Activation of Lysosomal Function and Reactive Oxygen Species Play Crucial Roles in SAR650984-Induced Direct Killing of Human Multiple Myeloma Cells with Mutated p53, Which Is Further Augmented By Pomalidomide
Authors: Jiang Hua , Acharya Chirag , Gang An Gang An , Feng Xiaoyan , Song Zhili , Yang Guang , Adrian Francisco , Qiu Lugui , Richardson Paul , Munshi Nikhil C. , Tai Yu-Tzu , Anderson Kenneth C 
Published: October 2019

Abstract
The anti-CD38 monoclonal antibody (mAb) SAR650984 (SAR) is showing promising clinical activities in ongoing trials in relapsed/refractory multiple myeloma (MM), a setting in which p53 is often mutated. Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define additional molecular mechanisms of SAR-directed MM cell death, and further delineate enhanced anti-MM activity triggered by combining SAR with pomalidomide (Pom) or lenalidomide (Len). Since CD38 expression on MM cell lines is not as high as on primary patient MM cells, we first used lentivirus to overexpress CD38 in 3 MM cell lines with lower CD38 levels and p53 mutations. Without Fc-cross-linking agents or effector cells, SAR, but not other anti-CD38 mAb, induced homotypic aggregation (HA)-associated MM cell death in these cell lines, which was dependent on CD38 level, membrane lipid raft, and actin cytoskeleton. SAR and its F(ab)'2 fragments triggered comparable caspase 3/7 activation in CD38-highly expressing MM cells, indicating that SAR-induced caspase-dependent apoptosis is both CD38-specific and Fc-independent. Most importantly, SAR and its F(ab)'2 fragments induce lysosome-dependent cell death (LCD) in CD38 overexpressing MM cells even with p53 mutations, evidenced by enlarged lysosomes and increased lysosomal membrane permeabilization (LMP) along with leakage of cathepsin B and lysosome associated membrane protein 1 (LAMP1), in the presence or absence of IL-6 or bone marrow stromal cells. SAR significantly induced mobilization of cathepsin B and LAMP1 from lysosomes to the cytosol, particularly at the intercellular junctions, further confirming that lysosomes relocalize to sites of cell adhesion following SAR treatment. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR stimulated reactive oxygen species (ROS), whereas the antioxidant N-acetylcysteine (NAC) blocks SAR-induced ROS and MM cell death. Since NAC does not inhibit HA or lysosome enlargement or LMP, SAR-induced ROS occurs downstream of HA and activation of lysosomal function. SAR does not induce autophagy, indicating that autophagy is not required for this unique mode of cell death. Finally Pom, more potently than Len, enhances SAR-induced direct and indirect killing of MM cells, even resistant to Pom/Len. Moreover, combined treatment of MM cells with SAR and Pom augments caspase 3/7 activity induced by either agent alone. Taken together, these data show that SAR is the first naked therapeutic mAb with convincing direct killing of MM cell lines and patient MM cells, including those with p53 mutations and drug resistance, mainly via lysosome activation and ROS. Coupled with augmented both caspase 3/7-dependent and effector-mediated MM cytotoxicity when combined with Pom, our data further provide the framework for combination clinical trials.

Disclosures
Song: Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.