Developmental Compound E61 Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma Cells By Targeting the Cellular Protein Folding Machinery

Proteasome inhibitors (PIs) are cornerstone agents in the treatment of Multiple Myeloma (MM). Although initially effective, resistance to PIs inevitably emerges, presenting an obstacle to sustained and durable treatment responses in the clinic. To address this limitation, we set out to discover new small molecules that are able to restore PI sensitivity in resistant MM cells. We screened multiple chemical libraries using a cell-based screening method that identifies synergistic combinations with the PI bortezomib (Btz). This method uncovered compound E61 that, in subsequent rounds of screening, demonstrated potent PI re-sensitizing activity. E61 synergistically enhanced the activity of multiple PIs, including Btz, carfilzomib, ixazomib, and oprozomib by 3-15 fold in a genetically diverse panel of PI sensitive and resistant MM cells. In addition, E61 exhibited strong anti-MM activity as a single agent after extended treatment times (48 hours) and demonstrated >10-fold selectivity for MM cell lines over normal peripheral blood mononuclear cells (PBMCs), normal lymphocytes, and a panel of normal fibroblast cell lines. Importantly, the PI sensitizing activity of E61 was also limited to MM cells, as the drug failed to enhance the activity of PIs in normal cells. For a hit stage molecule, E61 showed exceptional tolerability and activity in vivo, significantly improving animal survival and reducing the number of CD138+ MM plasma cells in the bone marrow of mice. We used a xenotransplant model where NOD-SCID IL2Rgamma^-/- (NSG) mice were injected via the lateral tail vein with PI resistant MM cells. Using this model, mice reliably reached the survival endpoint between weeks six and seven, with death being caused by the infiltration of mouse bone marrow by MM plasma cells and the development of bone lesions that closely resemble the human MM pathology. Continuous dosing with E61 (50 mg/kg, i.p., daily) was able to cure 38% of mice, with surviving mice showing only minimal residual disease (i.e., 1.0-1.5% CD138+ MM cells in the bone marrow) at the termination of the experiment. The molecular effects of E61 which lead to its anti-MM activity are characterized primarily by oxidative and endoplasmic stress responses. E61 induces reactive oxygen species (ROS) formation and oxidative damage to proteins, effects that are synergistically potentiated by the addition of PIs. This oxidative burst is critical to the anti-MM activity of E61, as the neutralizing of ROS with various molecular scavengers blocks the pro-apoptotic effects of E61. E61 triggers a robust induction of canonical ER stress markers including phospho-eIF2a, ATF4, XBP-1s, and CHOP. In order to identify the direct molecular target of E61, we chemically modified the molecule to enable copper-catalyzed azide-alkyne click chemistry coupling to fluorescent dyes and immobilizing agents. This strategy was used in tandem with peptide mass fingerprinting, which identified a small set of E61 protein binding partners that are principally involved in the proper folding of nascent polypeptide chains. Additionally, we have synthesized a small library of derivatives which retain the PI re-sensitization phenotype, that have increased metabolic stability against human liver microsomes by at least 5-fold. Ongoing efforts by our group are aimed at further validating and confirming this mechanism of action of E61 as well as further optimizing the chemical structure of E61 for enhanced potency and pharmaceutical properties toward the goal of clinical development. Overall, this work demonstrates the potential of developmental compound E61, a new class of small molecule with an apparent novel mechanism of action, as a new drug candidate for the treatment of refractory MM.