U2AF1 Mutations Research Articles

Association of Somatic Gene Mutations with Risk of Transformation into Acute Myeloid Leukemia in Patients with Myelodysplastic Syndrome: A Systematic Review and Meta-Analysis.

Objectives:we aim to conduct a systematic review and meta-analysis in population of adult MDS patients to elucidate the role of these genes in AML transformation risk. Materials and Methods:The protocol for this systematic review and meta-analysis was registered in the international prospective register of systematic reviews (PROSPERO) with ID number of CRD42020218581. Systematic literature search was conducted by all authors up to October 2021 on: (1) PubMed, (2) EBSCOhost, (3) Scopus, (4) JSTOR, and (5) grey literatures. Hand-searching for relevant articles was also conducted. The following keywords with their synonyms and combinations using Boolean operators were applied to all database: “myelodysplastic syndrome”, SRSF2”, “SF3B1”, “U2AF1”, “ASXL1”, “DNMT3A”, “TET2”, “IDH1”, “IDH2”, “RUNX1”, “acute myeloid leukemia progression”, and “leukemia free survival”. Outcome was measured using hazard ratio (HR). Results:We identified 14 articles to be used for this systematic review and meta-analysis. There was no statistically significant difference in AML transformation risk between U2AF1 mutant and U2AF1 wildtype MDS patients (HR: 1.41; 95% CI: 0.95–2.07, p=0.08, I2=0%). Pooled HR showed that patients with SRSF2 mutation had higher risk of AML transformation (HR 2.62; 95% CI: 1.54-4.45; p= .0004; I2= 55%). The pooled HR for SF3B1 was 0.48 (95% CI: 0.22–1.06, p=0.07, I2=55%). Mutations of TET2, ASXL1, and EZH2 were not associated with AML transformation. Meanwhile, DNMT3A mutations were associated with AML transformation with pooled HR of 2.73 (95% CI: 1.43-5.21; p= 0.08; I2: 67%). The pooled HR for IDH genes was smaller (HR: 2.92; 95%CI: 1.21-7.06; p=0.02; I2:65%). Patients with RUNX1 mutation were associated with AML transformation (HR: 1.85; 95%CI: 1.11-3.09; p=0.02; I2:38%). Conclusion:Based from our analyses, MDS patients with mutations of SRSF2, DNMT3A, IDH, and RUNX1 have higher hazard ratio for AML transformation.

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.

Identification of a novel HOOK3-FGFR1 fusion gene involved in activation of the NF-kappaB pathway

BackgroundRearrangements involving the fibroblast growth factor receptor 1 (FGFR1) gene result in 8p11 myeloproliferative syndrome (EMS), which is a rare and aggressive hematological malignancy that is often initially diagnosed as myelodysplastic syndrome (MDS). Clinical outcomes are typically poor due to relative resistance to tyrosine kinase inhibitors (TKIs) and rapid transformation to acute leukemia. Deciphering the transcriptomic signature of FGFR1 fusions may open new treatment strategies for FGFR1 rearrangement patients.MethodsDNA sequencing (DNA-seq) was performed for 20 MDS patients and whole exome sequencing (WES) was performed for one HOOK3-FGFR1 fusion positive patient. RNA sequencing (RNA-seq) was performed for 20 MDS patients and 8 healthy donors. Fusion genes were detected using the STAR-Fusion tool. Fluorescence in situ hybridization (FISH), quantitative real-time PCR (qRT-PCR), and Sanger sequencing were used to confirm the HOOK3-FGFR1 fusion gene. The phosphorylation antibody array was performed to validate the activation of nuclear factor-kappaB (NF-kappaB) signaling.ResultsWe identified frequently recurrent mutations of ASXL1 and U2AF1 in the MDS cohort, which is consistent with previous reports. We also identified a novel in-frame HOOK3-FGFR1 fusion gene in one MDS case with abnormal monoclonal B-cell lymphocytosis and ring chromosome 8. FISH analysis detected the FGFR1 break-apart signal in myeloid blasts only. qRT-PCR and Sanger sequencing confirmed the HOOK3-FGFR1 fusion transcript with breakpoints located at the 11th exon of HOOK3 and 10th exon of FGFR1, and Western blot detected the chimeric HOOK3-FGFR1 fusion protein that is presumed to retain the entire tyrosine kinase domain of FGFR1. The transcriptional feature of HOOK3-FGFR1 fusion was characterized by the significant enrichment of the NF-kappaB pathway by comparing the expression profiling of FGFR1 fusion positive MDS with 8 healthy donors and FGFR1 fusion negative MDS patients. Further validation by phosphorylation antibody array also showed NF-kappaB activation, as evidenced by increased phosphorylation of p65 (Ser 536) and of IKBalpha (Ser 32).ConclusionsThe HOOK3-FGFR1 fusion gene may contribute to the pathogenesis of MDS and activate the NF-kappaB pathway. These findings highlight a potential novel approach for combination therapy for FGFR1 rearrangement patients.

