Abstract A019: Role of MTI-101 in disrupting calcium homeostasis leading to cell death in multiple myeloma

Abstract

Abstract Introduction: Multiple Myeloma (MM) is a hematologic cancer characterized by neoplastic proliferation of the plasma cells in the bone marrow. Newly diagnosed MM patients typically respond to currently available treatments. However, the disease is not curable and unfortunately all patients eventually will relapse with drug resistant disease. Clinical data continues to indicate that novel treatment strategies are required to improve patient outcomes. Our group has developed a novel agent cyclic peptide called MTI-101 for the treatment of MM. MTI-101 was optimized form the D-amino acid linear peptide HYD-1 (1). HYD-1 and the more potent MTI-101 were discovered to induce necroptotic cell death by disrupting Ca2+ homeostasis in MM cell lines. Our laboratory previously reported that acquisition towards resistance to MTI-101 correlated with repression of genes predicted to activate SOCE including decreased PLCβ, SERCA, ITPR3, and TRPC1 expression (2). Moreover, our data showed that pharmacological inhibition of SOCE or reducing the expression of TRPC1 blocks MTI-101 induced cell death (2). Activation of SOCE is dependent on STIM1-ORAI1 recruitment to endoplasmic reticulum- plasma membrane junction (ER-PM junction), allowing an oscillatory Ca2+ flux via activation of ICRAC (Calcium Release Activated Calcium channel) (3). Kaczmarek et al showed that TRPC5 (TRPC1 heterodimer) activity can be positively regulated by calpain truncation (4). In this study, we sought to determine the role of TRPC1 in MTI-101 Ca2+ induced necrosis, as we hypothesize that MTI-101 induced prolonged SOCE activation is due to TRPC1 truncation by calpains activation, leading to disruption of Ca2+ homeostasis and cell death Methods: To evaluate determinants of activity of MTI-101, we developed an assay to measure intracellular Ca2+ levels and cell death by using the Ca2+ indicator Fluo-4 AM and DAPI as a marker for cell death. Further, to evaluate the role of MTI-101 on TRPC1 activation, MTI-101 treated cells were lysed using RIPA detergent, and the detergent resistant component was lysed using RIPA and 2% SDS, followed by sonication with an ultrasonic homogenizer. Both compartments were analyzed via western blot. Results: Our data showed that MTI-101-induced robust, delayed calcium flux is causative for cell death due to irreversible activation of SOCE, while thapsigargin causes rapid, transient, reversible activation. Further, the data indicated that MTI-101 treatment induces TRPC1 truncation and translocation to a detergent-resistant compartment of the cell. Moreover, inhibiting the Ca2+ activated proteases calpain1 and calpain2 abrogated MTI-101 induced Ca2+ flux and cell death, and interferes with TRPC1 truncation following MTI-101 treatment in a time dependent manner. Conclusions: MTI-101 induces a robust, and sustained activation of SOCE causative for cell death. Increased intracellular Ca2+ levels following MTI-101 treatment induces calpains activation, furthering TRPC1 truncation and activation leading to necroptotic cell death. Our future studies will focus on evaluating the role of mutants TRPC1 on MTI-101 activity, identify calpains consensus sites on TRPC1, and determine the role of ORAI1 in MTI-101 Ca2+ induced cell death. Citation Format: Osama M Elzamzamy, Karen Hayes, Werner Geldenhuys, Lori A Hazlehurst. Role of MTI-101 in disrupting calcium homeostasis leading to cell death in multiple myeloma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A019. doi:10.1158/1535-7163.TARG-19-A019