Splicing Factor Mutations Research Articles

Impact of U2AF1 mutations on circular RNA expression in myelodysplastic neoplasms.

Mutations in U2AF1 are relatively common in myelodysplastic neoplasms (MDS) and are associated with an inferior prognosis, but the molecular mechanisms underlying this are not fully elucidated. Circular RNAs (circRNAs) have been implicated in cancer, but it is unknown how mutations in splicing factors may impact on circRNA biogenesis. Here, we used RNA-sequencing to investigate the effects of U2AF1 mutations on circRNA expression in K562 cells with a doxycycline-inducible U2AF1S34 mutation, in a mouse model with a doxycycline-inducible U2AF1S34 mutation, and in FACS-sorted CD34+ bone marrow cells from MDS patients with either U2AF1S34 or U2AF1Q157 mutations. In all contexts, we found an increase in global circRNA levels in the U2AF1-mutated setting, which was independent of expression changes in the cognate linear host genes. In patients, the U2AF1S34 and U2AF1Q157 mutations were both associated with an overall increased expression of circRNAs. circRNAs generated by a non-Alu-mediated mechanism generally showed the largest increase in expression levels. Several well-described cancer-associated circRNAs, including circZNF609 and circCSNK1G3, were upregulated in MDS patients with U2AF1 mutations compared to U2AF1-wildtype MDS controls. In conclusion, high circRNA expression is observed in association with U2AF1 mutations in three biological systems, presenting an interesting possibility for biomarker and therapeutic investigation.

Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing factor mutations and mis-splicing fuel therapy-resistant leukemia stem cell (LSC) generation in adults, splicing deregulation has not been extensively studied in pAML. Herein, we describe single-cell proteogenomics analyses, transcriptome-wide analyses of FACS-purified hematopoietic stem and progenitor cells followed by differential splicing analyses, dual-fluorescence lentiviral splicing reporter assays, and the potential of a selective splicing modulator, Rebecsinib, in pAML. Using these methods, we discover transcriptomic splicing deregulation typified by differential exon usage. In addition, we discover downregulation of splicing regulator RBFOX2 and CD47 splice isoform upregulation. Importantly, splicing deregulation in pAML induces a therapeutic vulnerability to Rebecsinib in survival, self-renewal, and lentiviral splicing reporter assays. Taken together, the detection and targeting of splicing deregulation represent a potentially clinically tractable strategy for pAML therapy.

The eukaryotic translation initiation factor eIF4E reprograms alternative splicing

Aberrant splicing is typically attributed to splice‐factor (SF) mutation and contributes to malignancies including acute myeloid leukemia (AML). Here, we discovered a mutation‐independent means to extensively reprogram alternative splicing (AS). We showed that the dysregulated expression of eukaryotic translation initiation factor eIF4E elevated selective splice‐factor production, thereby impacting multiple spliceosome complexes, including factors mutated in AML such as SF3B1 and U2AF1. These changes generated a splicing landscape that predominantly supported altered splice‐site selection for ~800 transcripts in cell lines and ~4,600 transcripts in specimens from high‐eIF4E AML patients otherwise harboring no known SF mutations. Nuclear RNA immunoprecipitations, export assays, polysome analyses, and mutational studies together revealed that eIF4E primarily increased SF production via its nuclear RNA export activity. By contrast, eIF4E dysregulation did not induce known SF mutations or alter spliceosome number. eIF4E interacted with the spliceosome and some pre‐mRNAs, suggesting its direct involvement in specific splicing events. eIF4E induced simultaneous effects on numerous SF proteins, resulting in a much larger range of splicing alterations than in the case of mutation or dysregulation of individual SFs and providing a novel paradigm for splicing control and dysregulation.

A multimodal analysis of genomic and RNA splicing features in myeloid malignancies

RNA splicing dysfunctions are more widespread than what is believed by only estimating the effects resulting by splicing factor mutations (SFMT) in myeloid neoplasia (MN). The genetic complexity of MN is amenable to machine learning (ML) strategies. We applied an integrative ML approach to identify co-varying features by combining genomic lesions (mutations, deletions, and copy number), exon-inclusion ratio as measure of RNA splicing (percent spliced in, PSI), and gene expression (GE) of 1,258MN and 63 normal controls. We identified 15 clusters based on mutations, GE, and PSI. Different PSI levels were present at various extents regardless of SFMT suggesting that changes in RNA splicing were not strictly related to SFMT. Combination of PSI and GE further distinguished the features and identified PSI similarities and differences, common pathways, and expression signatures across clusters. Thus, multimodal features can resolve the complex architecture of MN and help identifying convergent molecular and transcriptomic pathways amenable to therapies.

RNA splicing factors in normal hematopoiesis and hematologic malignancies: novel therapeutic targets and strategies.

RNA splicing, a crucial transesterification-based process by which noncoding regions are removed from premature RNA to create mature mRNA, regulates various cellular functions, such as proliferation, survival, and differentiation. Clinical and functional studies over the past 10 y have confirmed that mutations in RNA splicing factors are among the most recurrent genetic abnormalities in hematologic neoplasms, including myeloid malignancies, chronic lymphocytic leukemia, mantle cell lymphoma, and clonal hematopoiesis. These findings indicate an important role for splicing factor mutations in the development of clonal hematopoietic disorders. Mutations in core or accessory components of the RNA spliceosome complex alter splicing sites in a manner of change of function. These changes can result in the dysregulation of cancer-associated gene expression and the generation of novel mRNA transcripts, some of which are not only critical to disease development but may be also serving as potential therapeutic targets. Furthermore, multiple studies have revealed that hematopoietic cells bearing mutations in splicing factors depend on the expression of the residual wild-type allele for survival, and these cells are more sensitive to reduced expression of wild-type splicing factors or chemical perturbations of the splicing machinery. These findings suggest a promising possibility for developing novel therapeutic opportunities in tumor cells based on mutations in splicing factors. Here, we combine current knowledge of the mechanistic and functional effects of frequently mutated splicing factors in normal hematopoiesis and the effects of their mutations in hematologic malignancies. Moreover, we discuss the development of potential therapeutic opportunities based on these mutations.

