Michelle L. Stewart

An Interactive Resource to Identify Cancer Genetic and Lineage Dependencies Targeted by Small Molecules

The high rate of clinical response to protein-kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell line (CCL) profiling to identify additional biomarkers of small-molecule sensitivities. We have quantitatively measured the sensitivity of 242 genomically characterized CCLs to an Informer Set of 354 small molecules that target many nodes in cell circuitry, uncovering protein dependencies that: (1) associate with specific cancer-genomic alterations and (2) can be targeted by small molecules. We have created the Cancer Therapeutics Response Portal (http://www.broadinstitute.org/ctrp) to enable users to correlate genetic features to sensitivity in individual lineages and control for confounding factors of CCL profiling. We report a candidate dependency, associating activating mutations in the oncogene β-catenin with sensitivity to the Bcl-2 family antagonist, navitoclax. The resource can be used to develop novel therapeutic hypotheses and to accelerate discovery of drugs matched to patients by their cancer genotype and lineage.

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2-interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence-specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.

A Competitive Stapled Peptide Screen Identifies a Selective Small Molecule that Overcomes MCL-1-Dependent Leukemia Cell Survival

Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochemically by an antiapoptotic surface groove that neutralizes the proapoptotic BH3 α helix of death proteins. Antiapoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Although targeting the BCL-2 antiapoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of molecules tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor molecule that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochemical lock-hold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence.

Inhibition of Pro-Apoptotic BAX by a Noncanonical Interaction Mechanism

BCL-2 is a negative regulator of apoptosis implicated in homeostatic and pathologic cell survival. The canonical anti-apoptotic mechanism involves entrapment of activated BAX by a groove on BCL-2, preventing BAX homo-oligomerization and mitochondrial membrane poration. The BCL-2 BH4 domain also confers anti-apoptotic functionality, but the mechanism is unknown. We find that a synthetic α-helical BH4 domain binds to BAX with nanomolar affinity and independently inhibits the conformational activation of BAX. Hydrogen-deuterium exchange mass spectrometry demonstrated that the N-terminal conformational changes in BAX induced by a triggering BIM BH3 helix were suppressed by the BCL-2 BH4 helix. Structural analyses localized the BH4 interaction site to a groove formed by residues of α1, α1-α2 loop, and α2-α3 and α5-α6 hairpins on the BAX surface. These data reveal a previously unappreciated binding site for targeted inhibition of BAX and suggest that the BCL-2 BH4 domain may participate in apoptosis blockade by a noncanonical interaction mechanism.

DiSCoVERing Innovative Therapies for Rare Tumors: Combining Genetically Accurate Disease Models with In Silico Analysis to Identify Novel Therapeutic Targets.

Purpose: We used human stem and progenitor cells to develop a genetically accurate novel model of MYC-driven Group 3 medulloblastoma. We also developed a new informatics method, Disease-model Signature versus Compound-Variety Enriched Response (“DiSCoVER”), to identify novel therapeutics that target this specific disease subtype. Experimental Design: Human neural stem and progenitor cells derived from the cerebellar anlage were transduced with oncogenic elements associated with aggressive medulloblastoma. An in silico analysis method for screening drug sensitivity databases (DiSCoVER) was used in multiple drug sensitivity datasets. We validated the top hits from this analysis in vitro and in vivo. Results: Human neural stem and progenitor cells transformed with c-MYC, dominant-negative p53, constitutively active AKT and hTERT formed tumors in mice that recapitulated Group 3 medulloblastoma in terms of pathology and expression profile. DiSCoVER analysis predicted that aggressive MYC-driven Group 3 medulloblastoma would be sensitive to cyclin-dependent kinase (CDK) inhibitors. The CDK 4/6 inhibitor palbociclib decreased proliferation, increased apoptosis, and significantly extended the survival of mice with orthotopic medulloblastoma xenografts. Conclusions: We present a new method to generate genetically accurate models of rare tumors, and a companion computational methodology to find therapeutic interventions that target them. We validated our human neural stem cell model of MYC-driven Group 3 medulloblastoma and showed that CDK 4/6 inhibitors are active against this subgroup. Our results suggest that palbociclib is a potential effective treatment for poor prognosis MYC-driven Group 3 medulloblastoma tumors in carefully selected patients. Clin Cancer Res; 22(15); 3903–14. ©2016 AACR.

