U2AF1 Mutations Research Articles

MDS-406 Prognostic Implication of the Molecular Profiles of Patients With Hypocellular Myelodysplastic Syndrome

Hypocellular myelodysplastic syndrome (hMDS) is a subset of MDS that has not been fully characterized. This study aimed to better identify the clinical characteristics and molecular profile of hMDS and study the prognostic impact of mutations in hMDS. We conducted a retrospective review of patients with newly diagnosed MDS in a single tertiary cancer center registry from 2010 to 2021. Of 1,899 patients with MDS, 176 (10%) patients had hMDS. The median follow-up period was 19.5 months. hMDS patients were more likely to be younger and have therapy-related disease and more thrombocytopenic and neutropenic than normo/hypercellular MDS (non-hMDS). All other clinical characteristics were comparable. The distributions of IPSS-R cytogenetic scores (≥3, or <3) were comparable. hMDS patients had similar overall survival (OS) to non-hMDS (median 28.3 vs. 29.0 months) or AML transformation rates (23% vs. 19%). These findings were maintained after multivariate adjustment. Hypocellularity did not affect survival in either de novo or t-MDS. We further analyzed 1,352 (71%) pts with mutation data available. Patients with hMDS had less frequent ASXL1, RUNX1, TET2, or IDH1 mutations, whereas the frequency of TP53 and RAS pathway mutations were comparable. In de novo MDS, hMDS patients had less frequent RUNX1, SRSF2, or TET2 mutations. In t-MDS, hMDS pts had less frequent TP53 or U2AF1 mutations. The survival of hMDS can be stratified by IPSS-R with some overlaps. In hMDS, mutations in the RAS pathway or TP53 were associated with inferior OS, whereas TET2 mutations correlated with superior OS. Based on the mutation data, patients were stratified into 3 groups: (1) with TET2 mutations, (2) with TP53 or RAS pathway mutations without TET2 mutations, and (3) without TET2/TP53/RAS mutations. The TET2 mutant group showed marginally better mOS (HR 0.50, P=0.081), and the TP53/RAS pathway group showed inferior mOS (HR 2.74, P=.005) compared with the group without TET2/TP53/RAS pathway mutation. Patients with hMDS had similar outcomes to patients with non-hMDS but exhibit distinct molecular profiles; hMDS had less frequent driver mutations commonly seen in non-hMDS. The integration of molecular profiles into risk stratification in hMDS could be a study of interest.

AML-440 Prognosis of Molecularly-Defined Secondary Acute Myeloid Leukemia Patients

Mutations in ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 were proposed as secondary AML (sAML) defining mutations independent from the patients' history. We evaluated the prognostic impact of molecularly-defined sAML (msAML) patients in the context of the European LeukemiaNet (ELN) risk categories. 459 adult newly-diagnosed AML patients (median age 54) with available genetic and follow-up data were included. Patients received standard induction and consolidation chemotherapy or underwent allogeneic hematopoietic cell transplantation (alloHCT). Patients were classified as de-novo AML (dnAML), msAML carrying ≥ 1 sAML-defining mutations, and clinically-defined secondary AML (csAML) based on previous medical history and cytogenetics. 208 (45%) patients had dnAML, 155 (34%) msAML, and 96 (21%) csAML. 62 msAML patients overlapped with csAML. Of msAML patients, 104 (67%) had 1, 39 (25%) 2, and 12 (8%) 3 or more msAML-defining mutations. The most frequently mutated msAML-defining genes were ASXL1 (n=48, 31%) and SRSF2 (n=44, 28%). The likelihood to reach complete remission (CR) was higher for dnAML compared to msAML patients (92% vs 79%, P =.003), but there was no difference between csAML and msAML patients. The median follow-up of all patients was 5.56 years. The transplantation rate in first CR was similar in dnAML, msAML and csAML patients (33%, 37%, 44%, respectively). Overall survival (OS) was significantly worse in msAML compared to dnAML patients (median OS 3.3 years vs not reached, HR = 1.7, 95%CI 1.2-2.2, P<.001), also when ASXL1 mutated patients were excluded (HR = 1.7, 95%CI 1.2-2.4, P<.001). OS was similar in csAML vs dnAML and in msAML vs csAML patients. In the ELN favorable and intermediate risk groups msAML patients (n=19 and n=52) had a significantly worse OS compared to dnAML patients (n=66 and n=80) (HR=3.1 95%CI 1.5-6.3, P=.001 and HR=2, 95%CI 1.2-3.2, P=.008, respectively). OS was similar in the ELN adverse risk group between msAML (n=79) and dnAML patients (n=31). Accordingly, the ELN risk groups did not stratify OS in msAML patients. msAML-defining mutations identify a subgroup of dnAML patients with poor prognosis and reclassify 10% of all patients in our cohort from favorable/intermediate to the adverse risk group.

MPN-215 Outcomes of Myelofibrosis Patients Post Allogeneic-HSCT at the University of Southern California, a Retrospective Study.

