U2AF1 Mutations Research Articles

Impaired Early Spliceosome Complex Assembly Underlies Gene Body Elongation Transcription Defect in SF3B1K700E

Introduction: Recurrent mutations in splicing factors (SFs) such as SF3B1, U2AF1 and SRSF2 are common in clonal myeloid disorders. These mutations are typically hemizygous, exclusive to each other, and non-synonymous, pointing to neomorphic functions. Molecular mechanisms connecting the mutations to disease pathogenesis remain unclear. Previous studies predominantly focused on the well-established role of splicing factors in pre-mRNA splicing, seeking to explain how mutations in these SFs leading to changes in the abundance of key regulatory genes through their effects on alternative splicing. These studies are limited by inconsistent and relatively low isoform changes across mutations and independent datasets. They also don't explain the mutual exclusivity of these mutations. In this study, we explored a novel mechanism that link SF mutations to disease biology: dysregulation of RNA Polymerase II (Pol2) transcriptional kinetics arising from impaired spliceosome assembly. Methods: We generated inducible isogenic K562 cell lines that express single mutant alleles (SF3B1 K700E and U2AF1 S34F,and corresponding wild-type controls). A novel combinatory approach of AAV-intron trap with CRISPR/Cas9 (Boddu et al, Comms Biol, 2021) was utilized for gene editing. RNAPII transcription kinetics changes were assessed, at an early post-induction time point of 4 days (Fig1A), using ChIP-seq, and nascent-transcriptome assays (including GRO-seq and transient transcriptome-time lapse sequencing or TT-TL seq). Spliceosome assembly kinetics were assayed with non-denaturing gels. Co-transcriptional splicing efficiency (CoSE) was assessed using nascent long-read sequencing (LRS) (Reimer, Mol Cell 2021). Key findings in K562 cell lines were validated in CD34+ progenitor populations from MDS-derived patient samples (and healthy volunteer controls). Results: ChIP-seq for Pol 2 (both total (Fig1B) and elongation-specific Ser2P (Fig1C) showed a redistribution of Pol2 into gene-bodies, suggesting a failure of Pol2 elongation in SF3B1 K700E. This Pol2 redistribution was dependent on gene length and intron number (long, multi-intron genes most affected). GRO-seq confirmed the ChIP-seq results (Fig1D). Additionally, spike-in normalized TT-seq showed significantly reduced nascent transcription in gene bodies in SF3B1 K700E (Fig 1E), consistent with a gene-body elongation rate defect. A similar redistribution of Pol2 into gene bodies was noted by ChIP-seq and TT-seq in U2AF1S34F, pointing to a convergent transcriptional response among distinct SF mutations. Genome-wide CoSE was noted to be lower in SF3B1 K700Eindicating an associated splicing assembly defect (Fig1F). Changes to transcription due to SF3B1 mutations were similarly observed, using low-input Pol2 CUT&RUN, in CD34+ progenitor populations isolated from human MDS patient samples (Fig1G). Of pathologic relevance, this transcriptional dysregulation led to generation of R-loops, DNA damage, and S-phase arrest. Given close coordination of splicing and transcription, and role of SF3B1 and U2AF1 in early spliceosome formation, we speculated if mutant SF3B1 leads to defective assembly of early spliceosomes. Compared to wild-type protein, SF3B1 K700E was noted to have significantly impaired interactions with U2AF1, U2AF2, HTATSF1 (Fig1H). We then determined how spliceosome assembly kinetics of SF3B1 K700E differed from those of SF3B1 WTusing an in vitro splicing assay. The transition from E- to A- complex intermediates was reduced in SF3B1 K700E(Fig1I). Furthermore, disrupting the early pre-spliceosome assembly (through loss of DDX46 or HTATSF1 using both siRNA and inducible shRNA (Fig1J) knockdown systems) resulted in a transcriptional response similar to SF3B1 K700E. Based on recent cryo-EM structures of Pol II-U1 snRNP interactions (Zhang et al, Science, 2021), we speculate that the impaired transition to A/B complex in SF3B1 K700E prevents the release of U1 snRNP and release of Pol2, leading to intron-predominant defective Pol II elongation (Fig1K). Conclusion: Here, we found that SF3B1 and U2AF1 mutations impair Pol2 transcription elongation along gene bodies, leading to pathologic R-loops and replication stress. This elongation results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1.

