The Zymogen Form of Caspase-1 Is Required to Finetune Excessive Cell-Intrinsic Inflammation in Myelodysplastic Syndromes

Dysregulated NF-κB signaling is implicated in the expansion of clonally derived mutant Myelodysplastic syndromes (MDS) hematopoietic stem and progenitor cells (HSPCs). Inflammatory mediators have been shown to favor the expansion of mutant HSPCs while suppressing normal hematopoiesis. More recent findings have supported the premise that dysregulated innate immune and inflammatory pathways in mutant HSPCs do not result in increased pathway activation, but rather alter their response to the microenvironment, favoring the competitive advantage of the mutant cells. Activation of NF-κBthrough elevation of the inflammatory cytokine IL-1β in HSPCs has been linked to clonal expansion and ineffective hematopoiesis in MDS. This implicates Caspase-1, a cysteine-aspartic acid protease responsible for cleaving the precursors of IL-1β and Gasdermin D, inducing pyroptosis. CASP1 is typically expressed as a zymogen (Pro-CASP1) that can be cleaved into an active enzyme (CASP1). There is an urgent need for targeted therapies that restore dysregulated innate immune and inflammatory signaling via NF-kB in mutant HSPCs, such as for MDS. Here we present a novel and unexpected role for Pro-CASP1 in regulating NF-κB signaling independent of its proteolytic function. 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.