Ryan Lena

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.

Mcl-1 Mediates TWEAK/Fn14-induced Non-small Cell Lung Cancer Survival and Therapeutic Response

Insensitivity to standard clinical interventions, including chemotherapy, radiotherapy, and tyrosine kinase inhibitor (TKI) treatment, remains a substantial hindrance towards improving the prognosis of patients with non–small cell lung cancer (NSCLC). The molecular mechanism of therapeutic resistance remains poorly understood. The TNF-like weak inducer of apoptosis (TWEAK)–FGF-inducible 14 (TNFRSF12A/Fn14) signaling axis is known to promote cancer cell survival via NF-κB activation and the upregulation of prosurvival Bcl-2 family members. Here, a role was determined for TWEAK–Fn14 prosurvival signaling in NSCLC through the upregulation of myeloid cell leukemia sequence 1 (MCL1/Mcl-1). Mcl-1 expression significantly correlated with Fn14 expression, advanced NSCLC tumor stage, and poor patient prognosis in human primary NSCLC tumors. TWEAK stimulation of NSCLC cells induced NF-κB–dependent Mcl-1 protein expression and conferred Mcl-1–dependent chemo- and radioresistance. Depletion of Mcl-1 via siRNA or pharmacologic inhibition of Mcl-1, using EU-5148, sensitized TWEAK-treated NSCLC cells to cisplatin- or radiation-mediated inhibition of cell survival. Moreover, EU-5148 inhibited cell survival across a panel of NSCLC cell lines. In contrast, inhibition of Bcl-2/Bcl-xL function had minimal effect on suppressing TWEAK-induced cell survival. Collectively, these results position TWEAK–Fn14 signaling through Mcl-1 as a significant mechanism for NSCLC tumor cell survival and open new therapeutic avenues to abrogate the high mortality rate seen in NSCLC. Implications: The TWEAK–Fn14 signaling axis enhances lung cancer cell survival and therapeutic resistance through Mcl-1, positioning both TWEAK–Fn14 and Mcl-1 as therapeutic opportunities in lung cancer. Mol Cancer Res; 12(4); 550–9. ©2014 AACR.

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 907: Bcl-xL dependence predicts response to alvocidib in chronic lymphocytic leukemia patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction. Chronic lymphocytic leukemia (CLL) patients may benefit from personalized strategies targeting specific therapies to individuals with favorable molecular profiles. Many therapeutics exhibit response in a subset of patients, and factors such as age or cytogenetics are insufficient to predict treatment success with high accuracy. We assessed mitochondrial functionality in apoptosis signaling for identification of CLL patients likely to exhibit clinical response to treatment with alvocidib. Effects of this agent have been shown to involve pathways of apoptosis, providing the rationale for our study. Patients and Methods. Relapsed/refractory patients comprising OSU0055 and Sanofi EFC6663 clinical trials were analyzed. Pretreatment peripheral blood mononuclear cell (PBMC) specimens were divided into training and validation sets for response-correlative assessment. Blinded to outcomes, we analyzed specimens by flow cytometry-based BH3 profiling, indirectly measuring induction of mitochondrial outer membrane permeabilization in response to treatment with BH3-only peptides (Bim, Noxa, Bad, Bmf, Hrk) as surrogates for Bcl-2 family functions. Findings were correlated with disease characteristics and treatment outcome in the proof-of-principal set and tested for confirmation in the validation set. Results. Response data were available for 62 patients; training and test sets comprised 30 and 32 patients, respectively. Regression analyses indicated a correlation between clinical response (ordered 3 categories; PD, SD, PR) and priming in training set with Bim(0.1) (p=.014) and Hrk (p=.0098), that later validated in the test set for both markers (p=.0051 and p=.015, respectively). In total (n=62), Bim regression displayed a p=.0027 and Hrk regression a p=.00046. When analyzed as PD/SD vs PR, the combined cohort yielded Bim p=.04, and Hrk p=.0039 by logistic regression. Using area under the receiver operating curve (AUC) to quantify the accuracy of outcome prediction, Bim AUC = 0.73 (CI[.60-.85]; p=.0004) and Hrk AUC = .73 (CI[.61-.86]; p=.0002). Hrk benefitted from adjustment for trisomy12 status (AUC=0.83; CI[.71-.95]; p<.0001). Analysis of BH3 profiling and tumor lysis syndrome (TLS) indicated correlation between TLS and Bad priming (p=.012 log regression; AUC=.75; CI[.60-.89]; p=.0007) that benefitted from inclusion of ECOG status and patient age (AUC=.85; CI[.73-.97]; p<.0001). Conclusion. Bim and Hrk are significantly associated with response and, thus, engaging Bcl-xL dependence may be a component of response to alvocidib in relapsed CLL patients. Interestingly, TLS is predicted by a distinct BH3 profiling peptide readout, Bad, consistent with alvocidib-associated TLS driven by Bcl-2-dependency. Taken together, these biomarkers may predict patient response to investigational CDK inhibitors in CLL and concurrently identify patients at risk for treatment-related toxicity. Citation Format: William E. Pierceall, Steven L. Warner, Ryan J. Lena, Camille Doykan, Noel Blake, Michael Elashoff, Daniel D. Von Hoff, David J. Bearss, Michael H. Cardone, Michael Grever, Mark C. Lanasa, John C. Byrd, Amy J. Johnson. Bcl-xL dependence predicts response to alvocidib in chronic lymphocytic leukemia patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 907. doi:10.1158/1538-7445.AM2014-907