CHIPping in: Clonal hematopoiesis clinics in community oncology.

499 Background: Clonal hematopoiesis of indeterminate prognosis (CHIP) is recently recognized as a pre-malignant condition and independent cardiovascular risk factor with doubled risk of coronary heart disease, cerebrovascular events, and worsened heart failure outcomes. Despite this recognition, multidisciplinary CHIP clinics are near-uniformly associated with tertiary centers where only a minority of oncology patients receive care. Tennessee Oncology is a large community oncology practice with over 200 providers at 35 sites of care throughout Tennessee and North Georgia. Here, we report the characteristics of two years of patients with common CHIP mutations as we prepare to launch our CHIP clinic. Methods: Patients with solid tumors who underwent either tissue- or blood-based comprehensive genomic profiling from May 2021 to May 2023 were reviewed. Patients with incidental discovery of DNMT3A, ASXL1, TET2, JAK2, IDH1/2, SRSF2, U2AF1, SF3B1, and ZRSR2 mutations are classified as having CHIP. High-risk mutation profile was defined as >1 mutation or a single mutation with variant allele frequency (VAF) of ≥10%. In patients sequenced >1x, only initial sequencing was used. The presence of cardiovascular disease or traditional risk factors was based entries into the patients’ “problem list” or use of medications commonly used to treat these conditions. Results: Over a two-year period, 3599 patients underwent sequencing and CHIP mutations were identified in 1069 (29.7%) patients. The median VAF was 1.58% and 585 patients had at least one mutation with a VAF of ≥2%. Median age at sequencing was 69.6 years. Distribution between males/females was similar. Lung cancer was the most common histology (n=328) followed by breast (n=122) and colon (n=121). DNMT3A mutations (n=829) were most frequently seen followed by TET2 (n=353), ASXL1 (n=335), SRSF2/U2AF1/SF3B1/ZRSR2 (n=147), JAK2 (n=138), and IDH1/2 (n=104). Nearly two-thirds of patients had a high-risk mutational profile with >1 mutation (n=479) or VAF ≥10% (n=185). Assessing traditional risk factors for cardiovascular and cerebrovascular disease, essential hypertension and type 2 diabetes were present in 56.4% (n=603) and 30.1% (n=322) of patients, respectively, and a minority of patients had established coronary disease (n=181) or heart failure (n=74). Twenty-three percent (245/1069) had no cardiovascular risk factors. Conclusions: In a large community practice, 29.7% of patients have common CHIP mutations – most of whom are considered high-risk based on current risk stratification criteria. Many patients also have traditional risk factors or established cardiac disease underscoring the strong association between CHIP and cardiovascular disease. A community-based, multi-disciplinary CHIP clinic would enhance these patients’ care and potentially lead to improved outcomes.

IRAK-4 inhibition: emavusertib for the treatment of lymphoid and myeloid malignancies.

Several studies have identified mutations in the MYD88L265P gene as a key driver mutation in several B-cell lymphomas. B-cell lymphomas that harbor the MYD88L265P mutation form a complex with phosphorylated Bruton's tyrosine kinase (BTK) and are responsive to BTK inhibition. However, BTK inhibition in B-cell lymphomas rarely results in a complete response and most patients experience eventual disease relapse. Persistent survival signaling though downstream molecules such as interleukin 1 receptor-associated kinase 4 (IRAK-4), an integral part of the "myddosome" complex, has been shown to be constitutively active in B-cell lymphoma patients treated with BTK inhibitors. Emerging evidence is demonstrating the therapeutic benefit of IRAK-4 inhibition in B-cell lymphomas, along with possibly reversing BTK inhibitor resistance. While MYD88 gene mutations are not present in myeloid malignancies, downstream overexpression of the oncogenic long form of IRAK-4 has been found in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), particularly in AML and MDS that harbor mutations in splicing factors U2AF1 and SF3B1. These data suggest that the anti-leukemic activity of IRAK-4 inhibition can be exploited in relapsed/refractory (R/R) AML/MDS. In this review article, we discuss the currently available pre-clinical and clinical data of emavusertib, a selective, orally bioavailable IRAK-4 inhibitor in the treatment of R/R B-cell lymphomas and myeloid malignancies.