A Lower Frequency of Spliceosome Mutations Distinguishes Clonal Cytopenias of Undetermined Significance From Low-Risk Myelodysplastic Syndromes, Despite Inherent Similarities in Genomic, Laboratory, and Clinical Features

Clonal cytopenias of undetermined significance (CCUS) are associated with an increased risk of developing a myelodysplastic syndrome (MDS); however, the mechanism and factors associated with evolution remain unclear. We propose that next-generation sequencing (NGS) of cytopenic cases with equivocal morphologic dysplasia will improve patient clinical care and that serial sequencing of such equivocal cases could identify the factors that predict evolution to MDS. We performed targeted NGS of samples from 193 individuals with confirmed or suspected MDS or MDS/myeloproliferative neoplasm, including sequential investigation for 28 individuals at the time of diagnosis and during follow-up. NGS facilitated the diagnosis of all suspicious cases as myeloid neoplasm (21%), CCUS (34%), or idiopathic cytopenias of undetermined significance (45%) when no variants were detected. We found that there was no significant difference in most measured clinical features or clonal phenotypes, such as cell counts, number of variants, variant allele frequencies, and overall survival, between CCUS and International Prognostic Scoring System-Revised–defined low-risk MDS. However, there was a significant difference in the types of variants between CCUS and low-risk MDS, with a significantly lower number of splicing factor mutations in CCUS cases (P < .001). Moreover, we observed an increased probability of evolution to MDS of individuals with CCUS compared with that in those with idiopathic cytopenias of undetermined significance over the first 5 years (P = .045). Our analyses revealed no conclusive pattern associating clonal expansion or the number of variants with the evolution of CCUS to MDS, perhaps further supporting the similarity of these diseases and the clinical importance of recognizing and formally defining CCUS as a category of precursor myeloid disease state in the next revision of the World Health Organization guidelines.

Splicing factor mutations in the myelodysplastic syndromes: Role of key aberrantly spliced genes in disease pathophysiology and treatment.

Mutations of splicing factor genes (including SF3B1, SRSF2, U2AF1 and ZRSR2) occur in more than half of all patients with myelodysplastic syndromes (MDS), a heterogeneous group of myeloid neoplasms. Splicing factor mutations lead to aberrant pre-mRNA splicing of many genes, some of which have been shown in functional studies to impact on hematopoiesis and to contribute to the MDS phenotype. This clearly demonstrates that impaired spliceosome function plays an important role in MDS pathophysiology. Recent studies that harnessed the power of induced pluripotent stem cell (iPSC) and CRISPR/Cas9 gene editing technologies to generate new iPSC-based models of splicing factor mutant MDS, have further illuminated the role of key downstream target genes. The aberrantly spliced genes and the dysregulated pathways associated with splicing factor mutations in MDS represent potential new therapeutic targets. Emerging data has shown that IRAK4 is aberrantly spliced in SF3B1 and U2AF1 mutant MDS, leading to hyperactivation of NF-κB signaling. Pharmacological inhibition of IRAK4 has shown efficacy in pre-clinical studies and in MDS clinical trials, with higher response rates in patients with splicing factor mutations. Our increasing knowledge of the effects of splicing factor mutations in MDS is leading to the development of new treatments that may benefit patients harboring these mutations.

RNA splicing dysregulation in hematological malignancies

Recurrent mutations in genes encoding key splicing factors, SF3B1, SRSF2, U2AF1, and ZRSR2 have been found in a variety of cancers, particularly in hematologic malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, acute myeloid leukemia, and chronic lymphocytic leukemia. Global mis-splicing of mRNAs targeted by aberrant splicing factors partly contributes to leukemogenesis through decrease protein expression of tumor suppressors and epigenetic modifiers, caused by mRNAs degradation of aberrantly spliced. Some of the mis-spliced mRNAs influence intracellular oncogenic pathways and cellular processes through a dysregulated expression of associated proteins, whereas others influence the function of co-mutated genes such as aberrant transcriptional regulators. Spliceosomal disruption is common in many cancers, making spliceosome an appealing therapeutic target. The findings that spliceosomal mutant cells rely on wild-type splicing machinery for survival and that splicing factor mutations occur in a mutually exclusive manner strongly suggest that inhibiting wild-type splicing machinery causes synthetic lethality in cancer cells with these mutations. We discuss the characteristics and oncogenic mechanisms of splicing factor mutations, as well as the development of novel treatment strategies targeting aberrant splicing factors in hematologic malignancies.

Splicing factor deficits render hematopoietic stem and progenitor cells sensitive to STAT3 inhibition

Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis. Mutations of pre-mRNA splicing machinery, especially splicing factor 3b, subunit 1 (SF3B1), are early lesions found in malignancies arising from HSPC dysfunction. However, why splicing factor deficits contribute to HSPC defects remains incompletely understood. Using zebrafish, we show that HSPC formation in sf3b1 homozygous mutants is dependent on STAT3 activation. Clinically, mutations in SF3B1 are heterozygous; thus, we explored if targeting STAT3 could be a vulnerability in these cells. We show that SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Cells carrying mutations in other splicing factors or treated with splicing modulators are also more sensitive to STAT3 inhibition. Mechanistically, we illustrate that STAT3 inhibition exacerbates aberrant splicing in SF3B1 mutant cells. Our findings reveal a conserved vulnerability of splicing factor mutant HSPCs that could allow for their selective targeting in hematologic malignancies.

Molecular Characterization of Clinical Response in Relapsed/Refractory Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome Patients Treated with Single Agent Emavusertib

Introduction Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are myeloid malignancies that exhibit a dynamic mutational landscape as the disease progresses. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of interleukin-1 receptor-associated kinase 4 (IRAK4), which is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Splicing factor mutations are frequently reported in AML and MDS and have been associated with disease progression and poor prognosis in relapsed/refractory (R/R) diseases. Emavusertib is a potent oral inhibitor of IRAK4 as well as Fms-like tyrosine kinase 3 (FLT3). The safety, clinical activity, and potential biomarkers of emavusertib in AML or high-risk MDS (HR-MDS) are investigated in the ongoing open-label, phase 1/2a TakeAim Leukemia trial (NCT04278768). Methods Eligible patients with R/R AML or HR-MDS were enrolled and treated with emavusertib. The exploratory objectives of the study are to assess the association between clinical activity and target-related biomarkers, gene expression signatures, and molecular subtypes. Bone marrow or peripheral blood were collected at the baseline and on treatment. To characterize the mutational profiles of enrolled patients and further define the targeted population, targeted next generation sequencing (NGS) of 68 genes was performed on genomic DNA from bone marrow or peripheral blood mononuclear cells. A minimum coverage of 100x, variant allele frequency (VAF) of >1%, and a VARSCAN-assigned variant p<0.001 were used as thresholds for single nucleotide variants (SNV) calling and presumed somatic driver mutations were identified as previously reported (Metzeler et al. Blood 2016). Mutations from patients’ molecular pathology reports were also documented. Results As of December 16, 2021, 49 patients have been treated with emavusertib monotherapy at one of dose levels of 200, 300, 400, or 500 mg BID, including 30 R/R AML and 19 R/R HR-MDS. The median number of prior anti-cancer therapies was 2 (range 1-5). This abstract is intended to add additional molecular findings to the patient subset presented in 2022 ASCO, of which 6 AML and 7 HR-MDS had splicing factor SF3B1 or U2AF1 mutations and 3 AML had FLT3 mutations, including 2 AML with both splicing factor and FLT3 mutations (hereafter referred to as "targeted mutations"). In the patients with targeted mutations, with available mutational profile data or molecular reports from sites, there were 6 responders: 1 CR, 1 CRh, and 4 mCR. In this responder population, the most common co-mutation was ASXL1. During emavusertib treatment, FLT3-mutated clones became undetectable in 2 of 3 (67%) FLT3-mutated AML patients. One of the 3 patients (33%) with a FLT3 mutation achieved CR and the remaining 2 patients achieved SD (1 with > 90% blast reduction). Additional gene mutations, such as ASXL1, RUNX1, CEBPA, ETNK1, ETV6, and SRSF2, became undetectable during emavusertib treatment. New mutations that emerged after ≥ 1 cycle of emavusertib treatment include NRAS, SETBP1, RUNX1, WT1, STAG2, KRAS, CCND2, DNMT3A, ASXL1, and NOTCH1. Analysis of genomic and transcriptomic profiles will be updated. Conclusion Emavusertib demonstrated anti-cancer activity in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1, SF3B1, or FLT3 mutations. Our preliminary NGS data is suggestive of molecular responses and disease-modifying activity of emavusertib. Patients with targeted mutations responded to emavusertib, even in the presence of co-mutations of ASXL1 that are typically associated with poor prognosis. We will continue to explore potential genomic biomarkers of emavusertib sensitivity and resistance