In myelofibrosis (MF) patients, the standard of care curative (SOC) option is through an allogeneic-hematopoietic stem cell transplantation (allo-HSCT); however, no SOC pre- and post-transplant management exists. To report outcomes of MF patients, post-allo-HSCT at our institution with consideration of patient characteristics, risks, and pre-transplant treatment. A retrospective chart review. Single institution: academic center hospital/county hospital Patients or Other Participants: 18-99YO M/F patients with MF diagnosis by bone-marrow biopsy since 2012-2020 and received allo-HSCT at our institution. Chart review. Primary: OS, PFS since time of transplant, stratified to JAK2 inhibitor use, MF type and associated patient characteristics/risks. Secondary: Patient/treatment characteristics, response/mortality rates. P≤ 0.05 is considered significant. In 18 patients, median age 60.7 years 61.1% White/Non-Hispanic, 16.7% Hispanic with 44.4% high, 55.6% intermediate-1/2 risk by DIPSS, 27.8% high and 33.3% very high risk by MIPSS70+. 66.7% had primary MF. At time of transplant, 11.1% was CR/PR, 38.9% SD, and 50% PD. 38.9% received MSD, 27.8% MUD, and 33.3% received haploidentical stem cells. 76.5% had RIC and 23.5% underwent MAC. 44.4% were +JAK2 V617F, 16.7% had either: ASXL1, EZH2, SRSF2, IDHL1/2, U2AF1. 11.1% had high-risk karyotype/cytogenetics. 61.1% received a JAK2 inhibitor, 72% received non-JAK2 inhibitor treatments pre-transplant. Post-transplant, 78% alive at 1 year. Since transplant, 3-year (3y) OS was 66.7% in primary MF patients, 55.6% in secondary (p=0.75) with similar EFS. 3y-OS in JAK2 V617F+ patients was 83.3% (p=0.13) with similar EFS. 3y-OS of those who received a JAK2 inhibitor pre-transplant was 47.7% (p=0.17) with similar EFS (p=0.16). Those without either ASXL1, EZH2, SRSF2, IDHL1/2, or U2AF1 mutations achieved a higher, 72.2%, 3y-OS (p=0.076) than those with any. High-risk karyotype/cytogenetics 3y-OS was 50% (p=0.43). OS/EFS rates were generally lower with higher DIPSS and MIPSS70+. Given most of our patients were high/very high risks, achieving 3y-OS of 66.7% in primary and 55.6% in secondary MF since the time of transplant is appreciable. Interestingly, even with higher OS/EFS for JAK2V+ patients, the OS/EFS is lower for those who received a JAK2 inhibitor. This could be from the low sample size. Further studies are necessary.

U2AF1 and EZH2 mutations are associated with nonimmune hemolytic anemia in myelodysplastic syndromes

AbstractHemolysis is a well-recognized but poorly characterized phenomenon in a subset of patients with myelodysplastic syndromes (MDS). Its pathobiological basis seems to underpin a nonimmune etiology whose clinical significance has not been adequately characterized. Hemolysis in MDS is often attributed to either ineffective intramedullary erythropoiesis or acquired hemoglobinopathies and red blood cell (RBC) membrane defects. These heterogeneous processes have not been associated with specific genetic subsets of the disease. We aimed to describe the prevalence of hemolysis among patients with MDS, their baseline characteristics, molecular features, and resulting impact on outcomes. We considered baseline serum haptoglobin <10 mg/dL a surrogate marker for intravascular hemolysis. Among 519 patients, 10% had hemolysis. The baseline characteristics were similar among both groups. Only 13% of patients with hemolysis were Coombs-positive, suggesting that hemolysis in MDS is largely not immune-mediated. Inferior survival trends were observed among lower-risk patients with MDS undergoing hemolysis. Decreased response rates to erythropoiesis-stimulating agents (ESA) and higher responses to hypomethylating agents (HMA) were also observed in the hemolysis group. U2AF1 and EZH2 hotspot mutations were more prevalent among those undergoing hemolysis (P < .05). U2AF1 mutations were observed in 30% of patients with hemolysis and occurred almost exclusively at the S34 hotspot. Somatic mutations encoding splicing factors may affect erythrocyte membrane components, biochemical properties, and RBC metabolic function, which underpin the development of atypical clones from erythroid precursors in MDS presenting with hemolysis. Future studies will explore the contribution of altered splicing to the development of acquired hemoglobinopathies.

The Biological and Clinical Consequences of RNA Splicing Factor U2AF1 Mutation in Myeloid Malignancies.

Simple SummaryU2 small nuclear RNA auxiliary factor 1 (U2AF1) is one of the most important RNA splicing genes involved in regulating the alternative splicing of pre-mRNA. U2AF1 mutation is a genetic driver event in the initiation of myelodysplastic syndromes (MDSs) and frequently occurs in myeloid malignancies. U2AF1 mutation can severely impair hematopoiesis, drive tumor progression, adversely affect disease prognosis, and promote leukemic transformation. This review summarizes the biological and clinical implications of the oncogenic role of U2AF1 mutation in myeloid tumors. Our work provides important and comprehensive insights into the development of the U2AF1 mutation as a novel prognostic biomarker and therapeutic target for myeloid malignancies.Mutations of spliceosome genes have been frequently identified in myeloid malignancies with the large-scale application of advanced sequencing technology. U2 small nuclear RNA auxiliary factor 1 (U2AF1), an essential component of U2AF heterodimer, plays a pivotal role in the pre-mRNA splicing processes to generate functional mRNAs. Over the past few decades, the mutation landscape of U2AF1 (most frequently involved S34 and Q157 hotspots) has been drawn in multiple cancers, particularly in myeloid malignancies. As a recognized early driver of myelodysplastic syndromes (MDSs), U2AF1 mutates most frequently in MDS, followed by acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). Here, for the first time, we summarize the research progress of U2AF1 mutations in myeloid malignancies, including the correlations between U2AF1 mutations with clinical and genetic characteristics, prognosis, and the leukemic transformation of patients. We also summarize the adverse effects of U2AF1 mutations on hematopoietic function, and the alterations in downstream alternative gene splicing and biological pathways, thus providing comprehensive insights into the roles of U2AF1 mutations in the myeloid malignancy pathogenesis. U2AF1 mutations are expected to be potential novel molecular markers for myeloid malignancies, especially for risk stratification, prognosis assessment, and a therapeutic target of MDS patients.

Correlation Analysis and Prognostic Impacts of Biological Characteristics in Elderly Patients with Acute Myeloid Leukemia.

