Targeting Autophagy to Overcome Resistance to Immunogenic Chemotherapy in High-Risk Multiple Myeloma

Despite the remarkable clinical success of chemo- and immunotherapies in multiple myeloma (MM), patients with high-risk (HR) disease still have a dismal prognosis. However, the poor clinical response of HR-MM patients is not always due to a specific resistance to drug-induced cytotoxicity, and the biological mechanisms underlying a poor outcome are still not completely understood. Bortezomib (BTZ) can induce immunogenic cell death (ICD), which stimulates anti-cancer immunity and improves clinical response. To investigate whether the immunogenicity of cell death impacts the poor clinical response of HR-MM patients, we investigated 361 newly diagnosed and clinically annotated MM patients with HR disease, including t(4;14), del(17p), del(1p) and gain(1q) (IFM/DFCI 2009). We found that the expression of the previously identified ICD signature was positively correlated with overall survival (p<0.05). Moreover, in HR patients with high ICD expression, there was an enrichment of pathways related to the activation of the anti-tumor immune response. Thus, ICD signature can further stratify HR patients into risk categories, and our results suggest that cytogenetic abnormalities within tumor cells may alter immunogenicity of cell death and contribute to tumor immune escape. Cell-surface exposure of calreticulin (CALR) by tumor cells is an obligate step in the ICD process, as it drives their phagocytosis by dendritic cells (DCs). Therefore, we reasoned that the genomic alterations of HR patients may mediate resistance to ICD by specifically altering CALR exposure. By overlapping genes that were differentially modulated in HR patients with proteins that interact with CALR after BTZ treatment, as assessed by Co-IP, we nominated GABA Type A Receptor-Associated Protein (GABARAP) as a novel regulator of ICD. In a publicly available dataset of scRNAseq of MM patients (n=12) and normal donors (n=5), we found a positive correlation between GABARAP and ICD expression. Importantly, the GABARAP gene locus is on chr17p13.1, and loss of chromosome 17p is the most important prognostic HR factor. Low levels of GABARAP were associated with inferior clinical outcome in MM patients (EFS, p=0.00067; IFM/DFCI 2009). We found a strong correlation between GABARAP protein levels and the intensity of CALR exposure after BTZ treatment in 10 cell lines (R2: 0.5; p=0.01). Overexpressing GABARAP in a GABARAPLOW cell line restored CALR exposure. Moreover, GABARAPKO in 3 ICD-sensitive cell lines abrogated the induction of ICD by BTZ, and add-back experiments using pre-treatment with recombinant CALR or GABARAP overexpression in KO clones restored ICD. In vitro, CyTOF confirmed that treating GABARAPKO cells with BTZ failed to activate an efficient T cell response. In vivo, BTZ failed to induce ICD and tumor regression in immunocompetent mice bearing GABARAPKO tumors. By analyzing the CALR interactome in GABARAPKO cells, we found that CALR is trapped in the endoplasmic reticulum and mitochondria after ICD, which prevents its exposure. Neither an ER stressor nor inhibitor of the PP1/GADD34 complex were able to restore CALR exposure in GABARAPKO cell lines (n=2). Interestingly, transmission electron microscopy showed that GABARAP loss rendered MM cells autophagy-deficient, as confirmed by basal downregulation of the LC3B protein in 3 GABARAPKO cell lines, which is consistent with GABARAP's role as an autophagy regulator and vesicular transporter. Interestingly, combining BTZ with autophagy inducers, including rapamycin and venetoclax, restored vesicular transport of CALR to the cell surface and MM phagocytosis by DCs. Altogether, our work identifies a unique mechanism of immune escape that may contribute to the poor clinical outcome observed in del17p HR-MM patients. It further suggests that combining an ICD inducer BTZ with an autophagy inducer, such as venetoclax, in this subset of patients may restore ICD and improve the outcome of HR-MM patients.