Multi-Omics Data Integration of Genomics and RNA-Splicing in Myeloid Neoplasia

The high occurrence of splicing factor mutations (SFMT) has suggested that RNA splicing dysfunction plays a key pathogenic role in myeloid neoplasia (MN). Nevertheless, transcriptome-wide analysis suggests that MDS is linked with a broad spectrum of splicing lesions also occurring in cases without SFMT. Due to such a vast complexity, traditional approaches for identification of splicing patterns taking into account features in unimodal fashion may not be adequate to dissect the pathophysiologic features in detail, being instead amenable to more complex machine learning (ML) strategies. Indeed, we hypothesize that splicing dysfunction in MDS may be more wide spread and not restricted to SFMT cases consistent with view that MDS is a global spliceopathy. We explore the geno-transcriptomic pattern of MN by merging multi-omics data to build an ML multimodal tool. An integrative analysis of omics data (RNA and WGS of 1258 whole bone marrows of MN patients) along with ML methods coupling variational autoencoders and canonical correlation analysis was applied to identify covarying features including genomic lesions (mutations, deletions, CNV), exon-inclusion ratio (percent spliced in, PSI) and gene expression (GE). CIBERSORT analysis was used to quantify the composition of clonal cells in bulk samples. Patterns of similarities and differences across patients were compared to 63 age-matched healthy controls and an internal validation of the model was also performed (Fig1). Combination of all features followed by unsupervised clustering identified 15 clusters (C-0 to C-14) resulting in discrete differences in MN subtypes distribution. A distinct definition of the clusters was also seen based on molecular signatures. In our cohort, 54% of patients carried SFMT, further stressing that the mere mutational status of a handful of SFs may not be sufficient to assess the real impact of spliceosome changes. Some clusters were dominated by a high frequency of specific mutations, suggesting a strong functional consequence imprinted by the mutational event. MDS patients were enriched in three clusters with SF3B1MT mainly included in C-1, C-7, C-12. C-1 and C-12 had two distinctive PSI patterns showing decreased splicing efficiency of various genes including DDX20 and KDM3B. SRSF2MT followed similar patterns of distinct PSI in the clusters they populated most (C-8, C-14). C-2 and C-13 contained mainly TET2MT and ASXL1MT. AML patients composed six clusters with two of them (C-9, C-10) consisting of >95% of AML diagnoses. Carriers of DNMT3A, TET2 and FLT3 hits were equally represented in C-0, C-5, C-6 while DNMT3AMT and FLT3MT were equal in C-10. PSI score differences based on ΔPSI (difference in PSI between pairwise clusters) showed high correlation for EZH2 in SRSF2MT, LUC7L2MT and PRPF8MT. PSI similarities were found in subclusters with 1) SF3B1MT, DNMT3AMT, and TET2MT; 2) SF3B1MT with other SFs (PRPF8, DDX41, LUC7L2); 3) TP53MT and DDX41MT possibly suggesting a process of phenocopying and common downstream targets. Using Limma for differential GE analysis we also identified mutational groups clustering together based on GE profiles (group 1: SRSF2, RIT1, JAK2, PTPN11, TET2; group 2: LUC7L2, SF3B1, BCORL1, CSF3R). Using Ward's criterion based hierarchical clustering, we created a map of defining splicing features per cluster. Combination of our multimodal ML with survival outcome in a continuous fashion by means of survival forests and permutation of latent features identified components with highest associations with OS. Focusing on MDS as the most paradigmatic group inherently characterized by a high heterogeneity, we stratified patients based on low and high risk IPSS-R and found that our ML model was effective in discriminating high risk IPSS-R clusters. Finally, we also found a further refinement in low risk IPSS-R cases, with C-12 (SF3B1MT cluster) showing significant separation from other two close clusters (C-7, C-11). This is the first study unifying different levels of multi-omics information to examine the genetic picture of MN as a global spliceopathy. The convergence of molecular and transcriptomic pathways might unveil subgroups of seemingly diverse patients sharing common pathogenetic routes and the identification of these pathways might guide rational therapies. To facilitate the use of such integrated approach intertwining PSI, mutations and GE, we created an interactive MutExPSIplot.1 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Splicing Analysis Unveils Convergent Transcriptomic Pathways in U2AF1 S34 and Q157 Mutant Myeloid Neoplasia