BackgroundThe significant heterogeneity of elderly AML patients’ biological features has caused stratification difficulties and adverse prognosis. This paper did a correlation study between their genetic mutations, clinical features, and prognosis to further stratify them.Methods90 newly diagnosed elderly acute myeloid leukemia (AML) patients (aged ≥60 years) who detected genetic mutations by next-generation sequencing (NGS) were enrolled between April 2015 and March 2021 in our medical center.ResultsA total of 29 genetic mutations were identified in 82 patients among 90 cases with a frequency of 91.1%. DNMT3A, BCOR, U2AF1, and BCORL1 mutations were unevenly distributed among different FAB classifications (p < 0.05). DNMT3A, IDH2, NPM1, FLT3-ITD, ASXL1, IDH1, SRSF2, BCOR, NRAS, RUNX1, U2AF1, MPO, and WT1 mutations were distributed differently when an immunophenotype was expressed or not expressed (p<0.05). NPM1 and FLT3-ITD had higher mutation frequencies in patients with normal chromosome karyotypes than abnormal chromosome karyotypes (p<0.001, p=0.005). DNMT3A and NRAS mutations predicted lower CR rates. DNMT3A, TP53, and U2AF1 mutations were related to unfavorable OS. TET2 mutation with CD123+, CD11b+ or CD34- predicted lower CR rate. IDH2+/CD34- predicted lower CR rate. ASXL1+/CD38+ and SRSF2+/CD123- predicted shorter OS.ConclusionThe study showed specific correlations between elderly AML patients’ genetic mutations and clinical features, some of which may impact prognosis.

Genetic mutations associated with blood count abnormalities in myeloid neoplasms

ABSTRACT Introduction Myelodysplastic syndromes (MDS) predominantly present with varying degrees of cytopenia, while myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) exhibit proliferative features. Genetic defects underlying different complete blood count (CBC) alterations remain to be defined. Objective We aimed to evaluate mutations and impacts on abnormal blood counts in MDS and MDS/MPN. Method MDS and MDS/MPN patients were recruited and sequenced by targeted next-generation sequencing. Clinical parameters, especially CBC, were evaluated for the association with genetic abnormalities and clinical outcomes. Results A total of 168 patients with myeloid neoplasms were recruited (92 cases of low-risk MDS, 57 cases of high-risk MDS and 19 cases of MDS/MPN). Compared to low-risk MDS and MDS/MPN, patients with high-risk MDS were presented with more severe neutropenia with 17.5% showing absolute neutrophil counts (ANC) lower than 0.5 × 109/L. Patients with MDS/MPN more commonly harboured mutations and had a higher number of mutations per case than low-risk MDS (94.7% vs. 56.5%; p < 0.001 and 3 vs. 1; p < 0.001, respectively). Patients with SF3B1 mutations showed lower haemoglobin levels than wild-type (7.9 vs. 8.4 g/dL, p = 0.02), but were associated with normal platelet counts (286 vs. 93 × 109/L; p < 0.001). Patients with U2AF1 mutations were associated with more severe leukopenia than wild-type (3 vs. 4.18 × 109/L; p = 0.02). KRAS mutations were associated with monocytosis (p < 0.001). Multivariate analysis revealed high-risk MDS, MDS/MPN, severe neutropenia (ANC < 0.5 × 109/L), and mutations in ASXL1 and SETBP1 were associated with inferior survival outcomes. Conclusion Certain mutations were related to more severe anaemia, lower white blood cell count or monocytosis in Asian MDS and MDS/MPN patients.

Whole-Genome DNA Methylation Sequencing Reveals Epigenetic Changes in Myelodysplastic Syndromes.

Epigenetic dysregulation of cancer-associated genes has been identified to contribute to the pathogenesis of myelodysplastic syndromes (MDS). However, few studies have elucidated the whole-genome DNA methylation in the initiation pathogenesis of MDS. Reduced representation bisulfite sequencing was performed in five de novo MDS patients and four controls to investigate epigenetic alterations in MDS pathogenesis. The mean global methylation in five MDS patients showed no significant difference compared with the four controls. In depth, a total of 1,459 differentially methylated fragments, including 759 hypermethylated and 700 hypomethylated fragments, were identified between MDS patients and controls. Targeted bisulfite sequencing further identified that hypermethylation of DLEU7, FOXR1, LEP, and PANX2 were frequent events in an additional cohort of MDS patients. Subsequently, LEP hypermethylation was confirmed by real-time quantitative methylation-specific PCR in an expanded cohort of larger MDS patients. In clinics, LEP hypermethylation tended to be associated with lower bone marrow blasts and was significantly correlated with U2AF1 mutation. Survival analysis indicated that LEP hypermethylation was associated with a markedly longer survival time but was not an independent prognostic biomarker in MDS patients. Functional studies revealed pro-proliferative and anti-apoptotic effects of leptin in the MDS cell line SKM-1, and it was significantly associated with cell growth and death as well as the Toll-like receptor and NF-kappa B signaling pathways. Collectively, our findings demonstrated that whole-genome DNA methylation analysis identified novel epigenetic alterations such as DLEU7, FOXR1, LEP, and PANX2 methylations as frequent events in MDS. Moreover, LEP might play a role in MDS pathogenesis, and LEP hypermethylation was associated with longer survival but not as an independent prognostic biomarker in MDS.

