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.