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Mechanistic Insights And Target Discovery For SRSF2 Mutations In Acute Myeloid Leukemia

Posted on:2024-05-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:M H SuFull Text:PDF
GTID:1524306938957519Subject:Internal Medicine
Abstract/Summary:PDF Full Text Request
Aims:SRSF2 mutation is a common splicing mutation in acute myeloid leukemia(AML)and myelodysplastic syndrome(MDS).This mutation is also one of the agerelated clonal hematopoiesis(ARCH)mutations,and carrying it greatly increases the risk of developing leukemia.Currently,there is still a lack of effective targeted therapies for splicing factor mutations in clinical practice.Therefore,we propose that the next key step towards preventing and treating related AML/MDS is to find a series of safe interventions and treatments that target hematopoietic stem cells carrying the SRSF2 mutation.Currently,most studies focus on the mechanism by which SRSF2 mutations create abnormal mRNA splicing changes that possibly lead to hematologic malignancies.However,we still do not fully understand the precise mechanism by which SRSF2 mutations confer clonal advantage to mutated hematopoietic stem progenitor cells,as well as other related pathogenic mechanisms and phenotypes.Therefore,in addition to seeking appropriate mutation-specific drugs,we also hope to discover unknown pathogenic mechanisms or phenotypes of SRSF2 mutations by searching for vulnerable therapeutic targets for SRSF2 mutations.Methods:We used the CRISPR/Cas9 system to generate several SRSF2 mutation cell lines(OCI-AML2,MOLM-14,K562,OCI-AML3,MARIMO)and screened 3,988 compounds with high-throughput drug screening using cell viability as the endpoint.RKI-1447,a Rho/ROCK pathway kinase inhibitor,was identified as a lead compound based on the in vitro screening results.We also validated the compound in vitro using iPSC models and in vivo using PDX models carrying SRSF2 mutations.To understand the underlying mechanisms and explore the possible unknown new pathogenic mechanisms and phenotypes of SRSF2 mutations,we analyzed the proteomics,transcriptomics,and phosphorylation proteomics of RKI-1447-treated and control-treated cells.Then,we used confocal microscopy and transmission electron microscopy(TEM)to observe and analyze the cell cytoskeleton(actin,microtubules),nuclear morphology of SRSF2 mutant cells and the effects of RKI-1447 treatment.We used atomic force microscopy(AFM)to study changes in the mechanical properties of mutant cells and the effects of RKI-1447 treatment on cell mechanical properties.Results and conclusions:We found that the ROCK inhibitor RKI-1447 can specifically inhibit SRSF2 mutant cells,and its effectiveness was validated in vitro using iPSC models.Through PDX models,we found that RKI-1447 can specifically and effectively inhibit the engraftment of pre-leukemic HSPCs and leukemic HSPCs with SRSF2 mutations in vivo.Through proteomic and transcriptomic analyses,as well as cell cycle assays,we found that cells treated with RKI-1447 produced G2/M arrest.Mutant cells treated with RKI-1447 showed significant increased of abnormal multipolar spindles and multi-nucleated cells,as well as cell apoptosis.Confocal imaging showed that the cytoskeleton of SRSF2 mutant cells differed from that of wild-type cells:more pronounced nuclear F-actin,with a more irregular cell cortical actin morphology;Mutant cells exhibited a "looser" physical mechanical state after drug treatment.The microtubule structure of mutant cells was more pronounced,and after drug treatment,the microtubule structure became even more pronounced and chaotic.Through confocal microscopy and TEM,we found that SRSF2 mutant cells produced more indentation/segmentation of cell nuclei,and the nuclear lamina on both ends of the cell nucleus often connected together to divide the cell nucleus into lobes.Compared to wild-type cells,the nuclear morphology of mutant cells was more irregular,and cell nuclear sphericity were significantly decreased after treatment with RKI-1447.We also observed "intrusion" of segmented nuclear cytoplasm into the cell nucleus,with a large number of microtubules enriched at the tip of the intruding nuclear membrane in the cytoplasm.After exposure to RKI-1447,the abnormal cell skeleton and nuclear morphology of SRSF2 mutant cells were enhanced to a level incompatible with cell survival.Consistent with the phenotype of the cytoskeleton,we found through AFM that mutant cells and cells treated with RKI-1447 were significantly softer.Through proteomic and phosphorylation proteomic analysis,we found differences in the phosphorylation levels of many microtubule-related proteins between mutant cells and wild-type cells,and their levels changed after treatment with RKI-1447.One of the most important mechanical structural proteins and physiological functional proteins in the nuclear lamina,lamin A,underwent specific dephosphorylation in mutant cells after treatment with RKI-1447.Phosphorylation-mimicking of lamin A can rescue the cell viability level of cells after treatment with RKI-1447.These findings suggest that by using RKI-1447 and possibly other cytoskeleton modifiers to target the abnormal cytoskeleton caused by SRSF2 mutations,opening up new avenues for the treatment of AML with this mutation.Our results also reveal a new phenotype of abnormal nuclear and cytoskeletal morphology and changes in cell Young’s modulus associated with SRSF2 mutations.Further research is needed to explore the potential of ROCK inhibitors and other cytoskeleton modifiers in the treatment of SRSF2 mutant AML.
Keywords/Search Tags:SRSF2, cytoskeleton, ROCK, nuclear morphology, cell cycle
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