Timothy L. Lochmann

Exploitation of the Apoptosis-Primed State of MYCN-Amplified Neuroblastoma to Develop a Potent and Specific Targeted Therapy Combination

Summary Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.

Epigenetic Modifications of the PGC-1α Promoter during Exercise Induced Expression in Mice

The transcriptional coactivator, PGC-1α, is known for its role in mitochondrial biogenesis. Although originally thought to exist as a single protein isoform, recent studies have identified additional promoters which produce multiple mRNA transcripts. One of these promoters (promoter B), approximately 13.7kb upstream of the canonical PGC-1α promoter (promoter A), yields alternative transcripts present at levels much lower than the canonical PGC-1α mRNA transcript. In skeletal muscle, exercise resulted in a substantial, rapid increase of mRNA of these alternative PGC-1α transcripts. Although the β2-adrenergic receptor was identified as a signaling pathway that activates transcription from PGC-1α promoter B, it is not yet known what molecular changes occur to facilitate PGC-1α promoter B activation following exercise. We sought to determine whether epigenetic modifications were involved in this exercise response in mouse skeletal muscle. We found that DNA hydroxymethylation correlated to increased basal mRNA levels from PGC-1α promoter A, but that DNA methylation appeared to play no role in the exercise-induced activation of PGC-1α promoter B. The level of the activating histone mark H3K4me3 increased with exercise 2–4 fold across PGC-1α promoter B, but remained unaltered past the canonical PGC-1α transcriptional start site. Together, these data show that epigenetic modifications partially explain exercise-induced changes in the skeletal muscle mRNA levels of PGC-1α isoforms.

Venetoclax Is Effective in Small-Cell Lung Cancers with High BCL-2 Expression

Purpose: Small-cell lung cancer (SCLC) is an often-fatal neuroendocrine carcinoma usually presenting as extensive disease, carrying a 3% 5-year survival. Despite notable advances in SCLC genomics, new therapies remain elusive, largely due to a lack of druggable targets. Experimental Design: We used a high-throughput drug screen to identify a venetoclax-sensitive SCLC subpopulation and validated the findings with multiple patient-derived xenografts of SCLC. Results: Our drug screen consisting of a very large collection of cell lines demonstrated that venetoclax, an FDA-approved BCL-2 inhibitor, was found to be active in a substantial fraction of SCLC cell lines. Venetoclax induced BIM-dependent apoptosis in vitro and blocked tumor growth and induced tumor regressions in mice bearing high BCL-2–expressing SCLC tumors in vivo. BCL-2 expression was a predictive biomarker for sensitivity in SCLC cell lines and was highly expressed in a subset of SCLC cell lines and tumors, suggesting that a substantial fraction of patients with SCLC could benefit from venetoclax. Mechanistically, we uncover a novel role for gene methylation that helped discriminate high BCL-2–expressing SCLCs. Conclusions: Altogether, our findings identify venetoclax as a promising new therapy for high BCL-2–expressing SCLCs. Clin Cancer Res; 24(2); 360–9. ©2017 AACR.

Epithelial-to-Mesenchymal Transition Antagonizes Response to Targeted Therapies in Lung Cancer by Suppressing BIM

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it. Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance. Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes. Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

Targeted inhibition of histone H3K27 demethylation is effective in high-risk neuroblastoma

High-throughput screening identified hypersensitivity of neuroblastomas to H3K27 demethylation inhibition. A different way to differentiate neuroblastoma Neuroblastoma is one of the more common pediatric solid tumors, and it can be difficult to treat. These tumors are characterized by the failure of neural crest precursor cells to differentiate and can sometimes be treated with compounds that induce differentiation. Unfortunately, these do not always work, and thus, Lochmann et al. performed a high-throughput drug screen in search of additional differentiating agents, particularly ones that work by altering tumor epigenetics. Through such screening, the authors identified a compound called GSK-J4, which inhibited histone demethylation, induced tumor cell differentiation, and blocked tumor growth in multiple models of neuroblastoma. In addition, GSK-J4 cooperated with two approved drugs, suggesting that it could be a valuable part of treatment for this lethal disease. High-risk neuroblastoma is often distinguished by amplification of MYCN and loss of differentiation potential. We performed high-throughput drug screening of epigenetic-targeted therapies across a large and diverse tumor cell line panel and uncovered the hypersensitivity of neuroblastoma cells to GSK-J4, a small-molecule dual inhibitor of lysine 27 of histone 3 (H3K27) demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), and histone demethylase Jumonji D3 (JMJD3). Mechanistically, GSK-J4 induced neuroblastoma differentiation and endoplasmic reticulum (ER) stress, with accompanying up-regulation of p53 up-regulated modulator of apoptosis (PUMA) and induction of cell death. Retinoic acid (RA)–resistant neuroblastoma cells were sensitive to GSK-J4. In addition, GSK-J4 was effective at blocking the growth of chemorefractory and patient-derived xenograft models of high-risk neuroblastoma in vivo. Furthermore, GSK-J4 and RA combination increased differentiation and ER stress over GSK-J4 effects and limited the growth of neuroblastomas resistant to either drug alone. In MYCN-amplified neuroblastoma, PUMA induction by GSK-J4 sensitized tumors to the B cell lymphoma 2 (BCL-2) inhibitor venetoclax, demonstrating that epigenetic-targeted therapies and BCL-2 homology domain 3 mimetics can be rationally combined to treat this high-risk subset of neuroblastoma. Therefore, H3K27 demethylation inhibition is a promising therapeutic target to treat high-risk neuroblastoma, and H3K27 demethylation can be part of rational combination therapies to induce robust antineuroblastoma activity.

