SRSF2 P95H

To survive carcinogen treatment, Srsf2 P95H+/- cells undergo substantial transcriptional rewiring and restore bi-directional gene expression. Thus, our study underscores SRSF2's importance in regulating transcription to orchestrate the cell cycle and the DNA damage response.

Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

Accordingly, Srsf2 delays myelofibrosis induced by the thrombopoietin receptor agonist Romiplostim in Jak2 wild-type animals. These results unveil JAK2 exon 14 skipping promotion as a strategy to reduce JAK/STAT signaling in pathological conditions.

Second, murine Srsf2P95H leukemias showed improved prolonged survival when treated with olaparib (PARPi) compared to vehicle treatment in vivo...In summary, our data establish a previously unknown link between R-loop-induced PARP1 response and RNA splicing perturbation and provide a mechanistic rationale to evaluate the clinical efficacy of PARP inhibitors in spliceosome-mutant malignancies. Furthermore, our study highlights a new therapeutic potential of targeting the R-loop tolerance pathways caused by different spliceosome gene mutations.

Three patients were treated with thrombopoietin receptor agonists (2 eltrombopag, 1 romiplostim) and had minimal transient platelet count increases not meeting the IWG MDS criteria for response. Three patients have received allogeneic hematopoietic stem cell transplant.Conclusion :This series describes a subset of CCUS patients with isolated thrombocytopenia associated with SRSF2 mutations. We also describe atypical megakaryocytic findings which do not meet current WHO diagnostic criteria for dysmegakaryopoiesis; however, the uniform presence of these findings in this cohort suggests an association with thrombocytopenia and CCUS.

Since several genes implicated in erythropoiesis and heme metabolism have been shown to be misspliced in SRSF2-mutant MDS, we are currently analyzing RNA-sequencing data from wild-type and Srsf2P95H/+ CFU-E cells, with the expectation that these data will provide insights into the underlying mechanisms driving anemia. These data may ultimately help identify novel therapeutic targets to alleviate anemia in SRSF2-mutant MPN and MDS, which is an area of unmet clinical need.

SRSF2P95H and SF3B1K700E cells showed increased sensitivity to olaparib and rucaparib (Fig. In summary, this study provides a pre-clinical rationale for therapeutic targeting of PARP1 in SF-mutant leukemia. Moreover, PARP and ATR inhibitor combination could emerge as a new therapeutic strategy in this genetically distinct disease subtype.

Strikingly, while RUNX1 deficiency was responsible for altered transcription in both single and double mutants, it also induced dramatic changes in global splicing, as seen with mutant SRSF2, and only their combination induced mis-splicing of genes selectively enriched in the DNA damage response and cell cycle checkpoint pathways. Collectively, these data reveal the convergent impact of a prototypic MDS-associated double mutant on RNA processing and suggest that aberrant DNA damage repair and cell cycle regulation critically contribute to MDS development.

CD34+ cells expressing SRSF2 P95H showed impaired neutrophil differentiation in response to G-CSF and was accompanied by increased levels of Class IV. Our findings suggest that SRSF2 P95H promotes Class IV splicing by binding to key ESE sequences in CSF3R exon 17, and that SRSF2, when mutated, contributes to dysgranulopoiesis.

We found that mutations associated with faster CH growth were also those associated with higher risk of progression to AML (adjustedR 2 =0.55, p=0.004) and were under the strongest selective pressure in AML and Myelodysplastic Syndromes (adjustedR 2 =0.19, p=0.002). Collectively, our findings characterize the lifelong natural history of CH and give fundamental insights into the interactions between somatic mutation, aging and clonal selection.

Surprisingly, we found that Srsf2 P95H/+ cells are more sensitive to five inhibitors targeting ADP-ribosyltransferases or PARP (olaparib, talazoparib, rucaparib, niraparib, veliparib) ( Figs 1A-B ). Collectively, these data provide a pre-clinical rationale that splicing factor mutant leukemias are preferentially sensitive to PARP1 modulation compared to their wildtype counterpart. Moreover, combining PARPi and ATRi may further sensitize spliceosome mutant cells and could represent a new therapeutic strategy in myeloid leukemia patients harboring these mutations ( Fig 1L ).

Importantly, the variant allele frequency (VAF) levels of SRSF2 mutations in remission at HSCT did not correlate with outcomes following HSCT consolidation, limiting the applicability of SRSF2 mutations as a marker for residual AML disease. Following allogeneic HSCT SRSF2 mutated AML patients experienced a 2-year overall survival of 77%, indicating that SRSF2 mutated AML patients may benefit from HSCT consolidation.

Here, we present a CRISPR-Cas9 -based in-vitro model for the investigation of R-loops as a novel therapeutic target in splicing factor mutated MDS. Furthermore, preliminary results confirm the sensitivity of splicing factor-mutated cells to ATR-inhibition. Immunostaining of R-loops and functional assays with AZD6738 and combination drugs including more edited cell lines with other splicing factor mutations are currently ongoing.

Following allogeneic HSCT SRSF2 mut AML pts did not have dismal outcomes with a 77% 2-year OS, indicating that SRSF2 mut AML pts may benefit from HSCT consolidation. Importantly, the VAF levels of SRSF2 mutations in remission at HSCT did not correlate with outcomes following HSCT consolidation, limiting the applicability of SRSF2 mutations as MRD marker in AML.

Based on the loss of guide RNAs targeting Cdk6, we identified that Palbociclib, a CDK6 inhibitor, showed preferential sensitivity in Srsf2P95H/+ cell lines and in primary non-immortalized lin-cKIT+Sca-1+ cells compared to wild type controls. Our data strongly suggest that the cell cycle and DNA damage response pathways are required for Srsf2P95H/+ cell survival, and that Palbociclib could be an alternative therapeutic option for targeting SRSF2 mutant cancers.

The patient did not use a JAK-STAT pathway inhibitor (ruxolitinib) but started on hydroxyurea and alpha-interferon and developed pruritus after 4 months of diagnosis and nasal hemorrhage 1 month later. We repeated NGS and found that three additional mutations were detected: ASXL1, PRKDC, MYOM2; variant allele frequency (VAF) of the prior mutations in CSF3R, SETBP1, and SRSF2 increased. The concurrence of CSF3RT618I, ASXL1, SETBP1, and SRSF2 mutation may be a mutationally detrimental combination and contribute to disease progression and AML transformation, as well as the nonspecific treatment of hydroxyurea and alpha-interferon, but the significance and role of PRKDC and MYOM2 mutations were not undetermined.

We conclude that splicing factor mutations do not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes.

This can be enhanced by combination treatment with low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association of R-loops and ATR sensitivity with the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.