U2AF1 pathogenic variants in myeloid neoplasms and precursor states: distribution of co-mutations and prognostic heterogeneity

We have previously recognized the genotypic and prognostic heterogeneity of U2AF1 mutations (MT) in myelofibrosis (MF) and myelodysplastic syndromes (MDS). In the current study, we considered 179 U2AF1-mutated patients with clonal cytopenia of undetermined significance (CCUS; n = 22), MDS (n = 108), MDS/acute myeloid leukemia (AML; n = 18) and AML (n = 31). U2AF1 variants included S34 (60%), Q157 (35%), and others (5%): corresponding mutational frequencies were 45%, 55%, and 0% in CCUS; 57%, 39%, and 4% in MDS; 61%, 33%, and 6% in MDS/AML; and 55%, 35% and 10% in AML (P = 0.17, 0.36 and 0.09), respectively. Concurrent mutations included ASXL1 (37%), BCOR (19%), RUNX1 (14%), TET2 (15%), DNMT3A (10%), NRAS/KRAS (8%), TP53 (8%), JAK2 (5.5%) and SETBP1 (5%). The two most frequent U2AF1 MT were S34F (n = 97) and Q157P (n = 46); concurrent MT were more likely to be seen with the latter (91% vs 74%; P = 0.01) and abnormal karyotype with the former (70% vs 62%; P = 0.05). U2AF1 S34F MT clustered with BCOR (P = 0.04) and Q157P MT with ASXL1 (P = 0.01) and TP53 (P = 0.03). The median overall survival (OS) in months was significantly worse in AML (14.2) vs MDS/AML (27.3) vs MDS (33.7; P = 0.001); the latter had similar OS with CCUS (30.0). In morphologically high-risk disease (n = 49), defined by ≥10% blood or bone marrow blasts (i.e., AML or MDS/AML), median OS was 14.2 with Q157P vs 37.1 months in the presence of S34F (P = 0.008); transplant-adjusted multivariable analysis confirmed the detrimental impact of Q157P (P = 0.01) on survival and also identified JAK2 MT as an additional risk factor (P = 0.02). OS was favorably affected by allogeneic hematopoietic stem cell transplantation (HR: 0.16, 95% CI; 0.04-0.61, P = 0.007). The current study defines the prevalence and co-mutational profiles of U2AF1 pathogenic variants in AML, MDS/AML, MDS, and CCUS, and suggests prognostic heterogeneity in patients with ≥10% blasts.

Myelodysplastic neoplasm-associated U2AF1 mutations induce host defense defects by compromising neutrophil chemotaxis

Myelodysplastic neoplasm (MDS) is a hematopoietic stem cell disorder that may evolve into acute myeloid leukemia. Fatal infection is among the most common cause of death in MDS patients, likely due to myeloid cell cytopenia and dysfunction in these patients. Mutations in genes that encode components of the spliceosome represent the most common class of somatically acquired mutations in MDS patients. To determine the molecular underpinnings of the host defense defects in MDS patients, we investigated the MDS-associated spliceosome mutation U2AF1-S34F using a transgenic mouse model that expresses this mutant gene. We found that U2AF1-S34F causes a profound host defense defect in these mice, likely by inducing a significant neutrophil chemotaxis defect. Studies in human neutrophils suggest that this effect of U2AF1-S34F likely extends to MDS patients as well. RNA-seq analysis suggests that the expression of multiple genes that mediate cell migration are affected by this spliceosome mutation and therefore are likely drivers of this neutrophil dysfunction.