Abstract 340: Mitochondrial priming of new targeted agents in acute myeloid leukemia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA As numerous molecularly targeted agents are entering clinical trials, predictive testing is highly desirable. We investigated if response to certain agents correlates with the recently reported method of BH3 profiling (Chonghaile TN et al, Science, 2011), a functional assay developed by Letais group that measures tumor cell mitochondrial priming by measuring mitochondrial outer membrane permeabilization (MOMP) following exposure to the peptide-mimicking BH3 domains of BH3-only proteins. Mitochondrial priming has been reported to be correlated with clinical responses to conventional chemotherapy in solid tumors and hematological malignancies (Vo TT et al, Cell, 2012, Pierceall W et al, Mol Cancer Ther, 2013). Twenty-two AML lines were tested. Cells were permeabilized with digitonin and exposed to standardized doses of BH3 peptides (BIM, PUMA, NOXA, BAD, BMF, HRK, or PUMA2A). JC-1 was used for detection of MOMP and served as a measure of sensitivity to each peptide (reported as %\[BH3 peptide]). Also, BCL-2, BCL-XL and MCL-1 expression levels were determined by Western blot. In addition to studies of untreated cells, treatment effects of different anti-leukemia drugs (AraC, Nutlin-3a, KPT-330 [Selinexor] and ABT-199) were determined over a wide dose range and denoted as ([specific apoptosis] = [(%Annexin V+ cells at each dose) - (%Annexin V+ cells at 0 μM)]/[100- (%Annexin V+ cells at 0 μM)]). Mixed linear models were used for analysis. As expected, ABT-199 sensitivity positively correlated with %[BAD]-%[HRK\] (|β| = 3.22, p < 0.001), which is compatible with BCL-2 dependency of ABT-199 (while BAD is binding to BCL-2 and BCL-XL, HRK is binding to BCL-XL only and the difference is therefore BCL-2-specific). This was supported by the observed correlation between %\[BAD]-%[HRK] and BCL-2 protein expression levels (r=0.619; p = 0.018). AraC sensitivity showed a similar correlation with %[BAD]-%[HRK\] (|β| = 1.61, P < 0.05). KPT-330 sensitivity in p53 wild-type cell lines positively correlated with %\[PUMA\] (|β| = 0.92, P = 0.054), consistent with the notion that KPT-330 induces PUMA through p53 activation. Unexpectedly, Nutlin-3a activity did not correlate with any of the BH3 peptides. Results indicate that ABT-199, KPT-330 and Nutlin-3a show different modes of action in terms of BH3 peptide dependency, supporting potential combination effects of these agents. For ABT-199, increased MCL1 levels were associated with diminished cytotoxicity (r=0.532; p=0.05), as expected. For Ara-C, a similar correlation with MCL-1 was noted (r=0.505; p=0.06), but no correlations were observed for Nutlin-3a and KPT-330. In conclusion, BH3 profiling is a promising tool to predict the BH3 peptide dependency of the BH3-mimetic ABT-199 and the XPO1-inhibitor KPT-330. Functional BH3-profiling appears to be superior to the static quantitation of Bcl-2 family protein levels in AML. Furthermore, mitochondrial priming may be useful for the rational design of new combination therapies. Citation Format: Jo Ishizawa, Kensuke Kojima, Seshagiri R. Duvvuri, Teresa McQueen, Vivian R. Ruvolo, Graciela M. Nogueras-Gonzalez, Xuelin Huang, William Pierceall, Michael Cardone, Ryan Lena, Camille Doykan, Sharon Shacham, Michael Kauffman, Marina Konopleva, Michael Andreeff. Mitochondrial priming of new targeted agents in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 340. doi:10.1158/1538-7445.AM2014-340

Abstract 338: Mcl-1 mediates TWEAK/Fn14-induced non-small cell lung cancer survival and therapeutic response