S129: TAKEAIM LEUKEMIA- A PHASE 1/2A STUDY OF THE IRAK4 INHIBITOR EMAVUSERTIB (CA-4948) AS MONOTHERAPY OR IN COMBINATION WITH AZACITIDINE OR VENETOCLAX IN RELAPSED/REFRACTORY AML OR MDS

Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) and FLT3. IRAK4 is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of IRAK4 and have been associated with disease progression and poor prognosis of high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML). Aims: Assessment of safety, clinical activity, and Recommended Phase 2 Dose (RP2D) of emavusertib as monotherapy or in combination with azacitidine (AZA) or venetoclax (VEN). Methods: This is an open-label, phase 1/2a dose escalation and cohort expansion trial (NCT04278768). In phase 1 Dose Escalation, patients with R/R AML or HR-MDS are treated with emavusertib monotherapy. Phase 1b includes 2 arms of combination therapy: emavusertib + AZA and emavusertib + VEN. The primary objectives of this study are to assess the safety, clinical activity, and identify the RP2D of emavusertib as monotherapy or in combination with AZA or VEN in R/R AML or HR-MDS. The Phase 2a Dose Expansion includes patients for emavusertib monotherapy: R/R AML with FLT3 mutation, or AML and HR-MDS R/R to HMA with U2AF1 or SF3B1 mutations. Results: As of December 16th, 2021, 49 patients have been treated in the phase 1 portion, of whom 43 started by September 30th, allowing 2 on-study disease assessments. The median number of prior therapies was 2 (range 1-5). Four monotherapy dose levels of emavusertib were tested (200 to 500 mg orally BID). No dose-limiting toxicities were observed at 200 mg and 300 mg BID. No Grade 4 or 5 treatment-related AEs (TRAEs) were reported, and all the TRAEs were manageable. Reversible, manageable Grade 3 rhabdomyolysis occurred in 1/26 (4%) patients at 300 mg BID, 2/17 (12%) at 400 mg BID, and 1/3 (33%) at 500 mg BID. RP2D was determined as 300 mg BID. Of 43 patients starting before Sept 30th, 2021, 14 had SF3B1, U2AF1 or FLT3 mutations and demonstrated more promising efficacy. In the 5 evaluable AML patients with spliceosome mutations, 40% reached CR/CRh (1 CR, 1 CRh), both with study duration >6 months. In the 7 spliceosome-mutated HR-MDS patients, 57% reached marrow CR, including 1 with RBC transfusion independence and 1 proceeding to HSCT. One of the three FLT3-mutated AML reached CR, and 2 became FLT3-negative. Among the 29 patients without SF3B1/U2AF1/FLT3 mutations, 1 reached CR and 2 PR. Phase 1b and Phase 2a are ongoing. RNA-seq on selected samples showed decrease in relative expression of IRAK4-long isoforms with response to emavusertib. Summary/Conclusion: Emavusertib is well tolerated and effective in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1/SF3B1/FLT3 mutations. No dose-limiting myelosuppression was reported, suggesting emavusertib may be a candidate for combination therapy. Accrual of Phases 1b and 2a is ongoing.

P388: PREFERENTIAL SYNERGISTIC EFFECT OF INDISULAM IN COMBINATION WITH VENETOCLAX AND AZACYTIDINE IN AML CELLS WITH SF3B1 MUTATIONS.

Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis characterized by a high rate of relapse after conventional combination chemotherapy, highlighting the need for improved therapeutic interventions. Genes involved in pre-mRNA splicing, namely SF3B1, U2AF1 and SRSF2, are frequently mutated in AML.Recent studies suggest that pharmacological targeting of the splicing machinery results in preferential lethality of splicing mutant AML cells, potentially providing a novel strategy for treatment of this disease. Existing anti- cancer sulfonamides have been shown to interfere with splicing and induce preferential killing of splicing mutant AML cell lines. A particular sulfonamide drug, indisulam, was previously studied in cancer clinical trials where it was found to be safe but demonstrated limited efficacy, most likely because neither the mechanism of action nor potential biomarkers of response were known. Aims: We investigated, in this study, the efficacy of indisulam, either alone or in combination with ABT-199 (venetoclax) and azacitidine, in cultured primary human AML cells. Methods: Bone marrow cells were collected from adults newly diagnosed with AML, non-treated. Cells were cultured and incubated for 48 and 72 hours with indisulam at a range of nanomolar concentrations. Additionally, cells were incubated with ABT-199 (10nM) and azacitidine (1μM), alone or in combination with indisulam. Cell viability and cell death were measured. For mutation profiling, a targeted myeloid gene panel was used. Results: We found that treatment with indisulam resulted in lethality of primary AML cells harboring mutations in SF3B1, but had minor or no effect on AML cells with other mutations including mutations in splicing factors U2AF1 and SRSF2. In SF3B1-mutated cells, a clear synergistic effect was further observed when indisulam was combined with ABT-199 and azacitidine. The correlation between efficacy of indisulam and synergy with ABT-199 and azacitidine in AML cells with different mutations will be shown and discussed. Summary/Conclusion: Our results indicate that indisulam is preferentially active and synergistic with venetoclax and azacitidine in SF3B1-mutated cells from newly diagnosed, non-treated AML patients.