Validation of the molecular international prognostic scoring system in patients with myelodysplastic syndromes defined by international consensus classification

Myelodysplastic syndromes (MDS) have varied prognoses and require a risk-adapted treatment strategy for treatment optimization. Recently, a molecular prognostic model (Molecular International Prognostic Scoring System [IPSS-M]) that combines clinical parameters, cytogenetic abnormalities, and mutation topography was proposed. This study validated the IPSS-M in 649 patients with primary MDS (based on the 2022 International Consensus Classification [ICC]) and compared its prognostic power to those of the IPSS and revised IPSS (IPSS-R). Overall, 42.5% of the patients were reclassified and 29.3% were up-staged from the IPSS-R. After the reclassification, 16.9% of the patients may receive different treatment strategies. The IPSS-M had greater discriminative potential than the IPSS-R and IPSS. Patients with high, or very high-risk IPSS-M might benefit from allogeneic hematopoietic stem cell transplantation. IPSS-M, age, ferritin level, and the 2022 ICC categorization predicted outcomes independently. After analyzing demographic and genetic features, complementary genetic analyses, including KMT2A-PTD, were suggested for accurate IPSS-M categorization of patients with ASXL1, TET2, STAG2, RUNX1, SF3B1, SRSF2, DNMT3A, U2AF1, and BCOR mutations and those classified as MDS, not otherwise specified with single lineage dysplasia/multi-lineage dysplasia based on the 2022 ICC. This study confirmed that the IPSS-M can better risk-stratified MDS patients for optimized therapeutic decision-making.

Reliability of flow-cytometry in diagnosis and prognostic stratification of myelodysplastic syndromes: correlations with morphology and mutational profile

Diagnosis and prognostic stratification of myelodysplastic syndromes (MDS) have been complemented by new techniques, including flow cytometry and NGS. To analyze the relationship between molecular and cytofluorimetric data, we enrolled in this retrospective study, 145 patients, including 106 diagnosed with MDS and 39 controls. At disease onset, immunophenotypic (IF), cytogenetic tests, and cytomorphological (CM) examination on bone marrow were carried out in all patients, while NGS was performed in 58 cases. Ogata score presented a specificity of 100% and a sensitivity of 59%. The detection of at least two phenotypic aberrancies in Ogata negative patients increased the sensitivity to 83% and specificity to 87%. Correlations were identified between IF aberrancies and mutations, including positive Ogata>2 and mutations in SRSF2 (p=0.035), CD15 and U2AF1 (0.032), CD56 and DNMT3A (p=0.042), and CD38 and TP53 (p=0.026). In multivariate analysis, U2AF1 mutations, associated with del(20q) and/or abnormalities of chromosome 7 (group 4 as defined by the EuroMDS score), significantly correlated with an inferior overall survival (p=0.019). These parameters and Ogata score>2 also showed a significant correlation with inferior event-free survival (p=0.023 and p=0.041, respectively). Both CM and FC features correlated with prognosis and mutational patterns. In an integrated MDS work-up, these tools may guide indications for mutational screening for optimal risk stratification.

Incidence and prognostic impact of U2AF1 mutations and other gene alterations in myelodysplastic neoplasms with isolated 20q deletion.

In myelodysplastic neoplasms (MDS), the 20q deletion [del(20q)] is a recurrent chromosomal abnormality that it has a high co-occurrence with U2AF1 mutations. Nevertheless, the prognostic impact of U2AF1 in these MDS patients is uncertain and the possible clinical and/or prognostic differences between the mutation type and the mutational burden are also unknown. Our study analyzes different molecular variables in 100 MDS patients with isolated del(20q). We describe the high incidence and negative prognostic impact of U2AF1 mutations and other alterations such as in ASXL1 gene to identify prognostic markers that would benefit patients to receive earlier treatment.

The genetics of transformation from myelodysplastic syndrome to secondary acute myeloid leukemia.

