Scaffolding-dependent CASP1 constrains excessive cell-intrinsic inflammatory signaling in leukemia [THP1]

The inflammatory cytokine Interleukin-1b (IL-1b is implicated in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). IL-1b processing is regulated by Caspase-1 (CASP1), a protease that is initially expressed as an inactive zymogen (Pro-CASP1) and can be cleaved into its active form. CASP1 cleaves both pro-IL-1b and Gasdermin-D (GSDMD), which mediates IL-1b secretion and consequently pyroptotic cell death. Despite its known role in inflammation, the contribution of CASP1 to leukemogenesis has not been adequately investigated. To address this, we generated Pro-CASP1 knockout (CASP1KO) MDS/AML cell lines and patient-derived samples. As expected, CASP1KO cells were unable to induce pyroptosis or produce IL-1b. Surprisingly, however, these cells also exhibited impaired leukemic cell expansion, increased myeloid differentiation, and significantly reduced leukemic burden when xenografted into mice. Similarly, Casp1-/- pre-leukemic and AML mouse cells were impaired in their clonogenic potential and leukemia development. Notably, restoring IL-1b expression did not rescue the growth defects, suggesting that IL-1b is not the primary driver of CASP1's role in MDS/AML. Mutation or inhibition of the CASP1 protease activity did not impact MDS/AML cells, indicating that CASP1's pro-leukemic function is independent of its proteolytic activity. Transcriptomic and functional analyses of CASP1KO cells revealed excessive NF-kB and inflammatory signaling. Suppression of NF-kB activity restored leukemic cell function and viability, indicating that CASP1 acts as a negative regulator of NF-kB activation. We performed a proximity proteomic and computational prediction analysis and identified RPTOR, a component of the mTORC1 complex, as a key interactor of CASP1. Knockdown of RPTOR in CASP1KO cells reduced excessive NF-kB activity and rescued the leukemic growth defect, suggesting that the CASP1–RPTOR interaction regulates NF-kB signaling in leukemia. To target the scaffolding function of CASP1, we developed a PROTAC degrader (dCASP1-55). dCASP1-55 induced NF-kB activation and reduced the colony-forming potential of MDS/AML cells, supporting its potential as a targeted therapy. CASP1 functions as a critical organizational center regulating NF-kB signaling through mTORC1 in MDS/AML, while IL-1b expression is a secondary contributing event. Overall design: Bulk RNAseq profiling of THP1-Cas9 cells transduced with Lentiviral delivery vectors specific for safe harbor control gene sgAAVS1 (WT), or sgCASP1 (KO), grown in liquid culture, and selected for by cell sorting, and validated by western blot.