Optimizing Mechanisms for Induction of Immunogenic Cell Death to Improve Patient Outcome in Multiple Myeloma
Authors: Gulla Annamaria , Morelli Eugenio , Hideshima Teru , Samur Mehmet Kemal , Bianchi Giada , Fulciniti Mariateresa , Munshi Nikhil , Anderson Kenneth C. 
Published: October 2019

Abstract
Multiple myeloma (MM) is an incurable clonal plasma cell malignancy with a remarkably complex and heterogeneous genome. Restoration of host anti-MM immunosurveillance may lead to long-term benefit, even in patient subgroups with high-risk cytogenetics and poor survival. One mechanism is via the induction of immunogenic cell death (ICD) whereby the release of endogenous danger signals (DAMPs) from dying cancer cells stimulates a potent immune-response against cancer cells via T-cell priming by dendritic cells (DCs). Recent data reported that bortezomib (BTZ) is able to induce ICD; however, little is known about the molecular mechanisms underlying ICD induction in MM, as well as genetic alterations that may impair ICD-induction by BTZ, particularly in MM patients with high risk disease.
First we validated the occurrence of ICD after BTZ treatment in MM. Among several DAMPs, the exposure of endoplasmic reticulum (ER) protein calreticulin (CALR) ("eat-me" signal) has been shown to define the immunogenicity of cancer cells, and our western blot (WB) analysis confirmed CALR expression in a panel of MM cell lines (n=9). We next examined effects on CALR triggered by BTZ (1-10 nM) and Melphalan (2-4 uM) treatment of human H929 and AMO1 MM cells. As expected, in both cell lines we found a correlation between induction of apoptosis, as measured by Annexin V/7AAD staining, with the translocation of CALR to the cell membrane. CALR translocation is due to induction of an ER stress response, leading to activation of Unfolded Protein Response (UPR) via induction of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway. Consistently, we found that BTZ and Melphalan induce phosphorylation of the translation initiation factor eIF2-α (p-eIF2-α), along with an increase of Activating Transcription Factor 4 (ATF4) and CCAAT-enhancer-binding protein homologous protein (CHOP) proteins. Next we assessed whether membrane-translocated CALR had a functional effect as an "eat me signal" for DCs cells. AMO1 cells, either pre-treated with BTZ or Melphalan or untreated, were incubated with immature monocyte-derived DCs (iMo-DCs) and two different in vitro experiments were performed: 1) flow cytometric analysis after 24h co-culture showed an increase of maturation markers CD83 and CD86 on Mo-DCs surface only in AMO1 cells pre-treated with BTZ; 2) phagocytosis assay by flow cytometric analysis after 4 hours co-culture showed that only BTZ pre-treated AMO1 cells were phagocytosed by Mo-DCs, as compared to untreated or Melphalan pre-treated cells. These findings confirmed that Bortezomib was able to induce ICD in vitro, and that CALR exposure does not represent a valid screening method to identify new potential ICD-inducers in MM. To assess potential clinical relevance of ICD, we identified genetic abnormalities that negatively affect overall survival (OS) after BTZ treatment and correlated these alterations with their impact on induction of ICD by BTZ in vitro. We analyzed RNA-seq data from newly-diagnosed patients (n=400) and identified a list of differentially expressed genes among patients with longer survival rate (>5 years) as compared to those with poor survival (<1.5 years) after BTZ-based treatment. Gene set enrichment analysis (GSEA) revealed that pathways which positively regulate immune and inflammatory responses were enriched in patients with longest OS as well as genes located on chr17p13. KMS11 cell line carries del (17p13), and our in vitro experiments showed that BTZ treatment did not lead to an increase of CALR exposure in KMS11 cells, even when inducing apoptosis. Likewise, flow cytometric analysis after 24h co-culture of BTZ-treated KMS11 and iMoDCs did not show an increase of maturation markers CD83 and CD86 on iMo-DCs surface, nor was there increased phagocytosis of KMS11 by MO-DCs after 4 hours co-culture. Importantly these results indicate an impairment of ICD induction in MM cells bearing del (17p).
In conclusions, our results identify a novel role of genes located on chr17p13 in MM in the induction of ICD; and conversely, that poor OS in patients with chr17p13 MM may be due, at least in part, to the lack of ICD. Importantly, our studies suggest combination strategies to restore ICD by BTZ to improve outcome even in the subgroup with high risk disease.

Disclosures
Munshi: OncoPep: Other: Board of director. Anderson:Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Takeda Millennium: Consultancy.