KRAS Genomic Status Predicts the Sensitivity of Ovarian Cancer Cells to Decitabine

Decitabine, a cancer therapeutic that inhibits DNA methylation, produces variable antitumor response rates in patients with solid tumors that might be leveraged clinically with identification of a predictive biomarker. In this study, we profiled the response of human ovarian, melanoma, and breast cancer cells treated with decitabine, finding that RAS/MEK/ERK pathway activation and DNMT1 expression correlated with cytotoxic activity. Further, we showed that KRAS genomic status predicted decitabine sensitivity in low-grade and high-grade serous ovarian cancer cells. Pretreatment with decitabine decreased the cytotoxic activity of MEK inhibitors in KRAS-mutant ovarian cancer cells, with reciprocal downregulation of DNMT1 and MEK/ERK phosphorylation. In parallel with these responses, decitabine also upregulated the proapoptotic BCL-2 family member BNIP3, which is known to be regulated by MEK and ERK, and heightened the activity of proapoptotic small-molecule navitoclax, a BCL-2 family inhibitor. In a xenograft model of KRAS-mutant ovarian cancer, combining decitabine and navitoclax heightened antitumor activity beyond administration of either compound alone. Our results define the RAS/MEK/DNMT1 pathway as a determinant of sensitivity to DNA methyltransferase inhibition, specifically implicating KRAS status as a biomarker of drug response in ovarian cancer.

Abstract 2954: A noncanonical mechanism for BCL-2 inhibition of pro-apoptotic BAX

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In response to cellular stress, pro-apoptotic BCL-2 family members such as BAX initiate apoptosis by forming toxic pores in the mitochondrial outer membrane. It is well established that BAX is inhibited by BCL-2 through a sequestering interaction between a surface groove on BCL-2 and the alpha-helical BH3 death domain of BAX. Indeed, BCL-2 overexpression is a cardinal mechanism exploited by cancer cells to resist apoptosis. Novel BCL-2 inhibitors, such as ABT-199, are showing promise as pharmacologic activators of apoptosis in BCL-2-dependent cancers. Emerging data suggest that a distinct interaction between the BH4 domain of BCL-2 and BAX may also account for BCL-2 inhibition of BAX. To dissect this putative interaction mechanism, we generated stabilized alpha-helices of BCL-2 domains (SAHBs) modeled after the BH4 motif of BCL-2 and assessed their capacity to inhibit BAX. We find that distinct BCL-2 BH4 SAHBs directly engage BAX and block BAX-mediated membrane poration. Mutagenesis studies defined key residues for the inhibitory activity of BCL-2 BH4 SAHBs and confirmed the sequence-dependent specificity of action. We extended our findings to full-length BCL-2, demonstrating that point mutagenesis within the BH4 domain disrupts co-immunoprecipitation of expressed BCL-2 and BAX proteins. Taken together, we find that a noncanonical interaction between BCL-2 and BAX contributes to the functional regulation of these critical apoptotic proteins and may serve as a new and complementary target for therapeutic reactivation of cell death in human cancer. Citation Format: Lauren A. Barclay, Susan Lee, Michelle L. Stewart, Craig R. Braun, Gregory H. Bird, Loren D. Walensky. A noncanonical mechanism for BCL-2 inhibition of pro-apoptotic BAX. [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 2954. doi:10.1158/1538-7445.AM2014-2954