e19073 Background: Gene mutations play key role in the transformation from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (s-AML). The aim of this study was to explore the clonal progression pattern from MDS to s-AML. Methods: A total of 663 patients diagnosed with MDS (n = 291), s-AML (n = 86), and primary AML (p-AML) (n = 286, M3 excluded) were enrolled in this study from May 2018 to December 2021, and the median age was 57 year-old (range 1~88). The diagnosis was established according to the WHO criteria. The MDS patients were divided into low-risk MDS ( n = 158) and high-risk MDS (n = 133) according to 2012 IPSS-R. Next generation sequencing was performed to detect the mutations of entire coding regions of 112 genes related with hematologic tumors in all patients. Results: It was found that mutations from 31 genes occurred in both MDS and AML, including DNA methylation ( DNMT3A, TET2, IDH1, IDH2, WT1), chromatin modification ( SETBP1, ASXL1, EZH2, PHF6), RNA splicing ( U2AF1, SF3B1, SRSF2, ZRSR2), transcription regulation ( TP53, CEBPA, RUNX1, ETV6, GATA2, BCOR, BCORL1), signal transduction pathways ( NRAS, KRAS, PTPN11, NOTCH1, FLT3, JAK2, KIT), and others ( NPM1, FAT, STAG2, DDX41). By enrichment analysis, those mutations were classified into two categories: type A mutations ( FLT3, IDH1, NPM1, NRAS, PTPN11, WT1, CEBPA) and type B mutations ( IDH2, KRAS, U2AF1, ZRSR2, SRSF2, STAG2, DNMT3A, EZH2, RUNX1, ETV6, TP53). The type A genes were significantly enriched in p-AML than in MDS, while some type B mutations, particularly RNA splicing, were significantly enriched in MDS than in p-AML. The VAFs of type A mutations were significantly lower than those of type B mutations. The longitudinal analysis of 10 patients with s-AML revealed two main models of clonal evolution, linear evolution and clone sweeping. Although the two models differed in the disease development, new sub-clones appeared in 9/10 patients. Interestingly, many of the new mutations were type A mutations (n = 7, 7/10), and 7 out of 10 patients carried type B mutations when they were at MDS state. The VAF data also showed that the preexisting mutations were generally higher than the newly emerging genetic alterations. Finally, an individual case was used to delineate the clone evolution. The patient was initially diagnosed with 5q syndrome and U2AF1 mutation (type B) was identified. After treatment, the patient experienced remission, and developed a new ETV6 mutation while the clonal size of U2AF1 decreased. When the patient relapsed, the clone size of ETV6 decreased while U2AF1 mutated clone size were amplified, a typical “clone sweeping” pattern. When the patient eventually transformed to s-AML, three type A mutations ( NRAS, KRAS and PTPN11) appeared simultaneously. Conclusions: This study revealed that the type A mutations are mainly responsible for the progression of MDS to AML, and the type B mutations are related to the clonal origin of MDS.

Molecular features of 109 patients with chronic myelomonocytic leukemia in a single center

Objective: To explore the molecular features of chronic myelomonocytic leukemia (CMML) . Methods: According to 2022 World Health Organization (WHO 2022) classification, 113 CMML patients and 840 myelodysplastic syndrome (MDS) patients from March 2016 to October 2021 were reclassified, and the clinical and molecular features of CMML patients were analyzed. Results: Among 113 CMML patients, 23 (20.4%) were re-diagnosed as acute myeloid leukemia (AML), including 18 AML with NPM1 mutation, 3 AML with KMT2A rearrangement, and 2 AML with MECOM rearrangement. The remaining 90 patients met the WHO 2022 CMML criteria. In addition, 19 of 840 (2.3%) MDS patients met the WHO 2022 CMML criteria. At least one gene mutation was detected in 99% of CMML patients, and the median number of mutations was 4. The genes with mutation frequency ≥ 10% were: ASXL1 (48%), NRAS (34%), RUNX1 (33%), TET2 (28%), U2AF1 (23%), SRSF2 (21.1%), SETBP1 (20%), KRAS (17%), CBL (15.6%) and DNMT3A (11%). Paired analysis showed that SRSF2 was frequently co-mutated with ASXL1 (OR=4.129, 95% CI 1.481-11.510, Q=0.007) and TET2 (OR=5.276, 95% CI 1.979-14.065, Q=0.001). SRSF2 and TET2 frequently occurred in elderly (≥60 years) patients with myeloproliferative CMML (MP-CMML). U2AF1 mutations were often mutually exclusive with TET2 (OR=0.174, 95% CI 0.038-0.791, Q=0.024), and were common in younger (<60 years) patients with myelodysplastic CMML (MD-CMML). Compared with patients with absolute monocyte count (AMoC) ≥1×10(9)/L and <1×10(9)/L, the former had a higher median age of onset (60 years old vs 47 years old, P<0.001), white blood cell count (15.9×10(9)/L vs 4.4×10(9)/L, P<0.001), proportion of monocytes (21.5% vs 15%, P=0.001), and hemoglobin level (86 g/L vs 74 g/L, P=0.014). TET2 mutations (P=0.021) and SRSF2 mutations (P=0.011) were more common in patients with AMoC≥1×10(9)/L, whereas U2AF1 mutations (P<0.001) were more common in patients with AMoC<1×10(9)/L. There was no significant difference in the frequency of other gene mutations between the two groups. Conclusion: According to WHO 2022 classification, nearly 20% of CMML patients had AMoC<1×10(9)/L at the time of diagnosis, and MD-CMML and MP-CMML had different molecular features.

