SRSF2 Is Essential For Hematopoiesis and Its Mutations Dysregulate Alternative RNA Splicing In MDS

Abstract Myelodysplastic syndromes (MDS) are a group of neoplasms that are ineffective in generating multiple lineages of myeloid cells and have various risks to progress to acute myeloid leukemia. Recent genome-wide sequencing studies reveal that mutations in genes of splicing factors are commonly associated with MDS. However, the importance of these splicing factors in hematopoiesis has been unclear and the causal effect of their mutations on MDS development remains to be determined. One of these newly identified genes is SRSF2, and its mutations have been linked to poor survival among MDS patients. Interestingly, most of SRSF2 mutations occur at proline 95 and the majority of these mutations change this proline to histidine (P95H). Given that SRSF2 is a well-characterized splicing factor involved in both constitutive and regulated splicing, we hypothesize that SRSF2 plays an important role in normal hematopoiesis and the SRSF2 mutations induce specific changes in alternative splicing that favor disease progression. We first examined the role of SRSF2 in hematopoiesis by generating Srsf2 null mutation in mouse blood cells via crossing conditional Srsf2 knockout mice (Srsf2f/f) with blood cell-specific Cre transgenic mice (Vav-Cre). The mutant mice produced significantly fewer definitive blood cells (10% of wild type controls), exhibited increased apoptosis in the remaining blood cells, and died during embryonic development. Importantly, we detected no hematopoietic stem/progenitor cells (lineage-/cKit+) in E14 fetal livers of Vav-Cre/Srsf2f/f mice. These results indicate that SRSF2 is essential for hematopoiesis during embryonic development. We next examined the role of SRSF2 in adult hematopoiesis by injecting polyIC into mice that carry a polyIC inducible Cre expression unit. Unexpectedly, after multiple polyIC treatments, the Srsf2f/f mice stayed alive during several months of observation. Time course genotyping analyses of polyIC treated mice revealed an increased rate of incomplete Srsf2 deletion in peripheral blood cells. These observations suggest that Srsf2 ablation did not cause immediate cell lethality in differentiated blood cells, but the gene is indispensable for the function of blood stem/progenitor cells. Since mutations of splicing factors are generally heterozygous in MDS patients, we also examined mice with Srsf2+/- blood cells. No obvious defect of hematopoiesis was observed under normal conditions or in response to stress with 5-FU treatment and sublethal irradiation. To gain molecular insight into the splicing activity of MDS-associated mutant forms of SRSF2, we performed large-scale alternative splicing surveys by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) previously developed in our lab, which offers a robust and cost-effective platform for splicing profiling. Compared to vector transduction controls, we found that overexpression of both wild type and P95H SRSF2 induced many, but distinct changes in alternative splicing in lineage-negative bone marrow cells, and importantly, we noted several changes in genes with known roles in hematopoietic malignancies that were uniquely induced by the mutant SRSF2. To further link the mutations to altered splicing in MDS patients, we also applied RASL-seq to a large number of MDS patient samples with or without mutations in SRSF2 or other splicing regulators. The data revealed a specific set of alternative splicing events that are commonly linked to MDS with splicing factor mutations. These findings strongly suggest that many of these mutations in splicing regulators are gain-of-function mutations that are causal to MDS. In conclusion, we report that SRSF2 plays an essential role in hematopoietic stem/progenitor cells and that the MDS-associated mutations in SRSF2 have a dominant effect on RNA alternative splicing. These findings provide functional information and molecular basis of SRSF2 and its MDS-related mutations in hematopoiesis and related clinical disorders. Disclosures: No relevant conflicts of interest to declare

Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome.

