Michael H. Cardone

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

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.

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. Mol Cancer Ther; 12(12); 2940–9. ©2013 AACR.

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs.

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.

Sequential azacitidine plus lenalidomide in previously treated elderly patients with acute myeloid leukemia and higher risk myelodysplastic syndrome

Abstract The outcome of sequential azacitidine with lenalidomide has not been reported in previously treated patients with acute myeloid leukemia (AML) and higher risk myelodysplastic syndrome (MDS). This study describes a phase 2 study evaluating the safety and efficacy of this combination in elderly patients with AML and MDS with prior hypomethylating agent (HMA) and/or immunomodulatory agent exposure. Patients were treated on a 42-day cycle with azacitidine at 75 mg/m2 SQ/IV daily on days 1–7, followed by lenalidomide 50 mg orally daily on days 8–28. The median number of treatment cycles on study was two (range = 1–11). Of 32 evaluable patients, the overall response rate was 25%. Neutropenic fever was the most common serious adverse event, but overall the combination was well-tolerated. The median overall survival (OS) for responders vs non-responders was 9.8 vs 4.0 months, respectively (HR = 0.36, p = 0.016). In conclusion, this combination demonstrated modest clinical activity in this poor risk population.

Reduced occurrence of tumor flare with flavopiridol followed by combined flavopiridol and lenalidomide in patients with relapsed chronic lymphocytic leukemia (CLL)

Flavopiridol and lenalidomide have activity in refractory CLL without immunosuppression or opportunistic infections seen with other therapies. We hypothesized that flavopiridol treatment could adequately de‐bulk disease prior to lenalidomide therapy, decreasing the incidence of tumor flare with higher doses of lenalidomide. In this Phase I study, the maximum tolerated dose was not reached with treatment consisting of flavopiridol 30 mg m−2 intravenous bolus (IVB) + 30 mg m−2 continuous intravenous infusion (CIVI) cycle (C) 1 day (D) 1 and 30 mg m−2 IVB + 50 mg m−2 CIVI C1 D8,15 and C2‐8 D3,10,17 with lenalidomide 15 mg orally daily C2‐8 D1‐21. There was no unexpected toxicity seen, including no increased tumor lysis, tumor flare (even at higher doses of lenalidomide) or opportunistic infection. Significant clinical activity was demonstrated, with a 51% response rate in this group of heavily pretreated patients. Biomarker testing confirmed association of mitochondrial priming of the BH3 only peptide Puma with response.Am. J. Hematol. 90:327–333, 2015.

Mcl-1 dependence predicts response to vorinostat and gemtuzumab ozogamicin in acute myeloid leukemia

Older adults with acute myeloid leukemia (AML) are commonly considered for investigational therapies, which often only benefit subsets of patients. In this study, we assessed whether BH3 profiling of apoptotic functionality could predict outcomes following treatment with vorinostat (histone deacetylase inhibitor) and gemtuzumab ozogamicin (GO; CD33-targeted immunoconjugate). Flow cytometry of BH3 peptide priming with Noxa (anti-apoptotic protein Mcl-1 modulator) correlated with remission induction (p=.026; AUC=0.83 [CI: 0.65-1.00; p=.00042]: AUC=0.88 [CI:0.75-1.00] with age adjustment) and overall survival (p=.027 logistic regression; AUC=0.87 [0.64-1.00; p=.0017]). This Mcl-1-dependence suggests a pivotal role of Bcl-2 family protein-mediated apoptosis to vorinostat/GO in AML patients.

Mitochondrial priming of chronic lymphocytic leukemia patients associates Bcl-xL dependence with alvocidib response.

Mitochondrial priming of chronic lymphocytic leukemia patients associates Bcl-x L dependence with alvocidib response

Abstract 2282: Decitabine response in FLT3-negative AML is associated with mitochondrial priming