The clinical and molecular spectrum of ETV6 mutated myeloid neoplasms.

ETV6 mutations are rare but recurrent somatic events in myeloid neoplasms and are negatively prognostic in myelodysplastic syndrome. We set out to examine the clinical and molecular characteristics of patients undergoing investigation for myeloid neoplasms, found to have deleterious ETV6 mutations. ETV6 mutations occurred in 33 of 5793 (0.6%) cases investigated and predominantly in high-risk disease entities including MDS with increased blasts, primary myelofibrosis and AML, myelodysplasia-related. In three cases, isolated iso (17q) karyotype was concurrently detected, an otherwise rare karyotype in myeloid neoplasms. ETV6 mutations were frequently subclonal and never occurred as an isolated abnormality with ASXL1 (n = 22, 75%), SRSF2 (n = 14, 42%) and SETBP1 (n = 11, 33%) the predominant co-mutations. Restricting to patients with MDS, higher rates of ASXL1, SETBP1, RUNX1 and U2AF1 mutations occurred in ETV6 mutated cases, relative to a consecutive control cohort with wild-type ETV6. The median OS of the cohort was 17.5 months. This report highlights the clinical and molecular associations of somatic ETV6 mutations in myeloid neoplasms, suggests their occurrence as a later event, and proposes further translational research questions for their role in myeloid neoplasia.

Sex Disparities in Myelodysplastic Syndromes: Genotype, Phenotype, and Outcomes.

Introduction/Background The impact of biological sex on the clinical phenotype, genotype, and outcomes among patients with MDS is not well characterized. Materials and Methods We retrospectively reviewed the clinical and genomic data from male and female patients included in our institutional MDS database at Moffitt Cancer Center. Results Among 4580 patients with MDS, 2922 (66%) were men and 1658 (34%) were women. Women were younger (mean age 66.5 vs. 69 years for men, P < .001) at diagnosis. There were more Hispanic/black women than men (9% vs. 5%, P =<.001). Women had lower hemoglobin and higher platelet counts than men. More women had del 5q/monosomy 5 abnormalities compared to men (P =<.001). Therapy related MDS were more common in women than men (25% vs.17%, P=<.001). On assessment of molecular profile, SRSF2, U2AF1, ASXL1, and RUNX1 mutations were more frequent in men. The median overall survival (mOS) was 37.5 months (mo) for females compared to 35 monthsfor males, (P=.002). The mOS was significantly prolonged for women in lower-risk MDS, but not in higher-risk MDS. Women were more likely to respond to immunosuppression with ATG/CSA than men (38% vs. 19%, P= 0.04).Conclusion Ongoing research is needed for understanding the impact of sex on phenotype, genotype, and outcomes in patients diagnosed with MDS.

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

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

Sex Differences in the Spectrum of Clonal Hematopoiesis.

Sex Differences in the Spectrum of Clonal Hematopoiesis.

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

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

Clinical and molecular spectrum and prognostic outcomes of U2AF1 mutant clonal hematopoiesis- a prospective mayo clinic cohort study

• U2AF1 mutant CH is infrequent in the spectrum of genes mutated in age-related CH (<10%) and commonly presents as clonal cytopenias of undetermined significance (CCUS). • U2AF1 CH is more common in males (67%) and frequently co-occurs with truncating BCOR mutations (29%; with 86% occurring in males). • U2AF1 mutant CH is associated with a high rate (25%) and short latency (17.5 months) for progression to myeloid neoplasms. • While U2AF1 mutant CH usually occurs in the elderly (median age 71 years), the context specific occurrence in younger patients with telomere biology disorders needs further assessment.