Failure to Detect Mutations in U2AF1 due to Changes in the GRCh38 Reference Sequence

The U2AF1 gene is a core part of mRNA splicing machinery and frequently contains somatic mutations that contribute to oncogenesis in myelodysplastic syndrome, acute myeloid leukemia, and other cancers. A change introduced in the GRCh38 version of the human reference build prevents detection of mutations in this gene, and others, by variant calling pipelines. This study describes the problem in detail and shows that a modified GRCh38 reference build with unchanged coordinates can be used to ameliorate the issue.

Clonal hematopoiesis in aplastic anemia and paroxysmal nocturnal hemoglobinuria

Similar with myelodysplastic syndromes (MDS), aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are the major bone marrow failure syndromes. Approximately 10-20% of patients with AA/PNH present with transformation into MDS. Clonal hematopoiesis in AA/PNH affected by karyotypic abnormalities and genetic mutations should be discriminated from MDS clone, which is sometimes difficult due to shared genetic events among these diseases. In patients with AA/PNH, clones with UPD6p and PIGA mutations are selected under autoimmune pressure, and those with DNMT3A, ASXL1, and TET2 mutations originated from clonal hematopoiesis of indeterminate potential (CHIP) frequently identified in elderly healthy individuals. In patients with cytopenia, a single CHIP mutation is insufficient for MDS presentation. However, TP53 and U2AF1 mutations, which are not in the list of typical CHIP mutations, are observed in patients with AA with future MDS transformation. Therefore, clonal hematopoiesis in AA/PNH, partially overlapping the MDS clone, is caused by autoimmunity and originates from CHIP, demonstrating distinct genetic profiles.

Effect of U2AF1 Mutation to Inflammatory Cytokine Expression in SKM-1 Cells through FOXO3a-Bim Signaling Pathway

To investigate the effect of U2AF1 gene mutation to inflammatory cytokine in SKM-1 cell of human myelodysplastic syndromes (MDS), and whether the above effects were mediated by FOXO3a-Bim signaling pathway. Wide-type U2AF1 and mutant U2AF1 (the serine residue 34 was replaced by phenylalanine, and named as S34F) recombinant expression plasmids were constructed. Lentiviruses were packaged and transfected into SKM-1 cells. The expression of FOXO3a was up-regulated by lentiviruses, and its transfection rate was investigated. The cell proliferation was detected by CCK-8 method. Flow cytometry was used to detect the apoptosis and cycle of the cells. The expression pro-inflammatory cytokine IL-1β, IL-6, TNF-α and anti-inflammatory cytokine IL-4 were detected by qRT-PCR. FOXO3a, Bim, Bcl-2 and Bax protein expression levels were detected by Western blot. Compared with the control group, the cell apoptosis rate, pro-inflammatory cytokine IL-1β and TNF-α transcription levels were significantly increased in the S34F group (P<0.05); cell cycle was blocked at the G2 phase; cell proliferation and the anti-inflammatory cytokine IL-4 transcription level were significantly decreased; the expression levels of FOXO3a, Bim and Bax protein were significantly increased (P<0.05); while the expression level of Bcl-2 protein was significantly decreased (P<0.05). The up-regulation of FOXO3a could significantly inhibited the proliferation and increased cell apoptosis of SKM-1 cells with U2AF1 S34F mutation; cell cycle was blocked at the S and G2 phases; the pro-inflammatory cytokine IL-1β and TNF-α transcription levels were significantly decreased (P<0.05), and the transcription level of anti-inflammatory cytokine IL-4 showed no statistically significant as compared with control group (P>0.05). U2AF1 S34F mutation can regulate inflammatory phenotype in SKM-1 cells, which may be mediated through FOXO3a-Bim signaling pathway.

A Myelodepletive Phenotype Is Associated with Distinctive Molecular Features and Adverse Outcomes in Patients with Myelofibrosis