P1051: A PHASE 2 STUDY OF IMG-7289 (BOMEDEMSTAT) IN PATIENTS WITH ADVANCED MYELOFIBROSIS

Background: Lysine-specific demethylase-1 (LSD1) is an activity critical for the self-renewal of malignant myeloid cells and maturation of megakaryocytes, cells central to the pathogenesis of MF. Bomedemstat is an orally active LSD1 inhibitor that in mouse models ameliorated the hallmarks of MPNs and improved survival (Kleppe et al. 2015; Jutzi et al. 2018). Aims: IMG-7289-CTP-102 is an ongoing, global, open-label Phase 2 study evaluating bomedemstat dosed QD in MF patients (NCT03136185). Key eligibility criteria include patients intolerant, refractory, resistant, or inadequately controlled by approved therapy, and platelet count ≥100 x 109/L. Key objectives are safety and reduction of spleen volume (SVR) by MRI/CT and total symptoms scores (TSS) using the MPN-SAF instrument. Methods: Serial bone marrow (BM) biopsies and imaging studies are read centrally. 261 genes are serially sequenced to quantify changes in the allelic frequencies of mutations (MAF) and identify new mutations. The starting dose is 0.6 mg/kg/d titrating, as needed, to a platelet count of 50-75x109/L. Results: At 89 patients, the study is now fully enrolled: 46% primary MF, 33% post-essential thrombocythaemia-MF, 21% post-polycythaemia vera-MF. Median age is 68 (35-88) with 52% males. Prior treatment with ruxolitinib was reported in 83% (74/89); 46% had also received at least 1 additional treatment. 30% of patients (27/89) had received ≥1 RBC transfusion prior to dosing. By IPSS, 53% were high-risk, 40% int.-2, and 7% int.-1. At screening (N=103), sequencing to a mean depth of >1000 bp, JAK2 was mutated in 69%, CALR in 21%, MPL in 6%; 59% had ≥2 mutations of which 71% were high-molecular risk mutations in ASXL1, IDH1/2, EZH2, U2AF1, TP53 and/or SRSF2. At data cutoff (3 Feb 2022), the median duration of treatment is 28 weeks (2-131). Of patients for whom TSS data is available at 24 weeks in those with baseline values ≥20, 72% (18/25) had a reduction in TSS; 24% (6/25) reported a ≥50% reduction. Of patients evaluable for SVR (N=50), 64% had a reduction in spleen volume from baseline; in 28%, SVR was ≥20%. Of evaluable patients (N=41), 90% had stable (∆ <±1.0 g/dL) or improved (≥1.0 g/dL) hemoglobin. Of patients with BM fibrosis scoring post-baseline (N=52), 31% improved by 1 grade and 50% were stable. CCL5 and S100A8/A9 cytokines were elevated at baseline in 50% (16/32) and 78%, respectively; at Day 84, 81% and 68%, respectively, showed reductions of at least 10% and of those, 100% (CCL5) and 47% (S100A8/A9) normalized. In follow-up sequencing at around Week 24, of 60 mutant alleles in 32 patients, the mean MAF fell by 39% in 48%. JAK2 MAFs fell by 31% [SD 5%] in 46% (N=24); ASXL1 MAFs fell 40% in 71%. Clones with JAK2 and/or ASXL1 mutations were most affected. No new mutations have been identified and no patient has transformed to AML. The most common non-hematologic AEs reported by patients was dysgeusia in 36% (32/90) and diarrhoea in 34% (31/90). All dysgeusia events were grade 1/2 and 1 led to treatment discontinuation. Of 14 related SAEs, 4 were Grade 2, 9 Grade 3 and 1 Grade 4 (thrombocytopenia). Twenty-nine patients remain on bomedemstat. Early terminations due to AEs occurred in 18 (20%) patients (9 related to bomedemstat), and 13 discontinued for other reasons. There have been no safety signals, DLTs, or deaths related to drug. Summary/Conclusion: In patients with advanced MF, bomedemstat alone had an acceptable tolerability profile, relieved symptoms, reduced spleen volume and mutation burden while improving fibrosis and anemia without safety signals.

Bone Marrow Fibrosis at Diagnosis and during the Course of Disease Is Associated with TP53 Mutations and Adverse Prognosis in Primary Myelodysplastic Syndrome.

Simple SummaryTo understand the role of bone marrow fibrosis and its molecular changes in myelodysplastic syndrome, we retrospectively analyzed data from 814 patients. Older age, lower hemoglobin, unfavorable karyotype and higher BM blast were more often observed in patients with moderate/severe fibrosis. Cases with bone marrow fibrosis had reduced overall survival. TP53, U2AF1 and KMT2D mutations were more frequent in patients with moderate/severe fibrosis. In addition, 15.1% of patients progressed to moderate/severe fibrosis during the follow-up interval. The clinical features, mutation landscape and prognosis of patients with progressed fibrosis were similar to those patients with moderate/severe fibrosis at diagnosis. We concluded that bone marrow fibrosis was associated with reduced overall survival in primary MDS and correlated with TP53 mutations both at the time of initial diagnosis and during the course of the disease.The prognostic significance of bone marrow fibrosis (MF) grade in patients with myelodysplastic syndrome (MDS) is still debated and the molecular changes remain unclear. In our large cohort, a normal reticulum was found in 211 (25.9%) patients, whereas MF1, MF2 and MF3 were detected in 478 (58.7%), 90 (11.1%) and 35 (4.3%) patients at initial diagnosis, respectively. Patients with MF often correlated with some poor prognostic characteristics, including older age, anemia, unfavorable karyotype, higher BM blast and a higher IPSS-R category. For the entire cohort, the median OS was not reached, 30, 16 and 15 months for patients with MF 0, 1, 2 and 3, respectively. After adjusting for IPSS-R, the hazard ratio for mortality was 1.56 (95% CI, 1.18–2.06) for patients with MF1, 2.29 (95% CI, 1.61–3.27) for patients with MF2 and 2.75 (95% CI, 1.69–4.49) for patients with MF3 compared with those with MF0. The mutational landscape of 370 patients showed that TP53, U2AF1 and KMT2D mutations were more frequent in patients with MF2-3. In addition, of the 408 patients with MF0-1, 62 patients (15.1%) progressed to MF2-3 during the follow-up interval. The clinical features, mutation landscape and prognosis of patients with progressed fibrosis were similar to those of patients with MF2-3 at diagnosis. We concluded that BM fibrosis (MF1, 2 and 3) was an adverse prognosis feature in primary MDS and correlated with TP53 mutations both at the time of initial diagnosis and during the course of the disease. Therefore, BM fibrosis should be included in the revised prognostic scoring system and carefully considered in treatment selection.

