Hydroxyquinoline-derived compounds and analoguing of selective Mcl-1 inhibitors using a functional biomarker.

Anti-apoptotic Bcl-2 family proteins are important oncology therapeutic targets. To date, BH3 mimetics that abrogate anti-apoptotic activity have largely been directed at Bcl-2 and/or Bcl-xL. One observed mechanism of resistance to these inhibitors is increased Mcl-1 levels in cells exposed to such therapeutics. For this reason, and because Mcl-1 is important in the onset of lymphoid, myeloid, and other cancers, it has become a target of great interest. However, small molecule inhibitors displaying potency and selectivity for Mcl-1 are lacking. Identifying such compounds has been challenging due to difficulties in translating the target selectivity observed at the biochemical level to the cellular level. Herein we report the results of an HTS strategy coupled with directed hit optimization. Compounds identified have selective Mcl-1 inhibitory activity with greater than 100-fold reduced affinity for Bcl-xL. The selectivity of these compounds at the cellular level was validated using BH3 profiling, a novel personalized diagnostic approach. This assay provides an important functional biomarker that allows for the characterization of cells based upon their dependencies on various anti-apoptotic Bcl-2 proteins. We demonstrate that cells dependent on Mcl-1 or Bcl-2/Bcl-xL for survival are commensurately responsive to compounds that genuinely target those proteins. The identification of compound 9 with uniquely validated and selective Mcl-1 inhibitory activity provides a valuable tool to those studying the intrinsic apoptosis pathway and highlights an important approach in the development of a first-in-class cancer therapeutic.

Acquired resistance to ABT-737 in lymphoma cells that up-regulate MCL-1 and BFL-1.

ABT-737 is a small-molecule antagonist of BCL-2 currently under evaluation in clinical trials in the oral form of ABT-263. We anticipate that acquired resistance to this promising drug will inevitably arise. To study potential mechanisms of resistance to ABT-737, we derived resistant lines from initially sensitive OCI-Ly1 and SU-DHL-4 lymphoma cell lines via long-term exposure. Resistance was based in the mitochondria and not due to an inability of the drug to bind BCL-2. Resistant cells had increased levels of BFL-1 and/or MCL-1 proteins, which are not targeted by ABT-737. Proapoptotic BIM was displaced from BCL-2 by ABT-737 in both parental and resistant cells, but in resistant cells, BIM was sequestered by the additional BFL-1 and/or MCL-1. Decreasing MCL-1 levels with flavopiridol, PHA 767491, or shRNA restored sensitivity to ABT-737 resistant cells. MCL-1 was up-regulated not by protein stabilization but rather by increased transcript levels. Surprisingly, in addition to stable increases in MCL-1 transcript and protein in resistant cells, there was a dynamic increase within hours after ABT-737 treatment. BFL-1 protein and transcript levels in resistant cells were similarly dynamically up-regulated. This dynamic increase suggests a novel mechanism whereby modulation of antiapoptotic protein function communicates with nuclear transcriptional machinery.

Aurora kinase A is a target of Wnt/beta-catenin involved in multiple myeloma disease progression.

Multiple myeloma (MM) is a cancer of plasma cells with complex molecular characteristics that evolves from monoclonal gammopathy of undetermined significance, a highly prevalent premalignant condition. MM is the second most frequent hematologic cancer in the United States, and it remains incurable, thereby highlighting the need for new therapeutic approaches, particularly those targeting common molecular pathways involved in disease progression and maintenance, shared across different MM subtypes. Here we report that Wnt/beta-catenin is one such pathway. We document the involvement of beta-catenin in cell-cycle regulation, proliferation, and invasion contributing to enhanced proliferative and metastatic properties of MM. The pleiotropic effects of beta-catenin in MM correlate with its transcriptional function, and we demonstrate regulation of a novel target gene, Aurora kinase A, implicating beta-catenin in G2/M regulation. beta-catenin and Aurora kinase A are present in most MM but not in normal plasma cells and are expressed in a pattern that parallels progression from monoclonal gammopathy of undetermined significance to MM. Our data provide evidence for a novel functional link between beta-catenin and Aurora kinase A, underscoring a critical role of these pathways in MM disease progression.