Lung cancer is the leading cause of cancer-related mortality in the world killing more than one million people each year. Therapeutic resistance remains a significant clinical hurdle, contributing to a five-year survival rate of less than 5% in advanced lung cancer. The mechanisms of therapeutic resistance remain poorly understood and thus difficult to combat therapeutically. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling axis is known to promote cancer cell survival via NF-κB activation and up-regulation of pro-survival Bcl-2 family members. Mcl-1 is over-expressed across many tumor types and correlates with poor patient prognosis and therapeutic resistance. We hypothesize that activation of the TWEAK-Fn14 signaling axis can induce therapeutic resistance through Mcl-1, and that Mcl-1 suppression can sensitize lung cancer cells to therapeutic insult. Here we demonstrate that both Fn14 and Mcl-1 are over-expressed and correlated in primary patient lung tumors. Mcl-1 expression correlates with both advanced stage lung cancer and poor patient prognosis. TWEAK stimulation of non-small cell lung cancer (NSCLC) cell lines induces Mcl-1 protein expression in a NF-κB-dependent manner. TWEAK exposure completely abrogates the cell killing effects of cisplatin or radiation treatment in NSCLC cell lines, and this TWEAK-induced protection is dependent on Mcl-1 expression. The suppression of Mcl-1 through RNAi or a novel Mcl-1 inhibitor (EU-5148) enhances the cell killing effects of cisplatin or radiation and completely blocks TWEAK-induced cell survival, whereas Bcl-2 and Bcl-xL inhibition have lesser effect on TWEAK-induced survival. The specificity of EU5148 is demonstrated through abrogation of the protein-protein interaction of Mcl-1 and Bim, with no effect on the interaction of Bcl-xL and Bim. Moreover, EU-5148 treatment inhibits cell survival across a panel of NSCLC cell lines, representing a range of histological subtypes and driver mutations, such as EGFR and KRAS. Collectively, these results position TWEAK-Fn14 signaling through Mcl-1 as a significant mechanism for NSCLC tumor cell survival, and open new therapeutic avenues to abrogate the high mortality rate seen in advanced NSCLC. Citation Format: Timothy G. Whitsett, Ian T. Mathews, Michael H. Cardone, Ryan J. Lena, William E. Pierceall, Michael Bittner, Chao Sima, Janine LoBello, Glen J. Weiss, Nhan L. Tran. Mcl-1 mediates TWEAK/Fn14-induced non-small cell lung cancer survival and therapeutic response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 338. doi:10.1158/1538-7445.AM2014-338

Abstract 2466: Characterization and development of on-target Mcl-1 inhibitors; BH3 profiling provides a valuable drug discovery tool.

Anti-apoptotic Bcl-2 family proteins are central to the regulation of the intrinsic apoptotic pathway, and as such constitute an important group of targets with great potential as oncology therapeutics. The Bcl-2 family protein Mcl-1 has been demonstrated to facilitate survival and chemoresistance in multiple myeloma, AML, and other cancers, and agents which affect this pathway have become highly sought after. Currently, however, no therapies exist which directly target Mcl-1. We have identified compounds that target Mcl-1 which may be characterized as both Mcl-1-selective and pan-Mcl-1/Bcl-2 inhibitors. This effort has been facilitated by utilization of the BH3 profiling technology to guide SAR. This assay allows for determination of the mitochondrial priming state of both cell culture samples and primary patient samples. We have demonstrated a correlation between myeloma and leukemia cell line response to treatment with our inhibitors and the mitochondrial priming state of such cell lines. Such correlations have also been shown with respect to the extent of cytochrome C release. In the case of the selective Mcl-1 inhibitor, we have shown that cytochrome C release occurs preferentially in leukemia cell lines which are highly primed for Mcl-1 rather than Bcl-2. In addition, our Mcl-1 selective inhibitor demonstrates enhanced cell killing ability in leukemia cells which have been engineered to selectively express Mcl-1, Bcl-2, and Bcl-xL. Our current lead candidate possesses excellent drug-like properties and displays impressive efficacy in a multiple myeloma disseminated xenograft model. This work demonstrates the utility of the BH3 profiling assay as providing a functional biomarker for drug discovery tool and its ability to validate the on-target activity of Mcl-1 and Bcl-2 inhibitors. Citation Format: David J. Richard, Nicole Carlson, William Pierceall, Ryan Lena, Thomas Bannister, Peter Hodder, Timothy Spicer, Michael Andreeff, Joseph Opferman, Brian Koss, Andrew Kung, Michael Cardone. Characterization and development of on-target Mcl-1 inhibitors; BH3 profiling provides a valuable drug discovery tool. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2466. doi:10.1158/1538-7445.AM2013-2466