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

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

U2AF1 Splicing Factor Mutations Affect RNA Subcellular Compartmentalization in Myeloid Malignancies

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

Differential Impact on Survival in Myeloid Neoplasm Patients with U2AF1 Mutations

Introduction:U2AF1 mutations (m) are associated with abnormal splicing of genes involved in functionally important pathways, such as cell cycle progression and RNA processing. The U2AF1 gene has specific hotspots for mutations primarily involving the S34F and Q157P residues within the ZnF1 and ZnF2 causing mis-splicing of genes critical for hematopoiesis. Most recently in European Leukemia Net 2022 guidelines, U2AF1m is categorized as high-risk disease with inferior survival. We analyze outcome of U2AF1m in myeloid neoplasms and co-mutation patterns in large cohort of patients treated at Mayo Clinic. Methods: We reviewed our myeloid neoplasms patients (pts) database who underwent NGS (MC OncoHeme 42 genes panel) testing between October 2015 and August 2021. We identified 121 myeloid neoplasm pts with U2AF1m, and these were analyzed for clinical characteristics, co-mutational patterns, and outcomes using SPSS statistical software. An appropriate IRB approved was obtained. Results: The median age at diagnosis was 71 years (range [R], 19-92) (Table 1). Sixty-one (51%) pts were diagnosed with MDS, 29 (24%) pts with myeloproliferative neoplasms (MPN), 23 (19%) pts with acute myeloid leukemia (AML), 12 (10%) pts with MDS/MPN overlap and 3 (2.5%) pts with clonal cytopenia of unknown significance (CCUS). Out of 61 pts diagnosed with MDS, 59 pts had risk stratification information based on the revised international prognostic scoring system (IPSS-R): 20 (33.9%), 18 (30.5%), 17 (28.8%) and 4 (6.8%) patients were stratified into low, intermediate, high, and very high risk, respectively. Thirty-one out of 102 (30%) pts with MDS/MPN/MDS-MPN overlap progressed to AML with median time to AML progression 14.6 months (R, 1-118). Forty-four percent and 32% of pts had mutations involving the S34F and Q157P hotspots respectively, while the remaining had Q157R (12%), S34Y (6%), R156H (5%) and R35L (1%). The median variable allele frequency (VAF) was 38% (R, 2-49). Isolated U2AF1m was seen in 15 (12.4%) patients, 9 of these patients were diagnosed as MDS. The rest of the patients (87.6%) had at least 1 co-mutation, the most common ones being ASLX1 (46%), JAK2 (19%), TET2 (17.5%), RUNX1 (14%), DNMT3A (14%) and BCOR (14%). We did sub-group analysis for overall survival (OS) in advanced MDS and AML patients harboring most frequent U2AF1 mutations (S34F, Q157P and Q157R). The mOS was 31.3, 20.5 and 8.8 months (mo) in MDS/AML pts with S34F, Q157P and Q157R mutations, respectively (p= 0.005, Figure 1A). While VAF <40% (OS 21.5 mo) vs VAF >40% (OS 24.5 mo) had no differential impact on OS (p= 0.98, Figure 1B). Conclusion: In this study, we have demonstrated that U2AF1 gene with S34F hotspot have better OS, while Q157R had significantly inferior OS in pts with MDS/AML. Contrary to earlier reports we did not observe significant survival difference in MDS/AML patients with VAF cutoff of 40%. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of Myelofibrosis Patients in the Era of Using JAK2 Inhibitor with Allogeneic-HSCT at the University of Southern California