Splicing factor mutations (SFMT) are frequent in MDS and their pathogenic consequences may include missplicing and changes in alternative splicing (AS). However, divergent sequence-sequence effects on misspliced genes were recorded in U2AF1MT at the two canonical hotspots (S34, Q157). Most of the current studies have been focused on the identification of distinctive features of splicing dysfunction between the two hotspots, unveiling various unique hotspot specific splicing events. Classical instances of missplicing events include those in ATG7,H2AFY and STRAP specific to U2AF1S34 MDS. However, whereas we and others have demonstrated certain unique clinical features of MDS patients carrying S34 vs Q1571, these two alterations produce a generally similar disease phenotype and likely rely on a common transformation mechanism. This conclusion suggests that missplicing events might converge in pathways relevant to MDS pathogenesis, which may be much more widespread than what is believed. While defining a topology of AS events (ASE), our analysis of the global landscape of RNA splicing in myeloid neoplasia (MN)2 also opened the possibility to search for subsets of patients sharing RNA splicing features and gene expression (GE). We now curated RNA-Seq data from diagnostic samples of 1258 MN patients by comparing RNA splicing profiles of clonal primary cells with S34 (n=19) and Q157 (n=26) (VAFs >10%, both) in search for overlapping ASE representing new cooperative mechanisms germane to leukemogenesis. ASE were filtered based on percent spliced in (PSI) (variation threshold > 0.01). Limma analysis for differential GE and PSI was applied. All data were analyzed on the genome assembly GRCh37/hg19. Patients without SFMT plus a cohort of 63 age-matched controls were used to detect exclusive ASE common in U2AF1 S34 and Q157. Pathway analysis showed an enrichment of chromatin remodeling, histone demethylases and signal transduction genes. A total of 17564 ASE were called with 30% (n=5106) of them found to be significant (qvalues ≤ .05 and inclusion level difference ≥ 5%). In-depth analysis of ASE showed that 70% of significant PSI changes resulted from exon skipping (ES) followed by intron retention (17%) and alternative 3′ and 5′ splice site events (14%). No significant mutually exclusive ASE was found. Considering that ES might result in truncated proteins and consequent lack of functional domains, we mapped ASE to protein domains. Top PSI changes which were common between S34 and Q157 were found in chromatin remodeling (EZH2,JMJD1C, ARID2/4B, CREBBP), O-linked β-N-acetylglucosamine transferase (OGT), a partner of TET2, GTPase activating (GNAS, ARHGAP9, HBS1L), RNA processing/ binding (RBM5, DDX21, DHX38,HNRNPM, PRPF3), unfolded protein response (XBP1), and ubiquitination (CUL3, NAE1, UBQLN1) genes. Lesions affecting chromosome 7 either structurally (cytogenetic alterations) or by mutations and CNV (EZH2 among others) are associated with adverse prognosis in MN. In total, 8% of the patients had deletion 7 and 4% carried EZH2MT. EZH2 mRNA expression levels were significantly lower in U2AF1MTvs controls (LFC=-0.88; P= 1e-11). Of note is that only 6 cases carried EZH2MT, of whom none harbored deletion 7. This suggests that EZH2 low expression levels might be due to other mechanisms. Looking at most relevant findings, we identified a significant and single ES (ex12) in EZH2 flanking the pre-SET domain (cysteine rich, CXC: aa503-605) crucial to maintain histone methyltransferase activity. Beside somatic mutations, EZH2 function can be altered by other mechanisms, e.g., cooperative mutations. The presence of an ES possibly suggests that an alternative mechanism of gene inactivation beside mutations in the CXC/SET domain and chromosome loss might play a role. Similarly, a CREBBP ES (ex11) was detected upstream the kinase inducible interacting (KIX) domain, a docking site for transcriptional activators (e.g., MLL, c-Myb sites). Finally, U2AF1MT have been also associated with deletion 20q or with a hyperactive long GNAS isoform. We found indeed an ES in GNAS and other GTPases, further emphasizing the broad dysfunctions in U2AF1 cooperative genes. In conclusion, our study describes convergent splicing features common to both U2AF1 S34 and Q157 to conceptualize the essential mechanisms contributing to clonal expansion due to lesions in U2AF1.

SF3B1, RUNX1 and TP53 Mutations Significantly Impact the Outcome of Patients with Lower-Risk Myelodysplastic Syndrome

Introduction: Prognosis of patients (pts) with myelodysplastic syndrome (MDS), particularly the group with lower-risk disease (LR-MDS) is very heterogeneous. In the last decade, different studies have reported the role of recurrent somatic mutations involved in multiple cellular pathways and incorporated them into clinical practice and current prognostic models, including all risk categories. Recently, the incorporation of genomic data to clinical parameters defined the new Molecular International Prognostic Scoring System (IPSS-M). Materials and Methods: A retrospective study evaluating the impact of molecular profile in a series of 181 pts with LR-MDS and non-proliferative chronic myelomonocytic leukemia (CMML) focused on survival estimates and the probability of leukemic progression. Genomic DNA was isolated from bone marrow mononuclear cells at diagnosis using DNA kit from Qiagen according to the manufacturer´s instructions. A PCR-based targeted was performed sequencing of 40 genes involved in myeloid neoplasms (Oncomine Myeloid Research panel, including hotspots of ABL, BRAF, CBL, CSF3R, DNMT3A, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NPM1, NRAS, PTPN11, SETBP1, SF3B1, SRSF2, U2AF1, WT1 and entire coding regions of ASXL1, BCOR, CALR, CEBPA, ETEV6, EZH2, IKZF1, NF1, PHF6, PRPF8, RB1, RUNX1, SH2B3, STAG2, TET2, TP53, ZRSR2). Sequencing was performed using the Ion Torrent S5 sequencing platform from Life Technologies. Survival (OS) was analyzed from diagnosis until death as a result of any cause or the last follow-up and calculated by the Kaplan-Meier method and log rank test. A Cox proportional hazard regression model was performed to analyze OS as endpoint. Analysis was performed by the IBMÒ SPSSÒ version 26.0.0. The probability of leukemic progression was analyzed using the Gray test in the framework of competing risk and considering death not attributable to AML as a competing event. This study was conducted at Hospital Universitario Virgen del Rocío, approved by the local ethics committee and was performed in accordance with the Declaration of Helsinki. Results: In total, 392 mutations were detected in the 181 pts analyzed with a median of 2 mutations per sample (range: 0-8). Sixty-seven pts (37%) harbored ³3 mutations. Table shows baseline characteristics of pts. Most frequent mutated genes were TET2, SF3B1, RUNX1, ASXL1, TP53, and SRSF2 (all of them present in >10% of pts). Epigenetic regulation was the most recurrently mutated pathway, followed by mutations in splicing factors. After a median follow-up of 47 months (95% CI, 37-56 months), 93 out of 191 pts had died (48%). Median OS for the whole cohort was 41 months (95% CI, 28-53 months). TP53 mutations were detected in 22 pts, with equal distribution of mono-allelic or multi-hit TP53 state. As no significant differences in median OS was observed between groups [23 months (95% CI, 12-53 months) in mono-allelic TP53 state vs. 17 months (95% CI, 11-22 months) in multi-hit cases ( P = 0.1)], TP53 mutation was analyzed as a single parameter. Mutations in SF3B1 were detected in 34 pts and the majority of them (31 out of 34; 91%) were classified as SF3B1a according to the recent segregation of SF3B1-mutated cases by the IPSS-M. In univariate analysis, RUNX1 or TP53mutations correlated with lower median OS (Table 2). In contrast, SF3B1 mutation was associated with prolonged median OS [95 months (95% IC, 32-157) vs. 33 months (95% CI, 19-46) in unmutated pts (P < 0.01)]. In a multivariate Cox regression model, RUNX1 mutations independently associated with shorter OS, while SF3B1 mutation retained its favorable impact on outcome (HR: 0.24, 95% CI, 0.1-0.5; P = 0.001). At last follow-up, 49 patients (25%) progressed to AML. Median time to progression was 16.2 months (range: 2-105 months). Subdistribution hazard ratios (sHRs) for leukemic progression were estimated to identify predictive covariates for progression. TP53 mutation (sHR, 6.3; 95% CI, 3-14); P < 0.001) and RUNX1 mutation (sHR, 6.9; 95% CI, 3.3-14; P < 0.001) were predictive for the probability of leukemic progression (Table 2). Conclusion: Incorporation of molecular testing in LR-MDS identified a subset of pts with expected poorer outcome, either due to lower survival or probability of leukemic progression. Results herein reported may help prognosis stratification in this set of pts with low-risk disease and support the recommendation of molecular testing in pts with MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Therapeutic Targeting of Spliceosome Mutant Myeloid Neoplasms Via PARP1 Inhibition