To date, acute myeloid leukemia (AML) remains a poor-prognosis disease with only approximately 25% of patients surviving five years after being diagnosed (SEER – 2005-2011). There are a wide variety of treatment options ranging from intense chemotherapy to targeted therapies. Beyond tolerability, identifying the ideal treatment strategy for each patient remains a difficult task. Mitochondrial profiling of AML patient samples using Bcl-2 homology domain (BH3) mimetics has proven to accurately identify patients that respond to several therapies in AML. In this method, cancer cells are exposed to BH3 mimetics designed to test for susceptibility to induction of apoptosis. The readout, called “BH3-Priming” indicates anti-apoptotic protein dependencies in cancer cells. In this study, samples from AML patients that were treated with at least one cycle of the hypomethylating agent, decitabine, at The Ohio State University were collected and BH3 profiled (n = 64). The patients were assessed for a complete response to the drug and were characterized by several molecular biomarkers including FLT3 mutational status. We found that the FLT3 mutation negative patients (n = 32) who responded to decitabine had significantly higher mitochondrial responses to the BIM and HRK mimetics (p = 0.04) compared with non-responders. We also found that FLT3 mutant patients (n = 12) had significantly (p = 0.02) higher priming than those patients lacking those lesions. Surprisingly, despite being apparently more predisposed towards apoptosis, these patients are less likely to respond to decitabine (42% versus 20%) in vivo than those without these mutations. This could indicate that additional non-mitochondrial inhibitory mechanisms are present in those FLT3 mutant leukemias that prevent response to decitabine. To further examine the interaction between FLT3 and mitochondrial priming, we profiled engineered cell lines with and without FLT3 mutations. We found that mitochondrial priming was significantly lower (p = 0.03) in BAF3 cells with unmutated FLT3 compared with five BAF3 cell lines containing FLT3 mutations (ITD and TKD mutations). Further, we examined the Broad Institute9s Cancer Therapeutics Response database (CTRP v 2.0) for cancer cell line response to decitabine and found that wild type FLT3 cell lines that responded to decitabine had significantly higher mitochondrial priming than those that did not respond to the drug (p = 0.04), consistent with the patient data. Taken together, we have discovered that response to decitabine in AML cell lines and AML patient samples depends on mitochondrial dependencies and FLT3 mutational status. Genetic lesions in FLT3; however, may be able to over-ride mitochondrial responsiveness to pro-apoptotic factors, illustrating the probable need to measure multiple attribute types including genetics and ex vivo functional testing to accurately determine the likelihood of response to AML therapies. Citation Format: Elisha Dettman, Camille Doykan, Jo Ishizawa, Weiguo Zhang, Alison Walker, William Blum, Rebecca Klisovic, Michael Cardone, Michael Andreeff, Amy Johnson. Decitabine response in FLT3-negative AML is associated with mitochondrial priming. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2282.

Abstract A60: Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: Therapeutic implications

Molecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown. The anti-apoptotic Bcl-2 family member Myeloid cell leukemia-1 (Mcl-1) is frequently amplified in BC; and elevated Mcl-1 levels have been correlated with poor prognosis. Here, we demonstrate for the first time strong correlation between Mcl-1 protein levels and hypoxia, predominantly in Her2-positive BC cells. Surprisingly, genetic depletion of Mcl-1 decreased Her2 and Hif-1α levels followed by inhibition of BC cell survival. In contrast, Mcl-1 protein levels were not downregulated after genetic depletion of Her2 indicating a regulatory role of Mcl-1 upstream of Her2. Indeed, Mcl-1 and Her2 co-localize within the mitochondrial fraction and form a Mcl-1/Her2- protein complex. Similar to genetically targeting Mcl-1 the novel small molecule Mcl-1 inhibitor EU-5346 induced cell death and decreased spheroid formation in Her2-positive BC cells. Of interest, EU-5346 induced ubiquitination of Mcl-1- bound Her2 demonstrating a previously unknown role for Mcl-1 to stabilize Her2 protein levels. Importantly, targeting Mcl-1 was also active in Her2-positive BC cells resistant to Her2 inhibitors, including a brain-primed Her2-positive cell line. In summary, our data demonstrate a critical role of Mcl-1 in Her2-positive BC cell survival under hypoxic conditions and provide the preclinical framework for the therapeutic use of novel Mcl-1- targeting agents to improve patient outcome in BC. Citation Format: Muhammad Hasan Bashari, Fengjuan Fan, Sonia Vallet, M. Arn, Michael H. Cardone, Philipp Beckhove, Andreas Schneeweiss, Martin Sattler, Joseph T. Opferman, Dirk Jager, Klaus Podar. Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: Therapeutic implications. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A60.