OAB-033: Loss-of-function of GABARAP drives tumor resistance to bortezomib-induced immunogenic cell death in multiple myeloma

Abstract

<h3>Background</h3> Resistance to immune approaches poses a major challenge to effective immunotherapy and long-term clinical outcome in multiple myeloma (MM). Here we identified loss-of-function of gamma-aminobutyric acid receptor-associated protein (GABARAP) as a tumor-intrinsic mechanism of resistance to bortezomib (BTZ)-induced immunogenic cell death (ICD), the immunogenic consequence of apoptosis resulting in specific anti-MM immunity via T-cell priming by dendritic cells (DCs). <h3>Methods</h3> We found that BTZ induces ICD in human and murine MM cell lines that is dependent on the exposure of calreticulin (CALR), which drives DCs-mediated phagocytosis of MM cells. DCs-phagocytosis is followed by a specific T cell activation, with a significant increase of CD4+ effector memory (EM), total CD8+, CD8+ EM, and CD8+ terminally differentiated EM cells. Isolated T cells after co-cultures showed the presence of MM specific CTLs that were able to efficiently induce lysis of MM cells. Our results were validated using primary cells from MM patients. Moreover, induction of a protective immune response was confirmed in vivo. Specifically, treatment of 5TGM1 tumors with BTZ induced a tumor regression in a syngeneic model; and injection of live 5TGM1WT two weeks after regression did not result in tumor development, consistent with induction of immunological memory as confirmed by ELISPOT of mouse splenocytes. We identified a specific ICD signature induced by BTZ in mice; and we found that increased expression of the human orthologs of this signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Notably, these functional immunologic sequelae were abrogated after BTZ treatment of CALRKO MM cells both in vitro and in vivo, confirming the obligate role of CALR exposure in the ICD process. <h3>Results</h3> By interrogating the IFM/DFCI dataset and focusing on genes involved in ICD processes that were correlated with MM patients clinical outcome, we found that low levels of GABARAP (chr17p13.1), an autophagy regulator and putative CALR binding partner, negatively impact MM patient clinical outcome (EFS, p=0.0032); even excluding HR patients with 17p deletion (EFS, p=0.018). Interestingly, KMS11 cells carrying monoallelic deletion of GABARAP were resistant to induction of ICD by BTZ; and sensitivity was restored after overexpression of the gene. Moreover, GABARAPKO in 3 ICD-sensitive cell lines abrogated the induction of ICD by BTZ; and add-back experiments by pre-treatment with recombinant CALR or GABARAP overexpression in KO clones restored ICD. Finally, CyTOF confirmed that treatment of GABARAPKO cells with BTZ failed to activate an efficient T cell response. <h3>Conclusions</h3> our study demonstrates that loss-of-function of GABARAP, particularly in HR patients with 17p deletion, contributes to tumor immune evasion and ICD resistance. These studies provide the framework for novel combination treatments to restore anti-MM immunity and improve patient outcome in HR MM.