BH3 profiling discriminates response to cytarabine-based treatment of acute myelogenous leukemia.

As acute myelogenous leukemia (AML) patient response to cytarabine-based standard-of-care treatment is variable, stratification into subgroups by biomarker-predicted response may lead to improved clinical outcomes. Here, we assess cell mitochondrial depolarization to proapoptotic signaling BH3-only peptides as a surrogate for the function of Bcl-2 family proteins to address clinical response to cytarabine-based therapy in patients with AML (N = 62). Peripheral blood mononuclear cell (PBMC) or bone marrow aspirate specimens were obtained from newly diagnosed patients with AML, viably preserved, and assayed by flow cytometry following BH3 profile assay with individual BH3 peptides. Mann-Whitney analysis indicates biomarker correlation with response to induction therapy: Notably, BIM priming was highly significant (P = 2 × 10(-6)) with a compelling sensitivity/specificity profile [area under curve (AUC) = 0.83; 95% confidence interval (CI), 0.73-0.94; P = 2 × 10(-10)]. Multivariate analysis indicates improved profiles for BIM readout + patient age (AUC = 0.89; 95% CI, 0.81-0.97) and BIM + patient age + cytogenetic status (AUC = 0.91; 95% CI, 0.83-0.98). When patients were stratified by cytogenetic status, BIM readout was significant for both intermediate (P = 0.0017; AUC = 0.88; 95% CI, 0.71-1.04) and unfavorable (P = 0.023; AUC = 0.79; 95% CI, 0.58-1.00) risk groups, demonstrating predictive power independent of cytogenetics. Additional analyses of secondary clinical endpoints displayed correlation between overall survival (P = 0.037) and event-free survival (P = 0.044) when patients were stratified into tertiles by BIM peptide response. Taken together, these results highlight the potential utility of BH3 profiling in personalized diagnostics of AML by offering actionable information for patient management decisions.

Proapoptotic BH3-only BCL-2 family protein BIM connects death signaling from epidermal growth factor receptor inhibition to the mitochondrion.

A subset of lung cancers expresses mutant forms of epidermal growth factor receptor (EGFR) that are constitutively activated. Cancers bearing activated EGFR can be effectively targeted with EGFR inhibitors such as erlotinib. However, the death-signaling pathways engaged after EGFR inhibition are poorly understood. Here, we show that death after inhibition of EGFR uses the mitochondrial, or intrinsic, pathway of cell death controlled by the BCL-2 family of proteins. BCL-2 inhibits cell death induced by erlotinib, but BCL-2-protected cells are thus rendered BCL-2-dependent and sensitive to the BCL-2 antagonist ABT-737. BH3 profiling reveals that mitochondrial BCL-2 is primed by death signals after EGFR inhibition in these cells. As this result implies, key death-signaling proteins of the BCL-2 family, including BIM, were found to be up-regulated after erlotinib treatment and intercepted by overexpressed BCL-2. BIM is induced by lung cancer cell lines that are sensitive to erlotinib but not by those resistant. Reduction of BIM by siRNA induces resistance to erlotinib. We show that EGFR activity is inhibited by erlotinib in H1650, a lung cancer cell line that bears a sensitizing EGFR mutation, but that H1650 is not killed. We identify the block in apoptosis in this cell line, and show that a novel form of erlotinib resistance is present, a block in BIM up-regulation downstream of EGFR inhibition. This finding has clear implications for overcoming resistance to erlotinib. Resistance to EGFR inhibition can be modulated by alterations in the intrinsic apoptotic pathway controlled by the BCL-2 family of proteins.