Defining Primary Marrow Microenvironment-Induced Synthetic Lethality and Resistance for 2,684 Approved Drugs Across Molecularly Distinct Forms of Multiple Myeloma
Authors: Murnane Megan , Dhimolea Eugen , Li Ruojing , Bariteau Megan A. , Wheeler Diamond D. , Akhurst Rosemary J. , Logan Aaron C. , Munster Pamela , Wiita Arun , Martin Thomas G. , Wolf Jeffrey L. , Liu Jun O. , Mitsiades Constantine S. , Aftab Blake T. 
Published: November 2019

Abstract

Multiple Myeloma (MM) is a prototypical neoplasm for the study of tumor-microenvironment interactions and influences on drug response. These interactions within the bone marrow (BM) alter the signaling state of MM cells and their relative dependence on pharmacological targets. Conversely, many efforts to identify and validate drug targets in MM are conducted outside of this context. This raises the possibility that systematic re-evaluation of the current pharmacopeia may identify drugs with previously unappreciated capacity for targeting MM cells within the marrow environment.
To this end, we utilized the compartment-specific bioluminescence platform (CS-BLI) to characterize the activity of 2,684 FDA-approved drugs from The Johns Hopkins Drug Library (JHDL) in three distinct MM subtypes, in the presence or absence of patient-derived CD138-negative bone marrow stromal cells (BMSCs). Anti-MM activity was evaluated at 100 nM concentrations over 72 h in MM1S (t(14;16), KRASG12A, TRAF3LOF), L363 (t(20;22), NRASQ61H, p53S261T), and OPM2 (t(4;14), FGFRK560E, p53R175H) lines. These lines demonstrate phenotypes of strong, medium, and low BMSC-induced growth enhancement, respectively. Active drugs were placed into 4 categories: type 1 - having potent anti-MM activity independent of BMSC interactions (no stromal effect), type 2 - having anti-MM activity only in the presence of BMSCs (stroma-dependent "synthetic lethality"), type 3 - having anti-MM activity that is decreased in the context of BMSCs (stroma-dependent resistance), and type 4 - otherwise inactive agents that demonstrate pro-MM activity in context of BMSCs (stroma-dependent stimulants).
In this study, for MM1S, L363, and OPM2, respectively, we identified 103, 118, and 108 type 1 drugs, 217, 105, and 76 type 2 drugs, 128, 75, and 16 type 3 drugs, and 124, 33, and 38 type 4 drugs. For each category of drug phenotype, we assessed overlap across the three MM cell lines. We observed high degree of overlap for type 1 drugs (67 drugs active in all three models), while more diversity between lines was evident across the 3 lines for type 2-4 drugs, whose activity is altered by interaction with BMSCs (Figure 1). Specifically, focusing on agents demonstrating BMSC-associated stimulation, adrenergic drugs consistently stimulated MM growth in context of BMSCs, while glucocorticoids consistently grouped as type 3 agents (demonstrating BMSC-associated resistance). Interestingly, carfilzomib was also subject to BMSC-associated resistance.
Despite differences in drugs demonstrating stroma-induced lethality across the MM cell lines, salicylates were commonly represented in this category. In addition to the salicylates, tofacitinib, a Janus kinase (JAK) inhibitor, demonstrated a strong capacity to elicit a stroma-dependent synthetic lethal phenotype and ruxolitinib, another inhibitor in the same class, showed a similar, yet distinct pattern of stroma-mediated sensitization.
In conjunction with our screen, we performed an RNA-seq analysis to assess differential gene expression between MM in monoculture vs. in co-culture with BMSCs. Expression analysis revealed 4.0 fold increase in JAK3 expression induced by co-culture with primary BMSCs, as well as induction of a STAT3 transcription factor fingerprint by ChIP-seq enrichment analysis. A detailed dose-response analysis of tofacitinib revealed no anti-MM activity against MM cells in isolation at physiological concentrations, but showed typical sigmoidal log-dose response dynamics in the presence of stroma and a dynamic range that completely abrogated the growth advantage attributable to stromal stimulation.
This phenomenon of BMSC-dependent pharmacology identifies tofacitinib as an intriguing candidate for repurposing as an agent demonstrating stroma-induced synthetic lethality against MM. Further evaluation of this agent in combination with other anti-MM agents, like bortezomib, is also warranted. Taken together, this study demonstrates specific anti-MM activity for a wide array of clinically relevant drugs and drug classes in the context of BM microenvironment interactions and provides context for further validation and potential suitability for repurposing to treat MM within the medullary compartment.
Figure 1. Figure 1.

Disclosures
Aftab: Cleave Biosciences, Inc.: Research Funding; Omniox, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Onyx Pharmaceuticals, Inc.: Research Funding. Off Label Use: The use of tofacitinib citrate and ruxolitinib will be discussed in preclinical contexts for treatment of multiple myeloma. Other approved drugs and drug classes will be generally presented in similar off-label context..