Somatic mutations in splicing factors (SF) SF3B1, SRSF2, U2AF1 are observed across a spectrum of clonal myeloid disorders including myelodysplastic syndromes (MDS). Previous studies identified increased R-loops in SF-mutant cells. Abnormal R-loop accumulation is associated with increased genomic instability and preferential sensitivity to ATR kinase inhibitor. Motivated by these observations, we set out to identify novel therapeutic targets in SF-mutant myeloid malignancies. Using MLLAF9 Srsf2WT and Srsf2P95Hcells, we performed a focused drug screen using inhibitors targeting different DNA damage response pathways and drugs that inhibit DNA replication or DNA metabolic processes. Unexpectedly, Srsf2P95H cells displayed increased sensitivity to inhibitors targeting PARP1/2 (Fig. 1A-C). Olaparib (PARPi) treatment induced higher apoptosis in Srsf2P95Hcells than in Srsf2WTcells (Fig. 1D). To ensure this phenomenon extends beyond Srsf2 mutation, we generated MLLAF9 leukemia on U2af1WT and U2af1S34Fbackgrounds. PARPi treatment in U2af1S34F cells resulted in reduced cell viability and increased apoptosis compared to U2af1WT cells (Fig. 1E-G). Next, we genetically inactivated Parp1 in Srsf2WT and Srsf2P95H cells. The absence of Parp1 did not affect the growth of Srsf2WT cells (Fig. 1H). In contrast, Parp1 deletion significantly reduced proliferation of Srsf2P95H cells (Fig. 1I).Finally, to confirm this phenotype in vivo, we transplanted established MLLAF9 Srsf2WTor Srsf2P95H leukemia into syngeneic mice. Eleven days post-transplant, mice received either vehicle or PARPi. Mice transplanted with Srsf2WT cells showed no survival advantage when treated with PARPi (Fig. 1J). In contrast, PARPi treatment significantly prolonged survival of mice transplanted with Srsf2P95H cells compared to vehicle-treated mice (Fig. 1K). To examine whether PARPi sensitivity is conserved in humans, we treated K562 cells expressing either SRSF2P95H or SF3B1K700E mutation. SRSF2P95H and SF3B1K700E cells showed increased sensitivity to olaparib and rucaparib (Fig. 1L-M). This observation was further validated in a panel of primary AML blasts carrying SRSF2 and U2AF1 mutations(Fig. 1N). Taken together, our results identify PARP1 as a novel therapeutic target in SF-mutant myeloid neoplasms. PARP inhibitors preferentially kill cells with defective homologous recombination (HR)-mediated DNA repair. We asked if the observed PARPi sensitivity in SF-mutant cells was due to defects in HR. To measure HR activity, we performed a CRISPR-Cas9/mClover assay. The assay measures HR-dependent insertion of an mClover-containing donor cassette into Cas9-mediated double-stranded breaks in the LMNA gene, which results in the expression of a green fluorescent Lamin A/C protein. There was no significant difference in the relative percentage of mClover positivity in SRSF2P95H or U2AF1S34F cells compared to control (Fig. 1O), suggesting that these cells are HR-proficient. Interestingly, SF3B1K700E cells showed significantly reduced HR efficiency, consistent with a published study. We next investigated cellular PARP1 activity in SRSF2- and U2AF1-mutant cells by monitoring Poly-ADP-ribosylated chain (PAR) levels. SRSF2P95H cells had elevated PAR level compared to SRSF2WT cells (Fig. 1P). Since PARP1 is known to associate with R-loops, we wanted to test whether increased PARP1 activity and PARPi sensitivity are R-loop dependent. We generated SRSF2-mutant cells that inducibly express RNaseH1, an enzyme that specifically cleaves the RNA moiety within the RNA:DNA hybrids. Induction of RNaseH1 reduced PAR level and PARPi sensitivity in SRSF2P95H cells, suggesting that SRSF2-mutant cells elicit a PARP1 response to promote survival in an R-loop-dependent manner (Fig. 1Q). Lastly, since SF-mutant cells are also sensitive to ATRi, we tested if combining PARPi and ATRi can further sensitize SF-mutant cells. Combined PARPi + ATRi treatment induced synergistic killing of Srsf2- and U2af1-mutant cells compared to wildtype cells in cell viability and colony forming assays (Fig. 1R-S). In summary, this study provides a pre-clinical rationale for therapeutic targeting of PARP1 in SF-mutant leukemia. Moreover, PARP and ATR inhibitor combination could emerge as a new therapeutic strategy in this genetically distinct disease subtype. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

U2AF1 Splicing Factor Mutations Affect RNA Subcellular Compartmentalization in Myeloid Malignancies

