Group leader Candiolo Cancer Institute FPO-IRCCS, United States
Introduction: The induction of immunogenic cell death (ICD) is a critical mechanism by which the proteasome inhibitor bortezomib stimulates an anti-multiple myeloma (MM) immune response. During ICD, the dying cancer cell releases danger signals, such as calreticulin (CRT), which attract dendritic cells (DCs) to phagocytose the dying cell and present its antigens to T cells. Cancer cells employ several strategies to prevent this, including interfering with the exposure of CRT on the cell surface. Such resistance mechanisms may underlie the relapse and poor clinical outcome observed in MM patients.
Methods: To find resistance mechanisms, we overlapped the CRT interactome after ICD induction with transcriptomic data from the IFM/DFCI dataset (n=360) and found that GABARAP both interacted with CRT and was associated with worse clinical outcomes at low expression levels (p=0.0017). We observed a strong correlation between GABARAP protein levels and the intensity of CRT exposure after BTZ treatment in 10 MM cell lines (R2: 0.62). Consequently, GABARAP KO in 4 ICD-sensitive cell lines impaired the exposure of CRT, thus diminishing ICD, as assessed by MM cell phagocytosis by DCs and T cell activation both in vitro and in vivo in an immunocompetent model. ICD was restored by add-back experiments using recombinant CRT or GABARAP overexpression.
Results: To confirm this mechanism is active in patients, we analyzed single-cell RNA-seq from 80 MM patients and found a positive correlation between the expression of GABARAP and the ICD signature. Interestingly, low intratumor expression of GABARAP was associated with a lower T cell infiltration both at the single-cell level and by IHC analysis of bone marrow patient samples (n=10). Importantly, this mechanism generalizes to all MM cells but is especially relevant for those carrying a high-risk deletion of the 17p chromosome, where the gene is located.
Mechanistically, proteomic analysis, confocal and transmission electron microscopy showed that GABARAP loss significantly altered the morphology of the Golgi apparatus, causing alterations in the vesicular transport systems and autophagy. This effectively stopped CRT transport to the surface, thus hindering ICD. By combining BTZ with autophagy inducers, we could restore vesicular transport of CRT to the cell surface and the subsequent MM-cell phagocytosis by DCs. Importantly, we also found this mechanism to be relevant in other cancer types, such as lung cancer cells.
Conclusions: We propose a model by which dying cancer cells become resistant to ICD when the autophagy regulator GABARAP is low. The autophagy defects then disrupt the Golgi apparatus's structure, which then cannot export CRT to the cell surface. Without CRT, the dying cell cannot trigger an immune response. Therefore, coupling an ICD inducer, like bortezomib, with an autophagy inducer, like rapamycin, may improve patient outcomes in a variety of cancers and, specifically, in MM, where del(17p) is common and leads to worse outcomes.