▪Introduction: Myelofibrosis -either primary (PMF) or post-polycythemia vera/essential thrombocythemia (collectively, secondary MF [SMF])- is characterized by biologic and clinical heterogeneity, with some patients (pts) presenting with features of myeloproliferation (MyP) and others exhibiting a myelodepletive (MyD) phenotype. Although not well defined, the latter is marked by cytopenias involving one or more hematopoietic lineages, in some respects mimicking a myelodysplastic or bone marrow (BM) failure syndrome. MyD features have been associated with poor prognosis in patients with MF, but cytopenias have been usually considered individually. In the current study, we aimed at investigating the phenotypic and prognostic correlates of a MyD phenotype in a large cohort of MF patients.Methods: After IRB approval, 704 consecutive patients with WHO-defined MF referring to CRIMM (Florence), were included in the study. There were 442 (63%) PMF and 262 (37%) SMF. Cytopenias at diagnosis were defined as follows: white blood cell (WBC) <4×10 9/L, hemoglobin (Hb) <11 g/dL for male and <10 g/dL for female, platelets (Plt) <100×10 9/L. A MyD phenotype was defined by the presence of at least one cytopenia in the absence of any increase of other blood cells (WBC >15×10 9/L, Hb >16.5 g/dL for male and >16 g/dL for female, Plt >450×10 9/L). Pts not included in the MyD group were considered as having a MyP phenotype. Mutational analysis by targeted NGS was available for 594/704 patients (PMF 368/442; SMF 226/262).Results: Overall, 166 pts were diagnosed as having a MyD MF, including 108 (24%) PMF and 58 (22%) SMF. Among PMF patients, leukopenia, sex-adjusted anemia and thrombocytopenia were present in 38%, 84% and 35%, respectively; in SMF, corresponding figures were 24%, 91% and 24%. Two or more cytopenias were found in 11% and 6% of PMF and SMF patients, respectively.In PMF, a MyD phenotype was associated with male gender (P=.0468), older age (P=.0002), lower peripheral blast count (P=.0006), higher prevalence of splenomegaly (P=0.0142), constitutional symptoms (P<.0001), and BM fibrosis grade ≥2 (P<.0001). The rates of thrombosis and bleeding were not different compared to MyP PMF. MyD pts were more likely to have very high risk (VHR) karyotype abnormalities (P=.0002) and to be triple negative (TN; P<.0001); there were no correlations with JAK2 mutation status and mutant burden, CALR and MPL mutations. Among non-driver mutations, a MyD phenotype was significantly enriched in ASXL1 (P=.0074), IDH1/2 (P=0.064), N/KRAS (P=.0014), U2AF1 (P<.0001), and CUX1 (P=.0002) mutations. Karyotype abnormalities (P=.0084), VHR cytogenetics (P=.0343), CBL (P=.0.171) and U2AF1 (P=.0148) mutations were significantly enriched in MyD pts with ≥2 cytopenias. On univariate analysis, OS was significantly shorter in pts with MyD vs MyP PMF (median, 55 vs 103 months, respectively; P<.0001), and not different between MyD pts with one (median, 65 months) and ≥2 (median, 44 months) cytopenias (Fig. 1A). After competing risk analysis, the cumulative incidence of leukemic transformation (LT) was not different between MyD and MyP phenotypes (16% vs 11% at 5 years, respectively).Phenotypic differences between MyD and MyP patients were less evident in SMF, a part for older age of the former (P=.0207). The molecular background was likewise different: a MyD feature was enriched in mutations in TP53 (P=.0024), U2AF1 (P<.0001), and SETBP1 (P=.0125). TP53 mutations were remarkably more frequent in MyD SMF pts with ≥2 cytopenias (19%). Pts with MyD SMF had a significantly shorter OS compared to the MyP counterpart (median, 44 vs 105 months; P<.0001). Median OS was significantly inferior in MyD patients with ≥2 cytopenias compared to one cytopenia (median, 27 vs 58 months, respectively; P<.0001) (Fig. 1B). The cumulative incidence of LT was not different: 16% vs 7% at 5 years, respectively, for SMF pts with MyD and MyP phenotype.Conclusion: The current study provides a comprehensive analysis of the MyD phenotype in pts with PMF and SMF, partly recapitulating findings of previous studies (Marcellino BK et al. Clin Lymphoma Myeloma Leuk 2020). We showed that the MyD phenotype is associated with high-risk clinical and molecular features, particularly in PMF, and correlates with inferior OS. Of interest, U2AF1 mutations emerged as a distinct molecular marker of MyD in both PMF and SMF, while mutations in TP53 appear prevalent in MyD SMF. [Display omitted] DisclosuresVannucchi: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

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.

Nationwide Laboratory Network for AML Cross-Validated NGS Studies: Results from a Real-Life Cohort of the Pethema Group

Nationwide Laboratory Network for AML Cross-Validated NGS Studies: Results from a Real-Life Cohort of the Pethema Group

Triple-Negative Myelofibrosis: Disease Features, Response to Treatment and Outcomes

Triple-Negative Myelofibrosis: Disease Features, Response to Treatment and Outcomes

The Impact of Somatic Mutations on Allogeneic Hematopoietic Cell Transplantation in Chronic Myelomonocytic Leukemia: A Center for International Blood and Marrow Transplant Research (CIBMTR) Analysis