Abstract 2159: PRMT5 inhibitor PRT543 displays potent antitumor activity in U2AF1S34F and RBM10LOF spliceosome-mutant non-small cell lung cancer in vitro and in vivo

Abstract PRMT5 (protein arginine methyltransferase 5) is a major Type II PRMT, which catalyzes the symmetric dimethylation of protein arginine residues (sDMA). As an epigenetic regulator, PRMT5 plays essential roles in promoting cancer growth and survival, including through mechanisms that control alternative splicing and RNA processing, and the expression of DNA damage repair genes. Spliceosome mutations have been suggested to represent a potential biomarker for PRMT5 inhibitors, and our previous work highlighted this sensitivity in SF3B1R625C/G expressing uveal melanoma cells. Here, we highlight in vitro and in vivo activity of PRT543, a potent, selective, and orally available PRMT5 inhibitor, in cancer cells harboring mutations in other spliceosome factors such as U2 small nuclear RNA auxiliary factor 1 (U2AF1) and RNA binding motif protein 10 (RBM10). Mutations in U2AF1 (including S34F hotspot mutations) and RBM10 (primarily loss of function (LOF) mutations) occur in 5-10% of all non-small cell lung cancers (NSCLC). Cell proliferation (10-day assay) was assessed in a panel of NSCLC cell lines treated with PRT543, either wild-type or harboring U2AF1S34F or RBM10LOF mutations. Strikingly, both U2AF1S34F and RBM10LOF cell lines were significantly more sensitive to PRT543 compared to wild-type cell lines. Furthermore, PRT543 induced significant dose-related tumor growth inhibition at well-tolerated doses in cell-line derived xenograft (CDX) models harboring the U2AF1S34F or RBM10LOF mutation. Consistent with our previous findings in other tumor types, PRT543 decreased expression of DNA damage repair-associated genes (e.g. BRCA1, RAD51AP1, FANCA, and FANCL) in U2AF1S34F or RBM10LOF mutant NSCLC cells. Combination with PRT543 increased the effectiveness of specific chemotherapeutic agents in both in vitro and in vivo (CDX) models of U2AF1S34F and RBM10LOF NSCLC. Efficacy studies in patient-derived xenograft (PDX) models, as well as genomic profiling of spliceosome-mutant cellular models in response to PRT543 are ongoing. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Jack Carter, Koichi Ito, Venkat Thodima, Monisha Sivakumar, Michael Hulse, Joseph Rager, Komali Vykuntam, Neha Bhagwat, Kris Vaddi, Bruce Ruggeri, Peggy Scherle. PRMT5 inhibitor PRT543 displays potent antitumor activity in U2AF1S34F and RBM10LOF spliceosome-mutant non-small cell lung cancer in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2159.

Abstract 868: Splicing factor mutant lung cancers: A comprehensive molecular and clinicopathologic characterization

Abstract Background: Splicing factor mutations can drive oncogenesis in different cancer types. More importantly, strategies to target splicing-dependent cancers have been developed, including PRMT5 inhibition which regulates alternative splicing and targets posttranslational modification; PRMT5 inhibitors are in ongoing phase I clinical trials. The characterization of splicing alterations in non-small cell lung cancer (NSCLC) has not been extensively studied and represents an unmet need. Methods: Based on previously known genes associated with splicing alterations, genomic and clinicopathologic data from NSCLC patients with hotspot or loss of function mutations identified by next-generation sequencing in the splicing factors SF3B1, SRSF2, U2AF1, ZRSR2, RBM10, and FUBP1 were explored. Results: Among 626 NSCLC patients with at least one splicing mutation, 85% (529/626) also harbored a co-occurring oncogenic driver, such as alterations involving KRAS (60%; 319/529), EGFR (25%;132/529), MET (5%; 27/529), BRAF(4%; 24/529), and ERBB2 (3%; 17/529). After excluding patients with concurrent drivers, 97 splicing-factor-mutant NSCLCs were identified, with 80 (82%) adenocarcinomas and 6 (6%) squamous cell carcinomas. The median age was 71 years (43-89). Forty-nine patients (51%) were female. Sixty patients (62%) were diagnosed with stage I-III disease. Eleven (11%) were never smokers, while 86 (89%) were former/current smokers, with a median of pack years of 30. The median tumor mutational burden was 7.9 mut/Mb (0.9-90.4). Regarding splicing factor mutations, sixty-two patients (64%) had nonsense or splice-site RBM10 mutations, 17 (17%) had missense hotspot SF3B1 mutations, 16 (16%) had hotspot missense U2AF1 mutations, and 7 (7%) had nonsense, splice-site, or hotspot missense FUBP1 mutation. No patients with ZRSR2 or SRSF2 were identified. Only three patients presented concurrent splicing mutations: one with two SF3B1 mutations; one with RBM10 and SF3B1 mutations; and one with U2AF1 and SF3B1 mutations. Conclusion: Most patients with splicing factor mutant NSCLCs are smokers whose cancers commonly harbor RBM10 mutations and higher TMB compared to other driver-positive cancers. The characterization of this profile is key to identifying appropriate patients for ongoing targeted therapy trials for this population. Citation Format: Guilherme Harada, Christina J. Falcon, Fernando C. Santini, Jason C. Chang, Soo R. Yang, Maria E. Arcila, Natasha Rekhtman, Alexander Drilon. Splicing factor mutant lung cancers: A comprehensive molecular and clinicopathologic characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 868.

