Evaluation of Mcl-1 Inhibitors in Preclinical Models of Multiple Myeloma

Abstract

Mcl-1, an anti-apoptotic Bcl-2 family member, is a known resistance factor to many cancer therapies, and a historically difficult target to drug. Recently however, we have made significant progress optimizing the potency and characterizing the mechanism of action of a novel class of selective Mcl-1 inhibitors. Here, we characterize the activity of one of our lead compounds and confirm on-target mechanistic activity in vitro and in vivo. Using an Mcl-1 binding assay we demonstrate very potent activity (IC50 < 3nM), while maintaining selectivity over other anti-apoptotic Bcl-2 family proteins, Bcl-2 (IC50 > 33 µM) and Bcl-xL (IC50 > 33 µM). In an Mcl-1 dependent multiple myeloma cell line, MOLP-8, we demonstrate rapid Mcl-1/Bak complex disruption with 10 nM inhibitor by 15 min followed by caspase 9-dependent apoptosis by 1 hr (EC50= 50 nM), consistent with an Mcl-1 dependent mechanism of action. Importantly, by depleting Bak from cells using siRNA, we also confirm that the observed apoptosis is through a Bak-dependent mechanism. Moreover, our Mcl-1 inhibitor exhibits broad submicromolar activity across a panel of multiple myeloma cell lines. In the absence of a validated selective Mcl-1 reference compound to benchmark in vivo activity, we engineered MOLP-8 cells to express the pro-apoptotic protein, Noxa, using a Tet-on expression system and grew these as subcutaneous tumors on the flanks of SCID mice. Within 24 hours of switching the mice to a diet containing doxycycline, we observe significant upregulation of Noxa, leading to an increase in cleaved caspase 3 (CC3) and cleaved PARP in tumor lysates, resulting in rapid tumor regression. In the same MOLP-8 xenograft model, we demonstrate that our Mcl-1 inhibitor induces rapid dissociation of Bak from Mcl-1 in tumors leading to accumulation of CC3 and cleaved PARP by 1 hr. Complete tumor regression was observed after a single 60 mg/kg or 100 mg/kg dose, while partial regression was observed after a single 30 mg/kg dose, and tumor growth inhibition was observed after a single 10 mg/kg dose. All doses were well tolerated with no significant body weight loss. Together, these data reinforce the potential utility of selective Mcl-1 inhibitors in hematological malignancies. Disclosures Belmonte: Astrazeneca: Employment, Equity Ownership. Adam:Astrazenenca: Employment, Equity Ownership. Borrelli:AstraZeneca: Employment, Equity Ownership. Bhavsar:AstraZeneca: Employment, Equity Ownership. Bebernitz:AstraZeneca: Employment, Equity Ownership. Hird:AstraZeneca: Employment, Equity Ownership. Secrist:AstraZeneca: Employment, Equity Ownership.

Discovery and biological characterization of potent myeloid cell leukemia‐1 inhibitors

“…Nevertheless, progress has been made toward this goal. Several groups have reported Mcl‐1 inhibitors that have a range of binding affinities and activity in cell‐based assays including, UM‐36 (University of Michigan) , EU5346 (Eutropics Incorporated, Cambridge, MA, USA) , Patent EP2886545A1 (Servier Research Institute of Medicinal Chemistry, Budapest, Hungary/Vernalis (R&D) Ltd., Cambridge, UK), Patent WO2016033486 (Amgen, Thousand Oaks, CA, USA), AZ‐Mcl1 (Astra Zeneca, Waltham, MA, USA) , Dual Mcl‐1/Bcl‐xl inhibitors (Takeda, Cambridge, MA, USA) , and the AbbVie inhibitor, A‐1210477 . We have also reported on the discovery of small molecules that bind to Mcl‐1 with high affinity that were discovered using fragment‐based screening and structure‐based design .…”