Background We recently discovered recurrent cathepsin S (CTSS) alterations, i.e., either somatic gain-of-function mutations (at the hotspot Y132) or amplification/overexpression of CTSS that result in aberrant hyperactivity of this lysosomal cysteine protease and promote tumor growth in follicular lymphoma (FL). In addition, CTSS is frequently overexpressed in other lymphomas and other cancers (e.g., Glioma, triple-negative breast cancer, renal cancer). Intriguingly, when released from the lysosome, CTSS is tightly linked with the regulation of cell death pathways.

Materials and Methods BH3 profiling allows determining, to what extent a cell is primed towards cell death. It measures the extent of mitochondrial membrane permeabilization (MOMP) in response to BH3 peptides. Thus, BH3 profiling can record the net response of the pro- and anti-apoptotic interactions of the >15 apoptotic BCL2 family members in high-throughput (384-well plate flow-cytometry). Using BH3 profiling, we aimed to exploit the therapeutic vulnerabilities of CTSS hyperactive tumors, with a focus on compounds that induce lysosomal membrane permeabilization (LMP).

Results Our preliminary data suggest that LMP is a promising novel therapeutic strategy in tumors with CTSS hyperactivity. Specifically, dose-response curves for cell viability indicated a higher sensitivity to LLOMe treatment in DG75 CTSS knock-out (CTSS^KO) lymphoma cells with re-expression of CTSS WT (KO+CTSS^WT), and highest sensitivity in DG75 with overexpression of CTSS Y132D (KO+CTSS^Y132D). Similar results were obtained in single-cell derived clones from Karpas422 lymphoma cells (CTSS KO vs WT vs Y132D). Release of cathepsins into the cytosol can lead to MOMP and apoptosis through the cleavage of BCL2 family members, including proteolytic inactivation of anti-apoptotic MCL1 and cleavage of BID which produces proapoptotic truncated BID (tBID). Of note, CTSS is the only cathepsin that remains enzymatically active at neutral pH for hours. Western blot confirmed increased cleavage of BID, MCL-1 and PARP-1 in LLOMe treated CTSS^WT and CTSS^Y132D-cells, indicating that CTSS release by LMP can more efficiently induce apoptosis in CTSS hyperactive lymphomas. BH3 profiling confirmed that CTSS hyperactivity is associated with a dependency on MCL-1 and BCL-2 following LMP. In addition, CTSS hyperactive glioblastoma cell lines U-87 and U-251 also showed MCL-1 dependency following LMP, indicating that cytosolic CTSS hyperactivity primes for MCL-1 dependency across many cancer types.

Conclusions Releasing CTSS from the lysosome is novel, attractive concept that can be combined with conventional, apoptosis-inducing therapies.

Disclosure Information J. Hildebrand: None. P. Rengasayee: None. M. Antoniolli: None. L. Hackett: None. L. Adolph: None. S. Häbe: None. M. Heide: None. S. Wilhelm: None. C. Ludwig: None. C. Strobl: None. V. Passerini: None. M. Bergwelt-Baildon: None. R. Koch: None. M. Davids: None. O. Weigert: None.