BackgroundSomatic mutations are an important prognostic factor in chronic myelomonocytic leukemia (CMML). However, limited data are available regarding their impact on outcomes after allogeneic hematopoietic cell transplantation (HCT). In this registry analysis conducted via the Center for International Blood and Marrow Transplant Research (CIBMTR) as well as its sample repository, we analyzed the landscape of somatic mutations in patients with CMML and their impact on post-HCT outcomes.MethodsUsing the CIBTMR database, adult patients with CMML who underwent HCT from 2001-2017 with an available specimen in the National Marrow Donor Program (NMDP) repository were identified; samples were collected prior to the start of conditioning. Patients who received umbilical cord blood, syngeneic, or haploidentical HCT or patients with disease transformation to acute myeloid leukemia prior to HCT were excluded. The primary endpoint was overall survival (OS); secondary endpoints included disease-free survival (DFS), relapse/progression, treatment-related mortality (TRM); survival outcomes were calculated from time of HCT. A mutation analysis for 131 genes recurrently mutated in myeloid disorders was performed.ResultsWe identified 313 patients across 78 centers who met inclusion criteria with available samples. The median age was 64, and 69% were male. By World Health Organization (WHO) criteria, 53%, 20%, and 9% of patients had CMML-0, -1, and -2, respectively. By French-American-British (FAB) criteria, 76% of patients had dysplastic (MD)-CMML and 24% had proliferative (MP)-CMML. The majority (61%) had received hypomethylating therapy prior to HCT, and 76% of HCT were from a matched unrelated donor. Distribution of patients by CMML-specific prognostic scoring system (CPSS) score was 29% low, 25% intermediate-1, 31% intermediate-2, and 4% high (11% missing). Distribution of patients by clinical/molecular CPSS (CPSS-Mol) score was 12% low, 17% intermediate-1, 38% intermediate-2, and 25% high (8% missing).Of the 313 patients studied, 290 (93%) had 1+ pathogenic mutation identified, and the median number of mutations was 3. The most frequently mutated genes were ASXL1 (62%), TET2 (35%), KRAS/NRAS (33% combined), and SRSF2 (31%); TP53 was mutated in 3% of patients (Figure 1). U2AF1 mutations were significantly correlated with MD-CMML, and ASXL1, EZH2, KIT, and SRSF2 mutations were significantly correlated with MP-CMML.The CPSS score was correlated with OS and DFS on univariate analysis, and the CPSS-Mol score was correlated with OS and TRM (Figure 2). Neither scoring system was predictive for progression/relapse.In multivariable analysis, as compared to a CPSS score of low, intermediate-2 (hazard ratio [HR]=1.46, p=0.049) or high (HR=3.22, p=0.0004) correlated significantly with overall survival (OS) (table 1a). When CPSS-Mol was applied, a high score (HR=2, p=0.0079) correlated significantly with OS (table 1b). In multivariable analysis, DNMT3A and TP53 mutations were associated with decreased OS (HR=1.70, p=0.0147; and HR=2.72, p=0.0042, respectively) while DNMT3A, JAK2, and TP53 mutations were associated with decreased DFS (HR=1.66, p=0.0138; HR=1.79, p=0.0293; and HR=2.94, p=0.0018, respectively). The only mutation associated with increased relapse was TP53 (HR =2.94, p=0.0201). DNMT3A was associated with increased TRM(HR=1.89, p=0.0388) and PTPN11 was associated with decreased TRM (HR=0.205, p=0.0300). Goodness of fit was calculated by Harrell's C-index for CPSS and CPSS-Mol for OS, DFS, relapse, and TRM. For CPSS, the results were 0.56, 0.55, 0.52, and 0.56, respectively while for the CPSS-Mol they were 0.57, 0.55, 0.55, and 0.58, respectively.ConclusionOur study is the largest analysis of CMML patients who underwent HCT with paired mutation analysis. Via registry data we have provided the mutational landscape in patients with CMML who underwent HCT and demonstrated a strong association between CPSS-Mol and transplant outcomes although without major improvement in the risk prediction beyond the CPSS. [Display omitted] DisclosuresMei: TG Therapeutics: Research Funding; EUSA: Honoraria; Epizyme: Research Funding; BMS: Research Funding; Morphosys: Research Funding; Janssen: Honoraria; Beigene: Research Funding. Sobecks: CareDX: Membership on an entity's Board of Directors or advisory committees. Scott: Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Saber: Govt. COI: Other.

Preclinical Activity of the Clinical Stage Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor PRT543 in Splicing Mutant Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