Abstract 3476: Mechanistic evaluation of VS-6766 (dual RAF/MEK inhibitor) and defactinib (FAK inhibitor) in low-grade serous ovarian cancer models with correlations to clinical response

Abstract Background: Low-grade serous ovarian cancer (LGSOC) constitutes up to 10% of all ovarian cancer and has clinical and molecular characteristics (resistance to chemotherapy, presence of RAS/RAF mutations, lack of TP53 mutations) distinct from high-grade serous ovarian cancer. Here, we characterized the effects of the dual RAF/MEK inhibitor VS-6766 and the FAK inhibitor defactinib on signal transduction and viability in LGSOC cell lines and patient-derived organoids. To correlate molecular characteristics with clinical response, we characterized genomic alterations in archival tumor samples from patients with LGSOC treated with the combination of VS-6766 and defactinib on a clinical trial (FRAME). Material and Methods: We exposed 5 LGSOC cell lines to clinical Cmax concentrations adjusted for protein binding of VS-6766 and defactinib. We quantified phospho- and total proteins (n=66) with an antibody-bead based assay normalized to GAPDH. We also studied growth inhibitory effects of the combination on KRAS mutant (mt) LGSOC patient-derived organoids. We performed next generation sequencing on archival samples from LGSOC patients treated with VS-6766 in combination with defactinib. Results: Signal transduction changes at 1 hr included reduction of p-FAK in 5/5 cell lines in response to defactinib. Cells exposed to VS-6766 showed a reduction in p-ERK and p-p90-RSK in 4/5 cell lines. Additionally, VS-6766 decreased p-cJUN and increased p-IκB in 4/5 cell lines, changes correlated with apoptosis. At 24 hrs, p-ERK and p-p90-RSK inhibition were maintained in 3/5 cell lines. Both drugs increased cleaved PARP in 4/5 cell lines and VS-6766 increased p-SMAD3 and BIM levels, indicating an increase in cell death/apoptosis. The combination of VS-6766 + defactinib showed synergistic growth inhibition in a KRAS mt LGSOC organoid model (combination index 0.51). The clinical combination of VS-6766 and defactinib (September 2021 cut-off) has shown an objective response rate (ORR) of 11/24 (46%) across all patients with LGSOC, and an ORR of 64% (7/11) for patients with KRAS mt LGSOC (n=11). In addition to mutations in KRAS, emerging data may suggest a correlation of U2AF1 and MED12 mutations with response. Conclusions: VS-6766, the dual RAF/MEK inhibitor, induces significant inhibition of ERK pathway signaling in addition to perturbations in TNF/NFκB signaling. Both defactinib and VS-6766 induce apoptosis in LGSOC models. The results provide mechanistic insights into the encouraging response rates observed in patients with LGSOC treated with VS-6766 and defactinib (NCT03875820). These data support the ongoing randomized phase II ENGOTov60/GOG3052/RAMP201 study (NCT04625270). Citation Format: Adam R. Stewart, Lisa Pickard, Ekta Paranjape, Victoria Sanchez Perez, Sanjib Chowdhury, Stephanie Lustgarten, Silvia Coma, Jonathan A. Pachter, Mark S. Carey, Gabriel DiMattia, Hannah C. Badham, Toby Prout, Mona Parmar, Muneeb Mahmud, Christina Yap, Matthew G. Krebs, Susana Banerjee, Udai Banerji. Mechanistic evaluation of VS-6766 (dual RAF/MEK inhibitor) and defactinib (FAK inhibitor) in low-grade serous ovarian cancer models with correlations to clinical response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3476.

Phase 1/2a study of the IRAK4 inhibitor CA-4948 as monotherapy or in combination with azacitidine or venetoclax in patients with relapsed/refractory (R/R) acute myeloid leukemia or lyelodysplastic syndrome.

7016 Background: CA-4948 is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) and FLT3. IRAK4 is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of IRAK4 and have been associated with disease progression and poor prognosis of high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML). Methods: This is an open-label, phase 1/2a dose escalation and cohort expansion trial (NCT04278768). In phase 1 Dose Escalation, patients with R/R AML or HR-MDS are treated with CA-4948 monotherapy. Phase 1b includes 2 arms of combination therapy: CA-4948 + azacitidine (AZA) and CA-4948 + venetoclax (VEN). The primary objectives of this study are to assess the safety, clinical activity, and identify the Recommended Phase 2 Dose (RP2D) of CA-4948 as monotherapy or in combination with AZA or VEN in R/R AML or HR-MDS. The Phase 2a Dose Expansion includes patients for CA-4948 monotherapy: R/R AML with FLT3 mutation, or AML and HR-MDS R/R to HMA with U2AF1 or SF3B1 mutations. Results: As of December 16th, 2021, 49 patients have been treated in the phase 1 portion, of whom 43 started by September 30th, allowing 2 on-study disease assessments. The median number of prior therapies was 2 (range 1-5). Four monotherapy dose levels of CA-4948 were tested (200 to 500 mg orally BID). No dose-limiting toxicities were observed at 200 mg and 300 mg BID. No Grade 4 or 5 treatment-related AEs (TRAEs) were reported, and all the TRAEs were manageable. Reversible, manageable Grade 3 rhabdomyolysis occurred in 1/26 (4%) patients at 300 mg BID, 2/17 (12%) at 400 mg BID, and 1/3 (33%) at 500 mg BID. RP2D was determined as 300 mg BID. Of 43 patients starting before Sept 30th, 2021, 14 had SF3B1, U2AF1 or FLT3 mutations and demonstrated more promising efficacy. In the 5 evaluable AML patients with spliceosome mutations, 40% reached CR/CRh (1 CR, 1 CRh), both with study duration &gt;6 months. In the 7 spliceosome-mutated HR-MDS patients, 57% reached marrow CR, including 1 with RBC transfusion independence and 1 proceeding to HSCT. One of the three FLT3-mutated AML reached CR, and 2 became FLT3-negative. Among the 29 patients without SF3B1/U2AF1/FLT3 mutations, 1 reached CR and 2 PR. Phase 1b and Phase 2a are ongoing. RNA-seq on selected samples showed decrease in relative expression of IRAK4-long isoforms with response to CA-4948. Conclusions: CA-4948 is well tolerated and effective in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1/SF3B1/FLT3 mutations. No dose-limiting myelosuppression was reported, suggesting CA-4948 may be a candidate for combination therapy. Accrual of Phases 1b and 2a is ongoing. Clinical trial information: 04278768.

