Overcoming MCL1 Resistance in Multiple Myeloma
Authors: Siu Ka Tat , Huang Cherrie , Panaroni Cristina , Mukaihara Kenta , Fulzele Keertik , Soucy Rosemary , Cidado Justin , Drew Lisa , Chattopadhyay Shrikanta , Raje Noopur 
Published: October 2019

Abstract
Disruption of the intrinsic apoptotic pathway by the aberrant expression of the BCL2 family members are frequent events in multiple myeloma (MM). The anti-apoptotic protein myeloid cell leukemia-1 (MCL1) is highly expressed in MM and plays a crucial role in disease progression. Inhibition of MCL1, thus, represents a unique therapeutic opportunity for the control of the disease. Currently, there is no FDA-approved drug with the ability to selectively target MCL1. Because of its pivotal role in MM, MCL1 is considered a high-value therapeutic target in the clinic.
In this report, we use a selective small-molecule inhibitor of MCL1, AZD5991, to examine the therapeutic consequences of MCL1 inhibition in MM. AZD5991 treatment resulted in dose-dependent cytotoxicity with EC50 values ranging from 64 and 417 nM at 24 hours for MCL1-sensitive cell lines (H929, MM.1S, RPMI-8226, U266, LP-1 and ANBL6VR). Two cell lines DOX40 and KMS-12-PE remained resistant to MCL1 inhibition. For the MCL1-sensitive MM.1S and H929 cells, the decrease in cell viability upon MCL1 inhibition was due to an increase in apoptosis. AZD5991 treatment led to 40-82% decrease in viability of CD138+ cells isolated from relapsed and refractory MM patients at a dose of 300 nM at 24 hours. These results indicate that AZD5991 has promising single-agent activity, but it would be prudent to study it in combination with other anti-MM therapies.
The BM microenvironment enhances tumor cell growth and survival in MM. We found that soluble factors produced during the MM-BMSC interaction reduced the sensitivity of MM cells to AZD5991, and direct MM-BMSC contact blunted the cytotoxic effect of AZD5991. A comprehensive cytokine array analysis revealed an enrichment of a panel of pro-survival cytokines and growth factors, with the cytokines IL-6, IL-8 and GROα/β/γ being among the most highly up-regulated proteins, upon cell-cell contact between MM.1S cells and BMSCs. Enrichment of these cytokines in the BM milieu, at least in part, confer a protective effect on MM cells and endow them with the ability to resist MCL1 inhibition.
A shift in the balance of BCL2 family members is often the primary reason for drug resistance. We found that the baseline BCL2 mRNA expression and the BCL2:MCL1 ratios in the MM cell lines examined are negatively correlated to their corresponding sensitivity to AZD5991. In other words, MM cells with a high BCL2 reservoir are more likely to circumvent cell death elicited by MCL1 inhibition. Treatment with AZD5991 alone leads to release of Bim from MCL1 and an increased Bim bound to BCL2. Venetoclax, a BH3 mimetic that selectively binds BCL2, treatment alone releases Bim from BCL2 and results in an increased Bim bound to MCL1. Bim binding to MCL1 and BCL2 was significantly diminished upon co-treatment. The free-floating Bim proteins subsequently activate the intrinsic apoptotic pathway by facilitating cytochrome c release. These results suggested that MM cells switch their survival dependency to BCL2 upon MCL1 inhibition, and that BCL2 blockade could be an effective way to overcome MCL1 resistance in MM.
Based on these observations, we combined AZD5991 with Venetoclax in MM cells. A significant decrease in cell viability was observed with the combined therapy compared with both drugs used alone. Isobologram analysis confirmed greater than additive or synergistic effect upon co-treatment. The enhanced cytotoxic effect of the combined therapy retains even when the MM cells are in co-culture with BMSCs. Remarkably, the pro-survival cytokines IL-6, IL-8 and GROα/β/γ, which were expressed at high levels in the co-culture settings, were among the ones being most reduced after the combined therapy.
Our results demonstrated that the combined AZD5991/Venetoclax therapy overcomes the inherent MCL1-resistance in MM via two independent mechanisms. First, the concomitant suppression of the anti-apoptotic proteins MCL1 and BCL2 prevent MM cells from escaping apoptosis by releasing Bim to trigger mitochondrial outer membrane potential and the subsequent release of cytochrome c to activate the intrinsic apoptotic pathway. Second, this combined therapy prevents MM cell growth by causing down-regulation of pro-survival cytokines and growth factors in the BM microenvironment. As a proof of concept, our data indicate combining therapeutics that selectively target the anti-apoptotic proteins MCL1 and BCL2 could be an effective therapy for MM patients.

Disclosures
Cidado: AstraZeneca: Employment, Equity Ownership. Drew:AstraZeneca: Employment.