Introduction: In myelofibrosis (MF) patients, the standard of care curative option is with allogeneic-hematopoietic stem cell transplantation (allo-HSCT) but no clear standard of care pre- and post-allo-HSCT management exists. Questions then arise such as whether use of JAK inhibitors pre- or post-transplant lead to better outcomes. In addition, further elucidation of patient characteristics/risks/response are needed due to scarcity of data post-transplant. In this study, we aim to provide further data concerning this field. Methods: This retrospective chart review included patients aged 18 years with MF diagnosed in 2012-2020 and received allo-HSCT at our institution. Primary objectives include over-all survival (OS) and progression-free-survival (PFS) since transplant, stratified by risk, peri-transplant JAK2 inhibitor use and conditioning + associated graft versus host disease prophylaxis (GVHD ppx) use. Secondary objectives include patient/treatment/transplant characteristics, response, survival and GVHD rates. Two-sided p value ≤ 0.05 was significant. RESULTS: 21 screened MF patients received allo-HSCT at our institution, median age 61.1 years (85.7% non-Hispanic and 14.3% Hispanic). 71.4% had primary MF. 9.5% had unfavorable/high risk karyotype/cytogenetics with 38.1% at high risk per Dynamic International Prognostic Scoring System (DIPSS) Plus, and 38.1% high risk, 28.6% very high-risk per Mutation-Enhanced International Prognostic Score System (MIPSS) 70+ v2.0. 52.4% had JAK2 V617F mutation, 28.6% triple negative (lacked JAK2/MPL/CALR mutations) with 23.8% as having any positivity in either ASXL1, EZH2, SRSF2, IDH1/2 and U2AF1 mutations. Pre-transplant, 100% had pancytopenia requiring transfusions. 9.5% had CR/PR, 42.9% had PD and 47.6% had SD. Of note, 57.9% had any peri-transplant JAK2 inhibitor use (defined as use within 3 months pre- and post-transplant) with or without other MF related-treatments. 33.3% underwent MSD, 38.1% MUD and 28.6% underwent HAPLO allo-HSCT. 80% had reduced intensity conditioning (RIC) with an equal distribution of fludarabine-based regimens. In terms of GVHD ppx, 55% received tacrolimus+ methotrexate and 35% had post-transplant cyclophosphamide (PTCY). 30% underwent HAPLO + PTCY. In terms of outcomes and adverse events, pancytopenia improved from 90.5% at d+30 to 57.1% at d+365. Transfusion dependency decreased from 60% from d+90 to 26.7% by d+365. None had leukemic transformation. Clinical improvement rates increased from 33.3% d+30 to 78.6% by d+365. CR/PR rate increased from 5% at d+90 to 28.6% at d+365. 14.3% had relapsed/PD, 37.5% had primary graft failure with 33.3% any deaths post allo-HSCT. 42.9% had any acute GVHD pre-d+100, 42.1% had any chronic GVHD post d+100. Of note, 100% of our population were alive at d+100, and 78.9% alive by d+365. Since transplant, 3y OS was 62.6%, 3y EFS at 59.2% with corresponding worsening of OS/EFS with higher DIPSS (p=0.19-0.33) and a 3y OS (40%) (p=0.29) and EFS (40%) (p=0.41) in patients with very-high risk per MIPSS 70+ v2.0. With peri-transplant JAK2 inhibitor use, 3y OS was 56.8% vs 75% without; 3y EFS was 51.1% vs 75% without (p=0.33-0.51) (Fig.1). For patients who underwent HAPLO+PTCY GVHD ppx, OS and EFS reached at 16 months while patients who underwent non-HAPLO+ any GVHD ppx did not reach their median OS yet and achieved an EFS median of 57 months. 2y OS and EFS was at 50% for those who underwent HAPLO+PTCY vs a 67.1% OS and a 61.9% EFS for those who had non-HAPLO+ any GVHD ppx (p=0.51-0.72) (Fig.2). CONCLUSIONS: Given that most of our patients had high/very high-risk scores, we achieved a good rate of clinical improvement with less transfusion dependency. A good survival rate was also achieved (100% survival at d+100 and 78.9% at 1y). Our 3y OS of 62.6% and 3y EFS of 59.2% is also appreciable even with the rates of GVHD. Our data also further supports that having a higher risk score per DIPSS/MIPSS leads to poorer survival. Interestingly, there may be no difference in OS/EFS with peri-transplant JAK2 inhibitor use. Also, those who underwent HAPLO+PTCY had their median OS/EFS reached first (16 months) with a possible lower survival trend compared to those who underwent MSD/MUD + any GVHD ppx combination treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal