K700E is characteristically found in myelodysplastic syndrome with ring sideroblasts (MDS-RS) and frequently occurs as an isolated mutation. However, our understanding of how this mutation drives MDS pathogenesis remains limited. To explore the downstream consequences of the SF3B1K700E mutation and its role in disease pathogenesis, we generated a panel of isogenic SF3B1K700E and SF3B1WT induced pluripotent stem cell (iPSC) lines from 3 MDS-RS patients with isolated SF3B1K700E mutation (3 SF3B1K700E and 3 SF3B1WT lines per patient, total 18). Upon hematopoietic differentiation, SF3B1K700E cells exhibited lower growth and colony-forming ability, compared to SF3B1WT cells, recapitulating hallmark phenotypes of MDS cells.To investigate the effects of the SF3B1K700E mutation on the transcriptome and chromatin landscape, we performed RNA- and ATAC- sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from the panel of the 18 isogenic SF3B1K700E and SF3B1WT iPSC lines. Principal component analysis (PCA) and hierarchical clustering based on gene expression grouped the iPSC lines primarily by genotype (SF3B1K700E vs SF3B1WT) and secondarily by genetic background. To assess the impact of the SF3B1K700E mutation at the exon, transcript and gene level, we developed an analytical framework integrating differential splicing with differential transcript usage and differential gene expression analyses. We thus discovered 59 splicing events linked to 34 genes (most statistically significant events that also mapped to differentially used transcripts and differentially expressed genes). This SF3B1K700E splicing signature includes genes previously reported as mis-spliced in SF3B1K700E cells (e.g BRD9, ABCB7), as well as novel genes. We tested this signature against a published dataset of primary MDS patient samples (Pellagatti et al.). PCA based on the inclusion level of the splicing events of our signature separated SF3B1-mutated MDS patients from patients without splicing factor mutations (SF-WT) or healthy individuals. Furthermore, it identified one patient erroneously annotated as SF-WT that clustered together with the SF3B1-mutated patients, who had a, previously overlooked, 6bp in-frame deletion spanning the K700E hotspot.By comparing the chromatin accessibility profiles of SF3B1K700E and SF3B1WT iPSC-HSPCs to those defined in primary human cell types along the hematopoietic hierarchy (Corces et al.), we found that the chromatin landscape of SF3B1K700E HSPCs resembled more this of megakaryocyte-erythroid progenitor cells (MEPs) and erythroid cells, whereas that of SF3B1WT HSPCs resembled more granulocyte-monocyte progenitors (GMPs) and monocytes. This finding may underlie the more prominent involvement of the erythroid lineage in the pathology and clinical presentation of MDS-RS. To interrogate transcriptional programs in SF3B1K700E mutant cells, we performed transcription factor (TF) motif enrichment analysis. Motifs enriched in ATAC-Seq peaks more accessible in SF3B1K700E cells that were linked to genes upregulated in SF3B1K700E cells, included motifs of several TFs with known roles in hematopoiesis (GATA, ETS, STAT, AP-1). Unexpectedly, motifs of the TEAD TFs were also enriched. The TEAD family of TFs are best known as effectors of the Hippo signaling pathway, with important roles in various biological processes and malignancies, albeit no clear links to adult hematopoiesis or hematologic disease. TEAD2 and TEAD4 were upregulated in SF3B1-mutant, compared to the WT, iPSC-HSPCs and TEAD transcriptional activity, measured with a luciferase reporter construct, was higher in SF3B1K700E, compared to SF3B1WT iPSC-HSPCs. We did not find expression or activation of YAP or TAZ, which bind to DNA as a complex with TEAD upon Hippo pathway activation. These results support a Hippo-independent increase of TEAD expression and activity in SF3B1K700E cells.In summary, we generated a panel of isogenic patient-derived iPSCs that allowed us to comprehensively characterize the transcriptome and chromatin landscape of SF3B1K700E HSPCs in an isogenic system, derive a splicing signature of SF3B1K700E and identify the TEAD TF as a new transcriptional regulator of SF3B1K700E mutant HSPCs.G. Asimomitis, A.G. Deslauriers: shared 1 st authorshipE. Papaemmanuil, E.P. Papapetrou: shared senior authorship DisclosuresDeslauriers: Novo Nordisk A/S: Current Employment. Hellström-Lindberg: Celgene: Research Funding. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy.

Spliceosome Mutant Myeloid Malignancies Are Preferentially Sensitive to PARP Inhibition

▪Somatic heterozygous mutations in spliceosome genes SRSF2, U2AF1, and SF3B1 commonly occur in patients with myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Moreover, SRSF2 and U2AF1 mutations associate with poor survival and high risk of progression to AML, representing a unique genetic vulnerability for targeted therapy. We and others previously found that R-loops, a group of transcription intermediates containing RNA:DNA hybrids and displaced single-stranded DNA, are a source of genomic instability induced by different spliceosome mutants. We further showed that inhibition of ATR kinase activity preferentially kills spliceosome mutant cells in an R-loop-dependent manner. Inspired by ATR inhibition results, we performed a focused drug screen with inhibitors targeting additional DNA damage response pathways to identify novel therapeutic vulnerabilities generated by spliceosome mutations. We generated a murine leukemia model by overexpressing the MLL-AF9 fusion oncogene on an Srsf2 P95H/+background, a mutational combination that is found in ~10% of MLL-rearranged leukemias. Surprisingly, we found that Srsf2 P95H/+cells are more sensitive to five inhibitors targeting ADP-ribosyltransferases or PARP (olaparib, talazoparib, rucaparib, niraparib, veliparib) (Figs 1A-B). Olaparib (PARPi)-treated Srsf2 P95H/+cells exhibited increased apoptosis compared to Srsf2 +/+ cells as determined by AnnexinV (Fig 1C). PARPi sensitivity was also observed in isogenic murine MLL-AF9 U2af1 S34F/+cells compared to MLL-AF9 U2af1 +/+ cells (Fig 1D). These data highlight that both SRSF2 P95H and U2AF1 S34F mutations create a common vulnerability that is dependent on PARP activity for survival.To evaluate PARP activity in cells, we used isogenic K562 leukemia cells expressing SRSF2 P95H and U2AF1 S34F mutations from their endogenous loci and monitored PAR (poly(ADP-ribose)) chain levels, a marker of PARP activity. Both SRSF2 P95H and U2AF1 S34F cells exhibited elevated PAR levels compared to wildtype cells (Figs 1E-F). PARPi treatment significantly suppressed PAR signals in SRSF2 P95H and U2AF1 S34F cells. PARP inhibitors target both PARP1 and PARP2 enzymes, of which PARP1 plays a key role in DNA damage response. We used CRISPR-Cas9 to knockout PARP1 gene to determine the major PARP responsible for elevated PAR level in these leukemia cells. PARP1 deletion abrogated elevated PAR levels in U2AF1 S34F (Fig 1G) and SRSF2 P95H cells (data not shown). Altogether, we demonstrated that SRSF2 P95H and U2AF1 S34F cells trigger a PARP1 response critical for cell survival.To test whether increased PAR level arises from U2AF1 S34F-induced R-loops, we generated U2AF1 S34F cells that inducibly express RNaseH1, an enzyme that specifically cleaves the RNA moiety within RNA:DNA hybrids. Induction of RNaseH1 in U2AF1 S34F cells significantly reduced PAR levels, showing that U2AF1 S34F-induced PAR chains is R-loop-dependent (Fig 1H). Moreover, RNaseH1 overexpression suppressed the growth inhibition of PARPi-treated U2AF1 S34F cells (Fig 1I). Collectively, these results suggest that U2AF1 S34F mutants induce R-loop accumulation and elicit an R-loop-associated PARP1 signaling to promote cell survival.We next tested whether combining ATR inhibitor (ATRi) can further exacerbate PARPi sensitivity in spliceosome mutant cells. To examine ATR activity, we monitored phosphorylated RPA (Replication Protein A, or pRPA), a known ATR substrate. pRPA level was enhanced in PARPi-treated SRSF2 P95H cells compared to PARPi-treated SRSF2 WT cells but was suppressed when treated with ATRi (Fig 1J), suggesting that splicing factor mutant cells are more reliant on ATR function in the context of PARPi. Importantly, the combination of PARPi with ATRi, but not with ATMi, significantly promoted cell growth inhibition in SRSF2 P95H cells compared to SRSF2 WT cells or to SRSF2 P95H cells treated with individual compounds alone (Fig 1K).Collectively, these data provide a pre-clinical rationale that splicing factor mutant leukemias are preferentially sensitive to PARP1 modulation compared to their wildtype counterpart. Moreover, combining PARPi and ATRi may further sensitize spliceosome mutant cells and could represent a new therapeutic strategy in myeloid leukemia patients harboring these mutations (Fig 1L). [Display omitted] DisclosuresGraubert: Calico: Research Funding; Janssen: Research Funding; astrazeneca: Research Funding.

Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition

Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1 mutant HSPCs using a multi-model approach of in vivo zebrafish and mouse systems, and in vitro assays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1 point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivo STATTIC treatment selectively depleted Sf3b1 mutant cells over wildtype in vivo. RNAseq of sf3b1 homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1 heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1 heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivo while sparing wildtype cells. We sought to rescue HSPCs in sf3b1 homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1 and SRSF2 heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666N cells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1 homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment. DisclosuresShastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.

CTX-712, a Novel Clk Inhibitor Targeting Myeloid Neoplasms with SRSF2 Mutation

Splicing factors (SFs) are among the most frequent mutational targets in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and a subset of acute myeloid leukemia (AML), designated as chromatin/spliceosome-mutated AML, where major SFs mutated include SF3B1, SRSF2, U2AF1, and ZRSR2. SF mutations are largely mutually exclusive and except for ZRSR2 mutations, heterozygous, suggesting synthetic lethality of homozygous or multiple mutations. Thus, SF functions might be a plausible target of therapy for MDS/AML. Of potential interest in this regard is serine/arginine-rich (SR) domains ubiquitously shared by many SFs, including U2AF1, SRSF2, and ZRSR2, which need to be phosphorylated for their nuclear translocation by evolutionally conserved kinases, known as CLK family of proteins. CLK family kinases regulate mRNA splicing by phosphorylating various SR proteins, and inhibition of CLK family kinases resulted in reduction of phosphorylation levels of SR proteins, induction of splicing alterations and protein depletion for multiple genes. In addition, a recent report showed that CLK inhibition can induce skipped exons, cell death, and cell growth suppression. Thus, CLK inhibitors might have a role in the therapeutics of SF-mutated MDS/AML, by further compromising RNA splicing.We have recently developed an orally available and highly potent CLK inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. When tested in human myeloid cell lines (K562 and MV-4-11), CTX-712 showed a strong inhibitory effect on cell proliferation (IC 50=0.15 and 0.036 μM, respectively). The anti-leukemic effect was also confirmed by survival assay using a total of 79 primary AML cells (the average of IC 50 was 0.078 μM). In addition, CTX-712 suppressed phosphorylation of multiple SR proteins including SRSF3/4/6, all of which bind to SRSF2. RNA-seq analysis revealed that CTX-712 induced global splicing changes, which typically resulted in skipped exon. Notably, the degree of splicing (percent spliced-in value) in skipped exon events induced by the drug was positively correlated with the sensitivity to the drug (IC 50) in primary AML cells (n=32, R=0.61, P=0.00018).To further investigate the effect of CTX-712 on tumor growth in vivo, we established 13 MDS/AML-derived xenografts (PDXs), which were treated with varying doses of CTX-712. Among the 13 PDX models, SRSF2 mutation was found in 2 cases, which had the SRSF2 P95H or P95L mutation. The SRSF2 P95H PDX showed a significant response to CTX-712 in a dose-dependent manner. Of note, 4 out of 5 mice treated using a high dose protocol (12.5 mg/kg) achieved complete remission (the tumor shrank completely to unmeasurable size). Two weeks after treatment, tumor volumes (mm 3) were 762 ± 147 (vehicle), 331 ± 64 (low dose of CTX-712: 6.25mg/kg, P=0.0395), and 39 ± 39 (high dose, P=0.0064) (N=5 each, mean ± SEM). Interestingly CLK inhibition induced aberrant splicing events including skipped exons in vivo, which were more strongly affected in the SRSF2-mutated model. In addition, CTX-712 efficacy was also confirmed in the model with the SRSF2 P95L mutation. The SRSF2 P95L model showed a significant reduction in tumor volumes (mm 3) 2 weeks after CTX-712 treatment; 406 ± 94 (vehicle) and 75 ± 17 (high dose, P=0.0162) (N=6 each, mean ± SEM). CTX-712 also significantly improved the survival of the SRSF2 P95L-mutated model (high dose, P=0.0030) (N=6 each).Overall, 10 out of 13 PDX AML/MDS models, including 2 SRSF2-mutated models, showed anti-tumor effect of CTX-712. Complete disappearances of tumors were obtained in the SRSF2 P95H mutation model. These results provide mechanistic insights of CLK inhibition and a rationale for further investigation of the novel CLK inhibitor in MDS/AML. DisclosuresYoda: Chordia Therapeutics Inc.: Research Funding. Morishita: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Mizutani: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Tozaki: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Satoh: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Miyake: Chordia Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company.