Abstract P24: GABARAP deficiency drives resistance to immunogenic chemotherapy in multiple myeloma
Authors: Turi Marcello , Morelli Eugenio , Johnstone Megan , Samur Mehmet K , Botta Cirino , Cifric Selma , Folino Pietro , Vinaixa Delaney , Barello Francesca , Clericuzio Cole , Favasuli Vanessa K , Maisano Domenico , Talluri Srikanth , Prabhala Rao , Bianchi Giada , Fulciniti Mariateresa , Wen Kenneth , Kurata Keiji , Liu Jiye , Penailillo Johany , Bragoni Alberto , Sapino Anna , Richardson Paul G , Chauhan Dharminder , Carrasco Ruben D , Hideshima Teru , Munshi Nikhil C , Anderson Kenneth C , Gulla Annamaria 
Published: March 2024

Abstract
In multiple myeloma (MM), the proteasome inhibitor bortezomib (BTZ) triggers immunogenic cell death (ICD), a critical mechanism by which the drug stimulates an anti-MM immune response. During ICD, the dying cancer cell releases danger signals like calreticulin (CRT), which attract dendritic cells (DCs) to engulf the dying cell and present its antigens to T cells. Cancer cells may employ several strategies to restrain the immunogenic outcome, including interfering with the exposure of CRT on the cell surface. Such resistance mechanisms hinder immune activation and might be responsible for the relapse and poor clinical outcomes observed in MM patients. To uncover resistance mechanisms, we integrated the CRT interactome after ICD induction with transcriptomic data from the IFM/DFCI dataset (n=360) and found that GABARAP interacts with CRT and its lower expression was associated with worse clinical outcomes (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. This mechanism was also confirmed in patients, by the single-cell RNA-seq analysis of 80 MM patients in which we 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, GABARAP gene locus is on chr17p13.1, therefore, while this mechanism is general for all MM cells, it appears especially relevant for those carrying a high-risk deletion of the 17p chromosome. 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 in GABARAP low MM cells and their phagocytosis by DCs. We propose a model by which low levels of GABARAP compromise the vesicular transport of CRT exposure. This impairment results in the inhibition of tumor immunogenicity and the overall outcome of ICD. Consequently, combining an ICD-inducing agent, like bortezomib, with an autophagy inducer, like rapamycin, may improve patient outcomes in MM, where del(17p) is common and leads to unfavorable outcomes.
Citation Format: Marcello Turi, Eugenio Morelli, Megan Johnstone, Mehmet K Samur, Cirino Botta, Selma Cifric, Pietro Folino, Delaney Vinaixa, Francesca Barello, Cole Clericuzio, Vanessa K Favasuli, Domenico Maisano, Srikanth Talluri, Rao Prabhala, Giada Bianchi, Mariateresa Fulciniti, Kenneth Wen, Keiji Kurata, Jiye Liu, Johany Penailillo, Alberto Bragoni, Anna Sapino, Paul G Richardson, Dharminder Chauhan, Ruben D Carrasco, Teru Hideshima, Nikhil C Munshi, Kenneth C Anderson, Annamaria Gulla. GABARAP deficiency drives resistance to immunogenic chemotherapy in multiple myeloma [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P24.