MDS and AML are generally incurable malignancies that need newer therapeutic options, as the disease-initiating stem cells are not eliminated by conventional therapies. Splicing factor mutations account for approximately 50% of mutations in MDS. Among those are SF3B1 and U2AF1 mutations, which are related to pathogenesis of disease by overactivation of oncogenic pathways, such as Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) signaling. Via activation of IRAK4 and other pathways, spliceosome mutations can lead to a block in differentiation and malignant proliferation. PRMT5 is an enzyme involved in spliceosome complex formation and fidelity and is over-expressed in patients with MDS/AML. The inhibition of PRMT5 may contribute to stem/progenitor cell differentiation rather than aberrant proliferation in an undifferentiated immature state. The objective of this study is to determine the activity of a clinical stage PRMT5 inhibitor, PRT543, in subtypes of MDS/AML using cell lines and primary samples. In preclinical studies PRT543 showed broad antitumor activity in vitro and in vivo (Bhagwat AACR 2020) and is currently under investigation in a Phase I clinical trial in patients with myeloid malignancies.We used a K562 cell line with CRISPR-introduced SF3B1 K700E mutation and isogenic control (K700K) in proliferation and myeloid differentiation assays with PRT543. The SF3B1 K700E mutant cells showed myeloid differentiation after treatment with the PRT543 PRMT5 inhibitor, as assessed by single cell colony assays and flow cytometry, while no substantial effects were observed in controls (K700K). We next evaluated PRMT5 expression in a large set of MDS CD34+ cells and observed substantial overexpression in SF3B1 mutant samples. Primary MDS/AML progenitors were cultured in methylcellulose colony forming unit (CFU) assays and treated with PRT543 at multiple concentrations versus vehicle controls. A majority of the SF3B1 patient samples showed a substantial increase in erythroid differentiation as assessed by colony formation and flow cytometry in the presence of 1nM and 5nM PRT543. In non-SF3B1 mutated patient samples, there was no clear difference in differentiation in the presence of PRT543.We next evaluated whether PRMT5 inhibition led to inhibition of oncogenic IRAK4 pathways. Retention of exon 4 of IRAK4 occurs in splicing mutant MDS and leads to production of an active long IRAK4 isoform. As measured by RNA-seq, PRMT5 inhibition led to decreased retention of exon 4 in IRAK4 transcripts. This decrease in the IRAK4 long form in response to PRT543 treatment was confirmed by immunoblotting, demonstrating reduction of this oncogenic signaling pathway.In summary, PRMT5 inhibition with PRT543 can release a differentiation block in MDS/AML, specifically in splicing mutant samples. PRMT5 inhibition decreases IRAK4-long isoform expression providing a potential mechanism for its activity in splicing factor mutant cases. DisclosuresRuggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Heiser: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Starczynowski: kurome Inc: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; Stelexis: Current equity holder in publicly-traded company; Celgene: Consultancy; Acceleron: Consultancy; Novartis: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Curis: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; BMS: Research Funding.

Gender Disparities in Myelodysplastic Syndromes: Phenotype, Genotype, and Outcomes

Gender Disparities in Myelodysplastic Syndromes: Phenotype, Genotype, and Outcomes

The 1.5 Million Platelet Count Threshold in Essential Thrombocythemia: Phenotype and Genotype Correlates and Relevance to Vascular Events

▪BackgroundCurrent NCCN and ELN guidelines lack supporting evidence in regards to initiation of cytoreductive therapy in essential thrombocythemia (ET) with extreme thrombocytosis (ExT) ≥1500 x 10 9/L (NCCN guidelines: myeloproliferative neoplasms, version 2. 2019, Barbui, Leukemia, 2018).; we sought to determine prevalence and establish phenotype and genotype correlates for ET with ExT and assess its impact on thrombotic and bleeding events, in the context of aspirin therapy or cytoreduction.Methods1,249 WHO-defined ET patients evaluated over five decades (1967-2021) were retrospectively recruited from the Mayo Clinic. Conventional criteria were used to define major arterial and venous thrombosis and major hemorrhage. Conventional statistical methods were applied using JMP Pro 14.0.0 software package, SAS Institute, Cary, NC.Results 1.i) Phenotype and genotype correlatesAmong 1,249 consecutive patients with ET, 104 (8%) displayed Ext ≥1500 x 10 9/L, at time of initial diagnosis; these patients were characterized by younger age (median 47 vs 59 years; p<0.001) female gender (80% vs 62%; p<0.001), CALR mutation (39% vs 25%; p=0.004), lower hemoglobin (median 13.1 vs 13.7 g/dl; p<0.0001), leukocytosis ≥11 x 10 9/l (42% vs 23%; p<0.0001), and palpable splenomegaly (22% vs 14%; 0.03); multivariable analysis confirmed associations with age, anemia, leukocytosis, and CALR mutation. Acquired von Willebrand syndrome was reported in 4/9 (44%) vs 27/78 (35%) tested patients, with or without Ext ≥1500 x 10 9/L, respectively (p=0.57); however, comparison using platelet ≥1000 x 10 9/L resulted in near-signficant difference (46% vs 29%; p=0.11). NGS was performed in 297 patients and showed a non-sigificant association with U2AF1 mutations (5% vs 0.4%; p=0.11). ExT ≥1500 x 10 9/L was present in 25/986 (3%), 42/891 (5%), and 15/558 (3%) in the validation cohort; the limited number of cases precluded comparative analyses. 1.ii )Correlation with vascular events at/prior to diagnosisArterial or venous thrombosis at/prior to diagnosis occurred in 190 (15%) and 111 (9%) patients, respectively, and major hemorrage in 50 (4%). Ext ≥1500 x 10 9/L was associated with major hemorrhage (HR 2.9; 1.3-6.3), but not with arterial (p=0.15) or venous (p=0.41) thrombotic events, at/prior to diagnosis. Instead, multivariable analysis for events at/prior to diagnosis identified male gender (p=0.02), JAK2 mutation (p=0.01), and cardiovascular risk factors (p=0.001) as risk factors for arterial thrombosis and JAK2 mutation (HR 2.8; 1.4-5.7) as a risk factor for venous thrombosis.iii )Correlation with vascular events after diagnosisAt median follow-up of 10 years, arterial or venous thrombosis occurred in 222 (18%) and 97 (8%) patients, respectively, and major hemorrage in 128 (11%). Ext ≥1500 x 10 9/L at diagnosis was not associated with arterial (p=0.35) or venous (p=0.23) thrombosis-free survival or hemorrhage-free survival (p=0.24). Instead, multivariable analysis identified age >60 years, presence of cardiovascular risk factors, and history of arterial thrombosis, as risk factors for arterial thrombosis-free survival; and male gender and history of venous thrombosis as risk factors for venous thrombosis-free survival; and age >60 years and leukocytosis of ≥11 x 10 9/L as risk factors for hemorrhage-free survival. In addition, aspirin therapy was idependently protective of venous thrombosis (HR 0.5; 0.2-0.9), and also of arterial thrombosis, in univariate analysis (HR 0.6; 0.3-0.9), without being associated with an increased risk of major hemorrhage (p=0.64); by contrast, beneficial effect from cytoreductive therapy was not evident for either arterial (p=0.37) or venous (p=0.77) thrombosis. In low-risk patients with Ext ≥1500 x 10 9/L, both aspirin and cytoreductive therapy were protective of venous events (Figure 1), without affecting hemorrhage-free survival.ConclusionExT ≥1500 x 10 9/L in ET was associated with major hemorrhage at diagnosis but did not affect thrombosis-free or hemorrhage-free survival. Initial therapy with aspirin and/or cytoreductive therapy was protective of venous events in ET patients with ExT ≥1500 x 10 9/L, including those with low-risk disease, without enhancing the risk of hemorrhage. The limited number of informative cases with ExT in the validation cohort, underscore the foreseeable challenges associated with controlled studies. [Display omitted] DisclosuresSzuber: Novartis: Honoraria. Vannucchi: AbbVie: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Barbui: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Heterogeneous Definitions of Secondary Acute Myeloid Leukemia (AML) Yield Distinct Outcomes in Response to First-Line Treatment with Hypomethylating Agents (HMA) and Venetoclax (Ven)

Background:The combination of HMA and venetoclax is now standard of care for patients with AML who are not candidates for intensive chemotherapy. Elderly patients are more likely to have secondary AML (sAML), although the presence of an antecedent hematologic malignancy is often not apparent by history. Lindsley et al (Blood, 2015) showed that a somatic mutation in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 is >95% specific for sAML and associated with worse outcomes. While outcomes with HMA/ven in patients meeting standard criteria for sAML have recently been reported (Pullarkat, ASCO 2021), we set out to conduct a real-world analysis of sAML patients receiving HMA/ven, including those with a secondary mutation profile (SMP) as described by Lindsley et al. We hypothesized that-when treated with HMA/ven-outcomes of patients with SMP may be most similar to those with de novo AML.Methods:Patients diagnosed with AML at Stanford Cancer Institute from 4/2017-3/2021 and treated with front-line HMA/ven were retrospectively reviewed. These included patients previously treated with HMA monotherapy for an antecedent hematologic malignancy and those who had previously received ≤ 3 cycles of HMA monotherapy for AML. Responses were classified per the modified International Working Group response criteria. Overall survival (OS) was assessed for all patients, and for patients who had a complete response (CR) or CR with incomplete hematologic recovery (CRi), duration of response (DoR) was also assessed. Statistical analyses were performed in R using the logrank test, with hazard ratios (HR) computed using the Cox proportional hazards model. For multivariate analyses, p-values for a specific variable were calculated using Cox proportional hazards regression.Results:82 patients met criteria for inclusion; 78 had valid response assessments and 49 (62.8%) had achieved a CR or CRi at first response assessment. Median age was 72 years, with 3 patients younger than 60. 62 patients were male, median ECOG performance status (PS) was 1, median Charlson Comorbidity Index (CCI) was 6, median time to death or end of follow-up from the start of treatment was 366 days, and 58% of patients had adverse risk AML per ELN guidelines. Fig 1a demonstrates demographics for de novo, sAML (excluding SMP), and patients with SMP AML. 13 patients met criteria for AML-MRC, 23 patients had prior history of antecedent hematologic malignancy (18 with MDS or CMML, 5 with MDS/MPN overlap or MPN), 12 had tAML, and 20 patients possessed a SMP and did not meet criteria for the other three categories of sAML. 14 patients with de novo AML were characterized by the absence of any of the above factors. Patients with de novo AML were less likely to have adverse risk disease (29% vs. 64% in others) and had lower CCI scores (mean 5.1 vs. 6.2) but had no significant differences in age, gender, follow-up time, or PS.There was no statistically significant difference in rates of CR/CRi between the different subgroups or the different types of sAML; 69% of patients with de novo AML, 79% of SMP patients, and 57% of patients with other types of sAML achieved a CR or CRi. However, SMP patients had response durations and OS patterns similar to patients with de novo AML (Fig 1b and 1c), and when grouped with de novo patients, both DoR (HR = 3.5, p = 0.047, Fig 1d) and OS (HR = 2.1, p = 0.042, Fig 1e) were significantly longer than those of the sAML patients. Neither DoR nor OS were significantly longer when the SMP patients were grouped with sAML patients (respectively: HR = 3.3, p = 0.22, Fig 1f; HR = 1.5, p = 0.37, Fig 1g). In multivariate Cox proportional regression adjusting for age, ELN risk category, CCI, and PS, worse OS for sAML patients was maintained relative to the SMP and de novo patients (HR 2.9, p = 0.036), although the difference in DoR was no longer significant (HR 4.4, p= 0.10).Conclusions:Patients meeting standard definitions of sAML had worse outcomes than those with de novo AML when treated with HMA/ven in a retrospective, real-world analysis. Although a secondary mutation profile as described by Lindsley et al may be helpful in identifying patients with sAML, when treated with HMA/ven, patients with this profile have outcomes that align more closely with those of patients with de novo AML. [Display omitted] DisclosuresMannis: Astex, Forty Seven Inc/Gilead, Glycomimetics, and Jazz Pharmaceuticals: Research Funding; AbbVie, Agios, Astellas Pharma, Bristol Myers Squibb, Genentech, MacroGenics, Pfizer, and Stemline: Consultancy.

Onset of Blast Crises in CML Patients in Treatment-Free Remission: Descriptive Analysis of 4 Cases

Aims: The onset of blast crisis (BC) in initially chronic phase (CP) CML patients that entered treatment-free remission (TFR) after TKI is an exceptional event, however, there is emerging evidence that this may occur in such patients (pts), although the pathogenesis remains unclear to date.Methods: Anonymous clinical case retrospective data collection from patients' datafiles, after written agreement of living patients, centralisation of available frozen nucleic acid collection from diagnosis and from blast crisis and reanalysis by Next-Generation Sequencing of samples (ASXL1, ASXL2, BCOR, CALR, CBL, CEBP alpha , CSF3R, DNMT3A, EP300, ETNK1, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2 JAK2, KIT, , KRAS,MPL, NPM1, NRAS, PHF6, PTPN11 , RAD2, RUNX1, SETBP1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG1, STAG2, TET2, TP53, U2AF1, WT1 and ZRSR2 genes analysed) on Illumina platform. CNV analysis were performed using VisCAp or in-house pipelines and/or by Multiplex Ligation Dependant Probe Amplification (MLPA). ABL1 tyrosine kinase domain mutations were screened by NGS on cDNA or directly on DNA. BCR-ABL1 transcripts are expressed in % (IS) with at least 32,000 copies of ABL1 as control. All patients discontinued their TKI after 2 years of MR4.5 and a TKI was resumed in case of MMR loss.Results: Along 15-year experience of TFR in our country and ≥ ~800 patients experiencing a TKI cessation attempt, informations from 4 (~0.5%) TFR CML patients entering BC have been collected. All patients harboured Major BCR transcripts.The chronic phase characteristics are mentioned in Table 1.All these long-lasting CP CML pts were ELTS risk score low, and 1 was harbouring ACA at diagnosis. One pt was mutated for ASXL2 and 2 mutations for EP300, found again at BC. Three pts had IFN-a prior to imatinib for all. Three out of 4 lost their MMR after a first cessation attempt at 12, 10 and 3 months after cessation. Pt #1 experienced a 2 nd cessation attempt 52 months after re-initiation of TKIs and entered lymphoid BC 6 months after a second resumption of TKI for a 2 nd MMR loss.The BC characteristics are mentioned in Table 2.Three out of 4 BC were lymphoid, one had ACA different from those at CP diagnosis. Two pts explored had multiple mutations in Runx1, U2AF1, EP300 and ASXL2 genes, not present at CP diagnosis, in addition to multiple ABL1 mutations in 2 out of 4 pts (2 T315I, 3 P-Loop mutations). All pts underwent chemotherapy + various TKI leading to complete remission (CR) in all and 3 out of 4 pts could be allotransplanted in CR, one relapsed shortly after transplant, and a second one 34 months after transplant. Overall 3 pts are alive with 1 in controlled relapse.Conclusions: The onset of BC after TFR for sustained deep molecular response remains an exceptional event and is probably not induced by this therapeutic procedure. These 4 cases underline the need for a sustained long-lasting molecular follow-up of pts in TFR, although the majority of these BC seem sudden. [Display omitted] DisclosuresBauduer: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rea: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding; Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; BMS: Honoraria.

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.