Abstract 4851: Selective small molecule inhibition of anti-apoptotic MCL-1

BCL-2 family proteins are key regulators of the mitochondrial apoptotic pathway in health and disease. Anti-apoptotic members such as BCL-2, BCL-XL, and MCL-1 have been implicated in the initiation, progression, and chemoresistance of human cancer. The anti-apoptotic BCL-2/BCL-XL groove that binds and sequesters pro-apoptotic BH3 death helices has been successfully targeted by small molecules and peptides. Such compounds induce tumor cell apoptosis and are being advanced in clinical trials as promising next-generation cancer therapeutics. Notably, selective antagonists such as ABT-737 are highly effective at inducing apoptosis in BCL-2/BCL-XL-dependent cancers, but are rendered inactive by overexpression of MCL-1, a formidable chemoresistance protein that lies outside the molecule9s binding spectrum. By screening a library of Stabilized Alpha-Helix of BCL-2 domains (SAHBs), we previously discovered that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor (Stewart et al., Nat Chem Biol, 2010). Here, we deployed this natural peptidic inhibitor of MCL-1 in a competitive binding screen to identify an MCL-1 inhibitor molecule (MIM) that displays exquisite selectivity in in vitro binding and functional assays. NMR analysis documented that MIM engages the canonical BH3-binding pocket of MCL-1. Importantly, MIM selectively triggers caspase 3/7 activation and apoptosis in a cancer cell line that is dependent on induced overexpression of MCL-1, but showed no activity in the isogenic cell line that is driven instead by overexpressed BCL-XL. Conversely, ABT-737 manifested activity in the BCL-XL-overexpressing cancer cells but had no effect on the corresponding MCL-1-dependent cells. Thus, we report the application of MCL-1 SAHB to identify a selective small molecule inhibitor of MCL-1 that exhibits pro-apoptotic activity in the specific context of MCL-1 dependence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4851. doi:1538-7445.AM2012-4851

Abstract 1775: Therapeutic reactivation of cell death in refractory hematologic cancers using a broad spectrum BIM BH3 death helix

BCL-2 family proteins are essential regulators of cellular life and death and, when deregulated, contribute to the development, maintenance, and chemoresistance of human cancer. Whereas multidomain anti-apoptotic proteins such as BCL-2 guard against apoptosis, the multidomain pro-apoptotic proteins BAX and BAK induce cell death through mitochondrial damage. The BH3-only members act as cellular antennae, poised to transmit signals of cellular injury to their multidomain counterparts, and deliver the death message via conserved alpha-helical BH3 domains. Interaction-based neutralization or genetic deletion of BCL-2 family death proteins is a common mechanism employed by cancer cells to mount an apoptotic blockade against chemotherapy and radiation treatments. The design of next-generation therapeutics based on the molecular architecture of the BIM BH3 helix offers the unique advantage of recapitulating BIM9s natural capacity to directly target the full complement of anti- and pro-apoptotic BCL-2 proteins. By inserting a hydrocarbon staple into BIM BH3, we developed a Stabilized Alpha-Helix of BCL-2 domain (SAHB) peptide capable of targeting the three modes of BCL-2 family-mediated apoptotic blockade, achieving (1) anti-apoptotic inhibition, (2) pro-apoptotic direct activation, and (3) BIM BH3 replacement. We demonstrate that BIM SAHB is helical and targets BCL-2 family proteins and their complexes with high affinity in vitro and in cells. BIM SAHB, but not a binding interface mutant, induces dose-responsive apoptosis of resistant hematologic cancer cells as measured by viability, annexin V binding, and caspase 3/7 activation assays. Importantly, we demonstrate by immunoprecipitation that reactivation of the death program correlates with MCL-1/BAK dissociation and BAX activation. Non-malignant mouse embryonic and human fibroblasts demonstrate relative resistance to BIM SAHB, indicative of a therapeutic window for treatment. To evaluate the therapeutic potential of BIM SAHB in vivo, we examined the effect of pharmacologic BIM BH3 replacement in the B-cell lymphoproliferative disease of Bim −/− bone marrow-reconstituted mice. Strikingly, animals treated with BIM SAHB displayed significant TUNEL positivity within the aberrant lymphoid infiltrates, whereas no effect was observed in mice treated with vehicle or the mutant control SAHB. In addition, the surrounding parenchymal tissue was unaffected by BIM SAHB treatment, highlighting the selectivity of action and therapeutic window in vivo. Thus, we find that broad and multimodal targeting of the BCL-2 family pathway by pharmacologic replacement of the BIM BH3 helix can overcome formidable pathologic barriers to cell death in vitro and in vivo, and represents a promising strategy for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1775. doi:10.1158/1538-7445.AM2011-1775