Session: 636. Myelodysplastic Syndromes: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research
CASP1 expression is significantly higher in MDS and MDS/MPN secondary AML patients compared to healthy hematopoietic controls, and elevated CASP1 expression correlates with shorter overall survival in MDS. To investigate CASP1's role in MDS, we generated Pro-CASP1 knockout (Pro-CASP1 KO) human isogenic cell lines in MDSL and in THP1 using CRISPR-Cas9. Unexpectedly, Pro-CASP1 KO cells exhibited a marked reduction in liquid culture growth and colony-forming potential in methylcellulose, which corresponded with elevated expression of myeloid cell activation markers and reduced expression of the immature HSPC markers compared to isogenic wild-type cells. Moreover, transplantation of Pro-CASP1 KO MDSL cells into immunocompromised NSGS mice resulted in significantly reduced leukemic cell burden and extended overall survival. These findings suggest that Pro-CASP1 is essential for maintaining the undifferentiated state of pre-leukemic HSPCs.
To delineate which scaffolding domains of Pro-CASP1 are essential for MDS cells, we performed an in vitro tiled CRISPR dropout screen of Pro-CASP1. Domain footprint mapping implicated the N-terminal CARD domain of Pro-CASP1, but not the proteolytic domain, as being essential in MDSL and THP1 cells. Isogenic cells expressing Pro-CASP1 lacking the CARD domain (CASP1-ΔCARD) failed to rescue the progenitor growth defects observed in Pro-CASP1 KO MDS/AML cells. These results indicate that Pro-CASP1's proteolytic function is dispensable, whereas the N-terminal CARD domain, a scaffolding region, is crucial for the clonal properties of MDS/AML cells.
To understand the molecular basis for Pro-CASP1 dependency in MDS/AML, we performed a transcriptomic analysis in isogenic Pro-CASP1 KO and WT MDSL and THP1 cells. Unexpectedly, loss of Pro-CASP1 led to significant upregulation of NF-κB target genes. Moreover, Pro-CASP1-deficient MDS/AML cell lines exhibited nuclear translocation and phosphorylation of p65/RelA, and constitutive activation of NF-κB-target genes. In contrast, suppressing NF-κB activity through expression of the IkBα super-repressor was sufficient to reverse the leukemic progenitor defect and restore cell viability in Pro-CASP1 KO cells, suggesting that Pro-CASP1 is necessary for fine-tuning canonical NF-κB activation. Excessive NF-κB activity in Pro-CASP1 KO cells was restored to baseline levels by both full-length and enzymatically dead mutants, but not by a Pro-CASP1 scaffolding or CARD domain mutants, indicating a specific dependency on the zymogen, scaffolding-competent form of Pro-CASP1. In summary, Pro-CASP1, independent of its catalytic function, serves as a critical signaling hub, restricting excessive NF-κB activation in leukemic cells. Loss of Pro-CASP1 is a novel strategy to selectively target the clonogenic potential of leukemic cells, such as in MDS.
Disclosures: Uible: JucaBio: Consultancy. Bolanos: Kurome: Consultancy, Research Funding. Starczynowski: Curis: Honoraria; Tolero: Research Funding; Kurome: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Kymera: Consultancy; Treeline Biosciences: Research Funding.
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