Prediction of Complete Remission and Survival in Acute Myeloid Leukemia Using Supervised Machine Learning

Prediction of Complete Remission and Survival in Acute Myeloid Leukemia Using Supervised Machine Learning

Immunogenetic, Molecular and Clinical Determinants of Clonal Evolution in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Immunogenetic, Molecular and Clinical Determinants of Clonal Evolution in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

U2AF1 and Other Splicing Factor Gene Mutations in Telomere Biology Disorders Are Associated with Hematologic Neoplasia and Worse Overall Survival

Introduction: Telomere Biology Disorders (TBD) are due to germline variants in telomere maintenance and repair genes. Clinical manifestations include bone marrow failure (BMF), liver and lung fibrosis, and risk of cancer, especially myeloid neoplasia. We characterized clinical phenotype, pathology, and clonal landscape of patients with TBD and myelodysplastic syndrome (MDS) or acute leukemia (AL). We assessed if somatic variants in myeloid cancer genes are associated with MDS/AL or overall survival (OS).Methods: Patients (n=109) from the National Institutes of Health and University of São Paulo (NIH/USP) (n=91) or MD Anderson (MDA) (n=18) met TBD clinical criteria or had a pathogenic/likely pathogenic germline variant in a telomere maintenance/repair gene. All patients gave informed consent. Clinical characteristics and details of hematologic malignancy were collected. MDS was defined using WHO 2016 criteria. In the NIH/USP cohort, somatic variants in myeloid cancer genes were assessed in peripheral blood using error-corrected DNA sequencing (ECS; minimum variant allele frequency [VAF] of 0.5%) and in the MDA cohort patients were screened by targeted amplicon-based next-generation sequencing (minimum VAF of 2%). Serial samples were available in 31 (41%) patients.Results: Eighteen out of 109 (17%) patients from both the NIH/USP and MDA cohorts had a diagnosis of MDS/AL, developing in patients with TERC (n=8/31; 25%), TERT (n=8/48; 16%), RTEL1 (n=1/5; 20%), and DKC1 (n=1/2; 50%). Karyotypes were normal diploid (n=6), chromosome 1 abnormality (n=5), trisomy 8 (n=2), deletion 20q (n=1), complex (n=2), monosomy 7 (n=1) and deletion 7q (n=1). Six patients had >5% blasts. Four patients had concurrent liver fibrosis and 9 had pulmonary fibrosis. Somatic mutation data at time of MDS/AL was available for 16/18 patients; splicing factor genes (n=14) were most frequent, seen in 11 (69%) patients, with U2AF1 predominating (n=7; 44% of patients) (Figure 1A). More than 1 mutation was present in 12/16 (75%) of patients. One patient developed AML 6 years after MDS diagnosis . U2AF1 variants were also present in 8 patients without MDS/AL; 5 had no sequential samples and 3 had stable mutations over 2 (n=2) and 4 (n=1) years. Recurrent variants in U2AF1 were present at p.S34 (n=12) and p.Q157 (n=2). Median U2AF1 variant allele fraction (measured in NIH/USP cohort only) was 23% in patients with MDS/AL compared to 7% in those without.Variant association analysis was confined to NIH/USP cohort due to DNA sequencing assay differences; data were available in 75 patients. Patients were divided into groups defined by specific variants at any time of sampling; “Clonal-hematopoiesis of indeterminate potential / aplastic anemia (CHIP/AA) associated” (DNMT3A, TET2, ASXLI, or BCOR/L1), “MDS-associated” (containing splicing factor genes, RUNX1, SETBP1, ETV1, KRAS, STAG2, GATA2/1, TP53), “PPM1D” (containing PPM1D), and a subgroup, “Splicing factors” (U2AF1, ZRSR2, SF3B1, SRSF2). Patients with MDS-associated variants (Figure 1A; p=0.02), particularly splicing factors (Figure 1B; p=0.007) more likely had or later developed MDS/AL, compared to patients without somatic variants or in other variant groups. MDS/AL was not associated with PPM1D or CHIP/AA-associated variants. OS from time of first detected somatic variant was lower in the MDS-associated variant group (Figure 1C p=0.013) compared to those without variants at time of first assessment; this was not the case for CHIP/AA-associated variants or PPM1D. Patients with somatic variants (median ages 36, 41, and 42 for PPM1D, CHIP/AA-associated, and MDS-associated respectively) were significantly older than patients without variants (median age 24) though ages were similar within variant groups.Conclusion: Splicing factor gene variants predominate the clonal landscape of TBD-associated hematologic malignancy; U2AF1 p.S34 is recurrent and can occur in the absence of neoplasia. Splicing factor gene variants such as U2AF1 in the peripheral blood may be potential biomarkers for hematologic malignancy in TBD patients. PPM1D, previously associated with therapy-related MDS, is commonly mutated in TBD but does not associate with MDS/AL. OS was decreased in patients with MDS-associated variants but not with PPM1D or CHIP/AA-associated variants. Further study of U2AF1 variants in TBD patients may give insight to the underlying drivers of MDS/AL. [Display omitted] DisclosuresDiNardo: Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Forma: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Takeda: Honoraria; Foghorn: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Calado: Novartis Brasil: Honoraria; Instituto Butantan: Consultancy; AA&MDS International Foundation: Research Funding; Alexion Brasil: Consultancy; Agios: Membership on an entity's Board of Directors or advisory committees; Team Telomere, Inc.: Membership on an entity's Board of Directors or advisory committees. Young: Novartis: Research Funding.