NPM1-mutated AML-MRC diagnosed on the basis of history of MDS or MDS/MPN frequently harbours secondary-type mutations and confers inferior outcome compared to AML with mutated NPM1

BackgroundAcute myeloid leukemia with myelodysplasia-related changes (AML-MRC) is a prognostically diverse disease. Owing to its favourable prognosis, AML-MRC with mutated NPM1 (NPM1MUT) diagnosed on the basis of multi-lineage dysplasia has been reclassified into “AML with mutated NPM1”. However, it remains unclear if NPM1MUT AML with antecedent MDS or MDS/MPN (AML-MRC-H) should also be reclassified into this subentity. The mutational landscape of NPM1MUT AML-MRC-H remains poorly defined. MethodsThe clinicopathological features, molecular profiles and outcomes of 241 AML-MRC-H and 332 normal karyotype (NK)-AML with mutated NPM1 patients were retrospectively analyzed. Fisher’s exact test, chi-square test and Wilcoxon rank-sum test were used to compare clinicopathological and molecular features. Overall survival and event-free survival were compared using the log-rank test. Multivariable survival analysis was performed using Cox proportional hazards regression. Results33 (14%) AML-MRC-H patients had an NPM1 mutation. By NGS, NPM1MUT AML-MRC-H patients had a significantly higher frequency of secondary-type mutations in U2AF1 and ASXL1 compared to NK-AML with mutated NPM1. NPM1MUT AML-MRC-H was significantly associated with inferior outcomes compared to NK-AML with mutated NPM1. Bone marrow transplantation had a favourable prognostic impact in NPM1MUT AML-MRC-H. ConclusionsNPM1MUT AML-MRC-H has inferior prognosis compared to NK-AML with mutated NPM1, likely due to the higher frequency of secondary-type mutations, and thus should still be included in the high-risk subentity of AML-MRC. NPM1MUT AML-MRC patients may benefit from bone marrow transplantation.

U2AF1 mutation connects DNA damage to the alternative splicing of RAD51 in lung adenocarcinomas.

The recurrent mutation (S34F) in splicing factor U2AF1 is frequently observed in lung adenocarcinoma, but its function remains largely unknown. To determine the mechanistic basis and consequences of U2AF1 mutations, we established non-small cell lung carcinoma A549 cell lines with exogenous expression of wildtype (U2AF1-WT) or mutant (U2AF1-S34F). Splicing analysis revealed that U2AF1-S34F mainly caused aberrant exon usage and affected splicing of numerous DNA damage repair genes. Compared to A549 cells expressing U2AF1-WT, cells expressing U2AF1-S34F showed enhanced DNA damage and cell death in response to ATR inhibitors (ATRi). Mechanistically, U2AF1-S34F induced mis-splicing and downregulation of a key homologous recombination protein RAD51. Overexpression of RAD51 could largely rescue the defective DNA damage response in cells expressing U2AF1-S34F. Moreover, A549 cells expressing U2AF1-S34F, but not U2AF1-WT, were highly sensitive to treatment even with low dose of RAD51 inhibitor on ATRi-induced DNA damage. Our results suggest that U2AF1-S34F causes mis-splicing of DNA damage repair factors in lung cancer and sensitizes cells to RAD51 inhibition.

Predictive and prognostic value of gene mutations in myelodysplastic syndrome treated with hypomethylating agents: a meta-analysis

Although the effect of gene mutations on overall response rate (ORR) and overall survival (OS) in myelodysplastic syndrome (MDS) treated with hypomethylating agents (HMAs) has been explored, the effect is still controversial. We performed this meta-analysis to investigate the effect. The pooled odds ratio (OR) and 95% confidence interval (CI) for ORR and the pooled hazard ratio (HR) and 95%CI for OS were chosen to estimate the effect. The pooled OR of TET2 was 0.73 (95%CI: 0.59–0.91, p = 0.005) and the pooled OR of ASXL1 was 1.38 (95%CI: 1.12–1.71, p = 0.003). As for prognosis, the pooled HR of RUNX1 was 1.45 (95%CI: 1.15–1.85, p = 0.002). The pooled HR of TP53 was 2.30 (95%CI: 1.83–2.90, p < 0.001) and the pooled HR of U2AF1 was 1.41 (95%CI: 1.15–1.74, p = 0.001). There was no statistical difference shown in other genes. Therefore, TET2 mutation and ASXL1 wild-type were the predictor of better response to HMAs. Mutations of TP53, RUNX1, and U2AF1 were associated with poor prognosis in MDS.

A predictive model for bone marrow disease in cytopenia based on noninvasive procedures

AbstractBone marrow specimens are the core of the diagnostic workup of patients with cytopenia. To explore whether next-generation sequencing (NGS) could be used to rule out malignancy without bone marrow specimens, we incorporated NGS in a model to predict presence of disease in the bone marrow of patients with unexplained cytopenia. We analyzed the occurrence of mutations in 508 patients with cytopenia, referred for primary workup of a suspected hematologic malignancy from 2015 to 2020. We divided patients into a discovery (n = 340) and validation (n = 168) cohort. Targeted sequencing, bone marrow biopsy, and complete blood count were performed in all patients. Mutations were identified in 267 (53%) and abnormal bone marrow morphology in 188 (37%) patients. Patients with isolated neutropenia had the lowest frequency of both mutations (21%) and abnormal bone marrow morphology (5%). The median number of mutations per patient was 2 in patients with abnormal bone marrow morphology compared with 0 in patients with a nondiagnostic bone marrow morphology (P < .001). In a multivariable logistic regression, mutations in TET2, SF3B1, U2AF1, TP53, and RUNX1 were significantly associated with abnormal bone marrow morphology. In the validation cohort, a model combining mutational status and clinical data identified 34 patients (20%) without abnormal bone marrow morphology with a sensitivity of 100% (95% confidence interval: 93%-100%). Overall, we show that NGS combined with clinical data can predict the presence of abnormal bone marrow morphology in patients with unexplained cytopenia and thus can be used to assess the need of a bone marrow biopsy.