Somatic mutations in the spliceosome gene ZRSR2-located on the X chromosome-are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3'-splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here we characterize ZRSR2 as an essential component of the minor spliceosome (U12 dependent) assembly. shRNA-mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns and RNA-sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns, while splicing of the U2-type introns remain mostly unaffected. ZRSR2-deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS

Oral azacitidine prolongs survival of patients with AML in remission independently of measurable residual disease status

Measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) in remission after intensive chemotherapy is predictive of early relapse and poor survival. Postremission maintenance therapy that prolongs MRD negativity or converts MRD+ patients to MRD- status may delay or prevent relapse and improve overall survival (OS). In the phase 3 QUAZAR AML-001 trial, oral azacitidine (oral-AZA; formerly CC-486), a hypomethylating agent, significantly prolonged OS and relapse-free survival (RFS) compared with placebo in patients aged >= 55 years with AML in first remission after intensive chemotherapy who were not candidates for hematopoietic stem cell transplantation. In this trial, MRD (>= 0.1% leukemic cells in bone marrow) was assessed by multiparameter flow cytometry in serial samples collected at baseline and on day 1 of every 3 cycles. As expected, baseline MRD status was significantly associated with both OS and RFS. Multivariate analyses showed oral-AZA significantly improved OS and RFS vs placebo independent of baseline MRD status. Oral-AZA treatment also extended the duration of MRD negativity by 6 months vs placebo and resulted in a higher rate of conversion from MRD+ at baseline to MRD- during treatment: 37% vs 19%, respectively. In the oral-AZA arm, 24% of MRD responders achieved MRD negativity >6 months after treatment initiation. Although presence or absence of MRD was a strong prognostic indicator of OS and RFS, there were added survival benefits with oral-AZA maintenance therapy compared with placebo, independent of patients' MRD status at baseline.Peer reviewe

Adjunctive Volasertib in Patients With Acute Myeloid Leukemia not Eligible for Standard Induction Therapy : A Randomized, Phase 3 Trial

In this phase 3 trial, older patients with acute myeloid leukemia ineligible for intensive chemotherapy were randomized 2:1 to receive the polo-like kinase inhibitor, volasertib (V; 350 mg intravenous on days 1 and 15 in 4-wk cycles), combined with low-dose cytarabine (LDAC; 20 mg subcutaneous, twice daily, days 1-10; n = 444), or LDAC plus placebo (P; n = 222). Primary endpoint was objective response rate (ORR); key secondary endpoint was overall survival (OS). Primary ORR analysis at recruitment completion included patients randomized >= 5 months beforehand; ORR was 25.2% for V+LDAC and 16.8% for P+LDAC (n = 371; odds ratio 1.66 [95% confidence interval (CI), 0.95-2.89]; P = 0.071). At final analysis (>= 574 OS events), median OS was 5.6 months for V+LDAC and 6.5 months for P+LDAC (n = 666; hazard ratio 0.97 [95% CI, 0.8-1.2]; P = 0.757). The most common adverse events (AEs) were infections/infestations (grouped term; V+LDAC, 81.3%; P+LDAC, 63.5%) and febrile neutropenia (V+LDAC, 60.4%; P+LDAC, 29.3%). Fatal AEs occurred in 31.2% with V+LDAC versus 18.0% with P+LDAC, most commonly infections/infestations (V+LDAC, 17.1%; P+LDAC, 6.3%). Lack of OS benefit with V+LDAC versus P+LDAC may reflect increased early mortality with V+LDAC from myelosuppression and infections.Peer reviewe

SF3B1-mutant MDS as a distinct disease subtype:a proposal from the International Working Group for the Prognosis of MDS

The 2016 revision of the World Health Organization classification of tumors of hematopoietic and lymphoid tissues is characterized by a closer integration of morphology and molecular genetics. Notwithstanding, the myelodysplastic syndrome (MDS) with isolated del(5q) remains so far the only MDS subtype defined by a genetic abnormality. Approximately half of MDS patients carry somatic mutations in spliceosome genes, with SF3B1 being the most commonly mutated one. SF3B1 mutation identifies a condition characterized by ring sideroblasts (RS), ineffective erythropoiesis, and indolent clinical course. A large body of evidence supports recognition of SF3B1-mutant MDSas a distinct nosologic entity. To further validate this notion, we interrogated the data set of the International Working Group for the Prognosis of MDS (IWG-PM). Based on the findings of our analyses, we propose the following diagnostic criteria for SF3B1-mutant MDS: (1) cytopenia as defined by standard hematologic values, (2) somatic SF3B1 mutation, (3) morphologic dysplasia (with or without RS), and (4) bone marrow blasts <5% and peripheral blood blasts <1%. Selected concomitant genetic lesions represent exclusion criteria for the proposed entity. In patients with clonal cytopenia of undetermined significance, SF3B1 mutation is almost invariably associated with subsequent development of overtMDS with RS, suggesting that this genetic lesion might provide presumptive evidence of MDS in the setting of persistent unexplained cytopenia. Diagnosis of SF3B1-mutant MDS has considerable clinical implications in terms of risk stratification and therapeutic decision making. In fact, this condition has a relatively good prognosis and may respond to luspatercept with abolishment of the transfusion requirement. (Blood. 2020;136(2):157-170)

Amplified EPOR/JAK2 Genes Define a Unique Subtype of Acute Erythroid Leukemia

ゲノム解析から急性赤白血病の変異プロファイルと治療標的を解明 --特定の遺伝子変異群の組み合わせと、特徴となる遺伝子の増幅が鍵--. 京都大学プレスリリース. 2022-08-05.Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia characterized by prominent erythroid proliferation whose molecular basis is poorly understood. To elucidate the underlying mechanism of erythroid proliferation, we analyzed 121 AEL using whole-genome/exome and/or targeted-capture sequencing, together with transcriptome analysis of 21 AEL samples. Combining publicly available sequencing data, we found a high frequency of gains/amplifications involving EPOR/JAK2 in TP53-mutated cases, particularly those having >80% erythroblasts designated as pure erythroid leukemia (10/13). These cases were frequently accompanied by gains/amplifications of ERG/ETS2 and associated with a very poor prognosis, even compared with other TP53-mutated AEL. In addition to activation of the STAT5 pathway, a common feature across all AEL cases, these AEL cases exhibited enhanced cell proliferation and heme metabolism and often showed high sensitivity to ruxolitinib in vitro and in xenograft models, highlighting a potential role of JAK2 inhibition in therapeutics of AEL

TP53 mutation status divides myelodysplastic syndromes with complex karyotypes into distinct prognostic subgroups

Risk stratification is critical in the care of patients with myelodysplastic syndromes (MDS). Approximately 10% have a complex karyotype (CK), defined as more than two cytogenetic abnormalities, which is a highly adverse prognostic marker. However, CK-MDS can carry a wide range of chromosomal abnormalities and somatic mutations. To refine risk stratification of CK-MDS patients, we examined data from 359 CK-MDS patients shared by the International Working Group for MDS. Mutations were underrepresented with the exception of TP53 mutations, identified in 55% of patients. TP53 mutated patients had even fewer co-mutated genes but were enriched for the del(5q) chromosomal abnormality (p 10%), abnormal 3q, abnormal 9, and monosomy 7 as having the greatest survival risk. The poor risk associated with CK-MDS is driven by its association with prognostically adverse TP53 mutations and can be refined by considering clinical and karyotype features

Adjunctive volasertib in patients with acute myeloid leukemia not eligible for standard induction therapy: a randomized, phase 3 trial

In this phase 3 trial, older patients with acute myeloid leukemia ineligible for intensive chemotherapy were randomized 2:1 to receive the polo-like kinase inhibitor, volasertib (V; 350 mg intravenous on days 1 and 15 in 4-wk cycles), combined with low-dose cytarabine (LDAC; 20 mg subcutaneous, twice daily, days 1–10; n = 444), or LDAC plus placebo (P; n = 222). Primary endpoint was objective response rate (ORR); key secondary endpoint was overall survival (OS). Primary ORR analysis at recruitment completion included patients randomized ≥5 months beforehand; ORR was 25.2% for V+LDAC and 16.8% for P+LDAC (n = 371; odds ratio 1.66 [95% confidence interval (CI), 0.95–2.89]; P = 0.071). At final analysis (≥574 OS events), median OS was 5.6 months for V+LDAC and 6.5 months for P+LDAC (n = 666; hazard ratio 0.97 [95% CI, 0.8–1.2]; P = 0.757). The most common adverse events (AEs) were infections/infestations (grouped term; V+LDAC, 81.3%; P+LDAC, 63.5%) and febrile neutropenia (V+LDAC, 60.4%; P+LDAC, 29.3%). Fatal AEs occurred in 31.2% with V+LDAC versus 18.0% with P+LDAC, most commonly infections/infestations (V+LDAC, 17.1%; P+LDAC, 6.3%). Lack of OS benefit with V+LDAC versus P+LDAC may reflect increased early mortality with V+LDAC from myelosuppression and infections