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders characterized by morphological dysplasia, cytopenias and high risk of progression to acute myeloid leukemia (AML). Despite recent improvements in treatment strategies, the 5-year relative survival rate in MDS remains only 31%, dropping to 24% in AML. This highlights the need for a deeper characterization of the pathogenic mechanisms driving disease development and progression. Splicing factor (SF) mutations are the most common genetic abnormality in MDS and secondary AML. These mutations, in particular U2AF1 mutations, are associated with increased risk of AML transformation and poor survival. U2AF1 S34F and Q157R mutations determine RNA splicing alterations that eventually result in ineffective hematopoiesis. However, molecular mechanisms and biological consequences underlying the clonal advantage of SF-mutant over wildtype cells remain to be fully elucidated. To address this aim, we recently combined high-resolution transcriptomics and imaging techniques in preclinical models of U2AF1-mutant and wildtype MDS/AML (Biancon et al., Mol Cell 2022). Our results showed that U2AF1 mutations cause widespread RNA binding and splicing alterations that directly affect the biology of RNA granules, i.e. dynamic condensates of RNAs and RNA binding proteins implicated in RNA metabolism, signaling and gene regulation. In particular, in U2AF1-mutant vs WT cell lines and primary cells we observed increased formation of stress granules (SGs), cytoplasmic RNA granules mediating cellular adaptation to stress conditions. Crucially, the enhanced stress response in U2AF1-mutant cells was associated with improved cell fitness, that was reverted by SG inhibition. Dysregulation of SGs, and potentially of other subcellular RNA condensates, may therefore represent a novel therapeutic vulnerability in MDS/AML patients carrying U2AF1 mutations. We here integrated single-cell (sc)RNA-seq data to further support the direct link between the U2AF1-mutant genotype and the increased SG phenotype in myeloid malignancies. We first isolated CD34+ blasts from bone marrow samples of MDS patients with (n=3) or without (n=3) U2AF1 S34F mutation. We then performed 10x Genomics scRNA-seq obtaining an average of 3,478 sequenced cells per sample for downstream analyses. Cells from different patients were integrated with Seurat v4.0.1 and annotated with the recently released Cell Marker Accordion (https://github.com/TebaldiLab/TheCellMarkerAccordion), confirming the expected skewing towards granulocyte-monocyte progenitor cells and erythroid lineage in S34F-mutant patients. Single cells were subsequently classified according to the detection of the S34F mutation (VarTrix v1.1.19), into: "WT" (WT cells in U2AF1-WT patients), "S34F_WT" (WT cells in U2AF1-S34F patients), "S34F" (S34F-mutant cells in U2AF1-S34F patients). Each cell was also associated to an "SG expression score", measuring the collective abundance of 149 transcripts enriched in SGs and aberrantly bound-spliced by mutant U2AF1 (Biancon et al., Mol Cell 2022). Importantly, S34F-mutant cells represented the category with the highest SG expression score (Figure 1A), consistently indicating an increased stress adaptation that might explain the clonal advantage of splicing factor-mutant cells in MDS. To further understand the effect of U2AF1-mutant binding and splicing alterations on other RNA condensates, we focused on Cajal bodies (CBs), nuclear granules involved in snRNA transcription and assembly of spliceosomal RNPs. Immunofluorescence (IF) staining for coilin, a known CB marker, followed by confocal imaging revealed a decrease in the number of CBs in U2AF1 S34F vs WT myeloid cell lines (Figure 1B). Of note, IF data were supported by RNA-seq data performed in the same cellular system (Biancon et al., Mol Cell2022), showing a significantly lower expression of snRNAs in U2AF1 mutant vs WT cells. The role of CBs in cancer is currently unknown, but the disruption of CBs may be associated to altered RNA splicing kinetics and increased genomic instability. Collectively, our results suggest that U2AF1 mutations affect the compartmentalization and aggregation properties of targeted RNAs, providing the rational to further investigate RNA granule perturbations in splicing factor-mutant myeloid malignancies for therapeutic potential. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nonsense-Mediated RNA Decay Disruption Induces Altered Splicing in Spliceosome Mutant Cells

Introduction: Expression of mutant splicing factors U2AF1(S34F), SF3B1(K700E), or SRSF2(P95H) alter RNA splicing in myeloid malignancies, including increased production of nonsense transcripts that rely on nonsense-mediated RNA decay (NMD) for clearance. Cell lines expressing spliceosome mutants are more sensitive to NMD inhibition than wild-type cells and the differential sensitivity is partially dependent on R-loop formation. The impact of NMD disruption on the viability of primary splicing factor mutant hematopoietic cells and the consequence on downstream RNA splicing are not known. Methods: Cell viability of primary mouse hematopoietic cells (untransformed and AML cells) and a human AML cell line that expresses mutant or wild-type U2AF1 after treatment with a highly-specific inhibitor of SMG1 (SMG1i), the only known protein kinase in the NMD pathway, was measured using a CellTiter-Glo assay. Gene expression and RNA splicing changes induced by mutant U2AF1 and/or SMG1 inhibition were identified by short and long-read RNA sequencing (RNA-seq). Results: We treated primary untransformed mouse U2AF1S34F or U2AF1WT expressing cKit+ hematopoietic cells ex vivo with SMG1 inhibitor and monitored cell viability. We found that SMG1i reduced the viability of primary mouse cells expressing mutant U2AF1S34F compared to U2AF1WT (IC50 52.6 nM vs 173.5 nM, respectively, p=<0.0001). Next, we tested the sensitivity of primary mouse AML cells expressing a doxycycline-inducible U2AF1S34F or U2AF1WT transgene to SMG1i that were generated using retroviral expression of the fusion oncogene MLL-AF9 to induce AML. We found that NMD disruption using SMG1i also reduced the ex vivo viability of primary U2AF1S34F compared to U2AF1WT expressing mouse AML cells (IC50 40.9 nM vs 102.2 nM, respectively, p=<0.0001), highlighting the potential utility of SMG1i treatment for spliceosome mutant myeloid malignancies. To explore the underlying mechanism for the sensitivity of spliceosome mutant cells to NMD inhibition, we generated, K562 cell lines expressing doxycycline-inducible U2AF1S34F or U2AF1WT and confirmed that NMD disruption using SMG1i reduced cell viability of mutant expressing cells in vitro (IC50 128.1nM vs 578.5nM at 72 hours, respectively, p≤0.0001). Next, we performed RNA-seq on K562 cells expressing U2AF1WT or U2AF1S34F that were treated with vehicle or 1uM SMG1i for 24 hours (n=3 replicates). We identified reduced expression of genes involved in the cell cycle, DNA replication/repair, telomere maintenance and mRNA splicing in SMG1i treated U2AF1S34F compared to U2AF1WT cells (GSEA; FDR<0.008). Next, we identified 3,393 RNA splicing alterations induced by SMG1i treatment of U2AF1S34F compared to U2AF1WT cells (rMATS; FDR<0.1, percent spliced-in >10%). Genes with altered splicing were enriched in similar pathways, including the cell cycle, DNA replication, and RNA splicing and processing pathways (Cluster Profiler; FDR≤0.02). Finally, we performed long-read RNA-seq (Nanopore) on RNA isolated from K562 cells (not expressing spliceosome mutants) that were treated with vehicle or SMG1i (0.5uM, 24hours, n=2) to directly identify full length transcripts containing premature termination codons (PTC) that are normal targets of NMD. Our preliminary data indicate that SMG1i significantly increases the number of detectable PTC containing transcripts (known and novel) compared to vehicle treatment (mean 19,291 vs 5,621 PTCs, respectively; p=0.008). In addition, PTC-containing transcripts that were overexpressed in SMG1i vs vehicle treated cells also occur in genes involved in the cell cycle, DNA repair and mRNA splicing (FDR£0.001), suggesting they are regulated by NMD under basal conditions. These gene pathways may be particularly vulnerable to further dysregulation in mutant treated cells because they are also altered by mutant U2AF1S34F expression. Conclusion: A vulnerability of primary hematopoietic cancer cells with spliceosome mutations to NMD inhibition suggests the possibility that NMD can be targeted for treating MDS with aberrant splicing. Excessive R-loop accumulation in SMG1i treated U2AF1S34F cells may contribute to the downstream gene expression and splicing alterations and reduced viability of mutant cells. Ongoing pre-clinical in vivo efficacy studies will test the potential for SMG1i to treat hematologic malignancies that have splicing factor mutations.

Mis-Splicing Due to Somatic U2AF2 Mutations in Myeloid Neoplasms

Background Recent genetic studies have identified frequent mutations affecting a number of genes encoding RNA splicing factors (SDs) in myelodysplastic syndromes (MDS) and related myeloid neoplasms (MNs). Among these, most frequently mutation are SF3B1, SRSF2, U2AF1, and ZRSR2, whose functions have been intensively studied. By contrast, U2AF2, another SF, is also recurrently mutated in MNs. U2AF2 is a component of the U2 auxiliary factor that forms a heterodimer with U2AF1 for the recognition of the 3’ splice site (3'SS). U2AF2 contains a sequence-specific RNA-binding region with two RNA recognition motifs and identifies polypyrimidine (Py) tract signals of nascent transcripts. However, compared to other splicing factor mutations, U2AF2 mutations are much rare, preventing the detailed analysis of their role in leukemogenesis. Methods To characterize the role of U2AF2 mutations, we systematically analyzed mutation spectrum in 6,369 with different MNs using targeted-capture sequencing. We also performed RNA sequencing of cKit(+) bone marrow cells from 6 U2AF2 mutated MDS cases along with 52 MDS patients without common splicing factor mutations and 25 healthy individuals. THP1 and HL60 leukemia cells with exogenous expression of wildtype or mutant U2AF2 were established to evaluate the splicing response. We also used clustered regularly interspaced short palindromic repeats /CRISPR-associated protein-9 nuclease (CRISPR/cas9) to introduce the p.190_195del mutation to U2AF2 in K562 and MOLM-13 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant U2AF2. Results In total, U2AF2 mutations were found in 34 (0.53%) of 6,369 MN cases, of which 29 had the recurrent p.190_195del mutation, the median age of U2AF2 mutations was 55 years (range 31-79) and 55% were diagnosed with MDS, 31% with CMML and 7% with AML. U2AF2-mutated patients showed a male predominance (90%). The most frequently co-mutated gene was ASXL1 with 52%, followed by SETBP1 (35%) STAG2 (21%),RUNX1 (21%), CSF3R, NRAS, DNMT3A, NF1, PTPN11, ETV6, and STAT3. To investigate the effects of the U2AF2 p.190_195del mutation on RNA splicing, we analyzed transcriptome sequencing, followed by the rMATS bioinformatics pipeline to determine alternative splicing (AS) events . Various types of AS events were identified, including skipped exons (SEs), alternative 5’ ss exons (A5SSs), alternative 3’ ss exons (A3SSs), retained introns (RIs), and mutually exclusive exons (MXEs). Among these, the skipping exons are the most frequent in both U2AF2-mutant primary MDS cases and cell lines. To explore these alternative splicing results in more detail, we next examined differentially skipped exons. Unsupervised uniform manifold approximation and projection (UMAP) analysis based on the standardized splicing ratio of skipping exons segregated U2AF2-mutated patients from other MDS cases without common splicing factor mutations and healthy individuals. To prioritize mutant U2AF2-induced alterative splicing events, we intersected significant skipping exons across 2 datasets: MDS patient samples with and without U2AF2 p.190_195del mutations, and MDS patient samples with U2AF2 p.190_195del mutations and healthy individuals. The differential skipping exon usages and differential gene expression profiles enables both consensus sequence analysis and pathway/gene-set enrichment analysis. Conclusions Our study revealed that the U2AF2 p.190_195del regulated aberrant alternative splicing facilitated MDS progression through perturbations in splice isoforms. A better understanding of the U2AF2 p.190_195del and its critical specific changes will provide novel insights into disease pathophysiology and potentially inform future treatment decisions.

Decreased Embryonic Splicing Repressor Expression Enhances Splicing Modulator Sensitivity in Pediatric Acute Myeloid Leukemia Stem Cells

Adults with myeloid malignancies frequently harbor mutations in splicing regulatory genes, such as SRSF2, U2AF1, and SF3B1. In pediatric AML (pAML), splicing factor mutations are relatively infrequent in bulk populations of cells. However, by performing whole exome sequencing, single cell proteogenomics (Tapestri®) as well as whole transcriptome RNA sequencing (RNA-seq) analyses of FACS-purified hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we discovered an increased frequency of exon skipping, typical of embryonic splicing patterns, in pAML HSCs and HPCs. By employing single cell proteogenomics (Tapestri®) analysis, we discovered SF3B1 splicing factor mutations at the single cell in pAML CD34+ cells that were not readily detectable by whole exome sequencing in bulk populations thereby suggesting that clonal expansion of minor clones may fuel leukemic stem cell (LSC) generation. Further RNA-seq analysis demonstrated that both pAML HSCs and HPCs harbored decreased expression of a repressor of embryonic splicing, RNA-binding Fox protein 2, RBFOX2(also known as RBM9). By employing, rMATS, an algorithm that detects splicing events, we discovered differential splicing of genes involved in cell cycle regulation, DNA repair and chromatin organization in pAML more than in non-leukemic (NL) samples. In addition, differentially spliced RBFOX2 target genes were found to be involved in mRNA processing. In pAML HSCs, we observed differential splicing of THOC7, which regulates mRNA 3' end processing while in pAML HPCs, we uncovered a cluster of splicing regulators, such as SRSF7, SRSF11 and U2AF2. Thus, pAML is typified by widespread splicing deregulation in the setting of RBFOX2 downregulation. Previous reports suggest that splicing deregulation represents a therapeutic vulnerability in a broad array of malignancies. Thus, we examined the response of pAML cell lines in vitro and in lentiviral splicing reporter humanized AML mouse models to Rebecsinib (17S-FD-895; Crews et al, Cell Stem Cell, 2016), a small molecule splicing modulator that is currently undergoing IND enabling studies. In these studies, Rebecsinib treatment reduced pAML cell survival at doses that altered splicing reporter activity and spared normal hematopoietic stem and progenitor cells (HSPCs). Notably, shRNA knockdown of RBFOX2 in cord blood CD34+ HSPCs phenocopied aspects of pAML in that it significantly increased clonogenic capacity and sensitivity to Rebecsinib in replating assays. In summary, decreased RBFOX2 expression and reversion to an embryonic splicing program typified by differential exon usage in pAML HSPCs was associated with increased sensitivity to selective splicing modulation and may represent a new therapeutic paradigm for pediatric patients with relapsed or refractory AML.

Splicing Gene Mutations Do Not Affect Outcomes in Newly Diagnosed Patients with Acute Myeloid Leukemia

Background: Recent studies have shown that splicing factor (SF) gene mutations in AML implicate a negative prognostic significance associated with secondary AML and are now included as adverse in the 2022 ELN guidelines (Dohner et.al., Blood 2022). However, the contribution of these mutations to the prognosis of patients (pts) with AML, relative to their age, prior history of myeloid disorders, cytogenetics (CTG), concurrent mutations and frontline (FL) therapy requires further characterization. Methods: We retrospectively analyzed all pts with newly diagnosed (ND) AML treated at our center over the last 5 years (yrs) and compared outcomes for patients with and without SF mutations (Smut= SF3B1, SRSF2, U2AF1 and ZRSR2). FL therapy was classified as intensive (INT) and low intensity (L-I), with/without venetoclax (VEN). Relapse free survival (RFS) was calculated from the time of response to relapse or death and overall survival (OS) was calculated from the time of therapy initiation to death; both were censored at last follow up (F/U) Results: From January 2017 to April 2022, 994 ND pts with AML were reviewed; 266 (27%) had Smut in ≥ 1 of the SF genes while the rest were wild type (SWT). The most common Smut was SRSF2 (53%). Only 3% pts had >1 Smut (Table 1). The median age of Smut group was 72 yrs compared to 65 yrs in the SWT group, and significantly more pts in the former had secondary/therapy related AML (s/t AML) (51% vs 41%). The Smut group had less complex karyotype (CK), non CK adverse risk CTG and did not include any pts with core-binding factor (CBF) leukemias. Fewer pts in the Smut had a pathogenic TP53mut but had higher frequencies of RUNX1 and ASXL1 mutations. Overall, 71% Smut pts were classified as ELN 2017 adverse risk compared to 54% SWT pts (p<0.0001). 20% pts in the Smut group vs. 44% SWT pts received INT therapy (p<0.0001). Of the Smut pts who received L-I therapy, 62% received a hypomethylating agent (HMA) and 66% received a VEN- based combination, frequencies similar to the SWT group. At a median F/U of 26 months (mos), the median RFS and OS was 9.6 vs. 12.4 and 13.1 vs 13.3 mos for the Smut and SWT groups respectively. Given the significant age heterogeneity between the 2 groups we analyzed by young (<60 yrs) and older (≥60 yrs) pts with non-CBF AML and compared survival outcomes (Fig.1). Both Smut and SWT pts had similar RFS and OS in their respective age groups. In a small subgroup of older pts with de novo AML with CK treated with L-I regimens, the RFS and OS were especially dismal for those with Smut (RFS= 1.5 vs 5.7 mos, p=0.14 and OS= 2.6 vs 9.3 mos, p=0.003). For other subgroups (stratified based on ELN risk stratification, de novo or s/t AML, CK, TP53mut) outcomes of Smut and SWT were similar in both young and older pt groups treated with INT or L-I therapies. Smut pts who received VEN in the L-I arm had better RFS and OS, especially if they had denovo AML (RFS= 19 vs 5.5 mos and OS 25 vs 4 mos). On univariate (UV) analysis using age, CTG [adverse risk (AR) /intermediate risk (IR)], therapy arm (INT/L-I), Smut/SWT status and concurrent mutations (TP53, ASXL1, RAS, RUNX1, NPM1, FLT3-ITD) as covariates for overall response (OR= CR/CRi/MLFS): lower age, IR-CTG, TP53wt, RASwt ,FLT3mut, NPM1mut and use of INT therapy were favorable while all the factors other than FLT3mut , age and therapy arm remained significant on the multivariate (MV) model. Within the Smut cohort, the presence of SF3B1mut alone negatively affected the odds of OR (0.65, 95%CI=0.3-0.98) on UV but not on MV analysis. Cox regression analysis demonstrated adverse risk CTG, TP53mut and s/t AML had higher hazards of relapse, while the above factors along with the presence of RASmut increased the hazards of death. On stratifying by specific Smut, no individual mutation impacted OS. Cox regression analysis of the above covariates affecting RFS and OS in Smut pts who received L-I therapy showed VEN based combinations significantly reduced the hazards for relapse (0.5, 95% CI=0.3-0.8) and death (0.5, 95%CI=0.3-0.7). Overall, the presence of Smut did not affect response rates to therapy, RFS or OS in both young and older pts treated with L-I (majority VEN based) or intensive therapy. Conclusion: Smut are more common in elderly pts with AML and in s/t AML. Smut does not necessarily affect response rates and survival outcomes compared to SWT pts when controlled for these covariates, and with current therapeutic options including HMA + VEN based regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SRSF2P95Hand FLT3ITDSynergistically Drives Leukemic Transformation in Zebrafish

Internal Tandem Duplication of human FMS-like tyrosine kinase (FLT3-ITD) is one of the most frequent genetic alterations in de novo acute myeloid leukemia (AML) and is often acquired during transformation of myelodysplastic syndrome (MDS) into AML, predicting an unfavorable prognosis. RNA splicing factor mutations (e.g. hot spot mutations in serine and arginine rich splicing factor 2 - SRSF2) are often found in MDS and chronic myelomonocytic leukemia (CMML). However, the clinicopathologic features of co-mutated FLT3-ITD and SRSF2 in MDS/CMML and transformation to AML are presently unclear. Here we investigated whether FLT3-ITD cooperates with SRSF2-P95H to promote leukemogenesis in zebrafish. Transgenic zebrafish carrying human FLT3-ITD or SRSF2-P95H as driven by Runx1 promoter were generated by Tol2 transgenesis. Tg(Runx1:FLT3ITD) was outcrossed with Tg(Runx1: SRSF2P95H) to generate Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish. The presence of human FLT3-ITD or SRSF2-P95H was confirmed through Sanger sequencing, and the expression of each transcript was detected by qRT-PCR in both embryos and adult kidney marrow. Their clinicopathologic features were examined. At the embryonic stage, the number of leukocytes (l-plastin+) was significantly increased in Tg(Runx1: FLT3ITDSRSF2P95H) embryos compared with their single mutant and wildtype (WT) siblings, whereas the abundance of neutrophils (SBB+) was unaffected. At six months of age, Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish showed monocytosis in peripheral blood (PB) (1 out of 4), but not in the single mutant and WT siblings. In kidney marrow (KM), there was expansion of both immature and mature monocytes, and dysplasia of granulopoiesis and erythropoiesis. At 9 months of age, all four Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish showed neutropenia and substantial increase in monocytosis. At 1 year of age, one out of the nine Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish showed a significant increase in blasts in KM, reminiscent of human AML, and the other eight Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish demonstrated phenotypes consistent with human CMML-like diseases. Increased monocytic compartment of KM was confirmed by α-naphthyl acetate esterase sensitive to fluoride inhibition. In addition, both the spleen area and cellularity were expanded in Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish compared with Tg(Runx1: SRSF2P95H) single mutant and WT siblings. Taken together, our findings demonstrated that FLT3-ITD cooperates with SRSF2-P95H to drive the initiation and progression of CMML and the subsequent transformation to AML. Understanding the molecular underpinnings of these events will shed light to the diverse clinicopathologic features in CMML patients.