Coamplification of miR-4728 protects HER2-amplified breast cancers from targeted therapy

Significance In HER2-amplified breast cancers, HER2 inhibitors have been very successful as adjuvant therapy but not as monotherapy. Here, we demonstrate that coamplification of a HER2 intronic miRNA causes intrinsic resistance to HER2 inhibitors by indirectly down-regulating the pro-apoptotic NOXA. Importantly, coinhibition with MCL-1 inhibitors overcomes this resistance. HER2 (ERBB2) amplification is a driving oncogenic event in breast cancer. Clinical trials have consistently shown the benefit of HER2 inhibitors (HER2i) in treating patients with both local and advanced HER2+ breast cancer. Despite this benefit, their efficacy as single agents is limited, unlike the robust responses to other receptor tyrosine kinase inhibitors like EGFR inhibitors in EGFR-mutant lung cancer. Interestingly, the lack of HER2i efficacy occurs despite sufficient intracellular signaling shutdown following HER2i treatment. Exploring possible intrinsic causes for this lack of response, we uncovered remarkably depressed levels of NOXA, an endogenous inhibitor of the antiapoptotic MCL-1, in HER2-amplified breast cancer. Upon investigation of the mechanism leading to low NOXA, we identified a micro-RNA encoded in an intron of HER2, termed miR-4728, that targets the mRNA of the Estrogen Receptor α (ESR1). Reduced ESR1 expression in turn prevents ERα-mediated transcription of NOXA, mitigating apoptosis following treatment with the HER2i lapatinib. Importantly, resistance can be overcome with pharmacological inhibition of MCL-1. More generally, while many cancers like EGFR-mutant lung cancer are driven by activated kinases that when drugged lead to robust monotherapeutic responses, we demonstrate that the efficacy of targeted therapies directed against oncogenes active through focal amplification may be mitigated by coamplified genes.

Increased synthesis of MCL-1 protein underlies initial survival of EGFR mutant lung cancer to EGFR inhibitors and provides a novel drug target

Purpose: EGFR inhibitors (EGFRi) are effective against EGFR-mutant lung cancers. The efficacy of these drugs, however, is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M. We recently demonstrated that T790M can arise de novo during treatment; it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells [referred to as drug-tolerant cells (DTC)] prior to acquiring secondary mutations like T790M. Experimental Design: We have developed DTCs to EGFRi in EGFR-mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR-mutant lung cancer tumors in vivo. Results: We demonstrate surviving EGFR-mutant lung cancer cells upregulate the antiapoptotic protein MCL-1 in response to short-term EGFRi treatment. Mechanistically, DTCs undergo a protein biosynthesis enrichment resulting in increased mTORC1-mediated mRNA translation of MCL-1, revealing a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors. Moreover, MCL-1 is a key molecule governing the emergence of early EGFR-mutant DTCs to EGFRi, and we demonstrate it can be effectively cotargeted with clinically emerging MCL-1 inhibitors both in vitro and in vivo. Conclusions: Altogether, these data reveal that this novel therapeutic combination may delay the acquisition of secondary mutations, therefore prolonging therapy efficacy. Clin Cancer Res; 24(22); 5658–72. ©2018 AACR.

Abstract B31: A protein synthesis switch underlies initial survival of EGFR-mutant lung cancer to EGFR inhibitors

EGFR inhibitors (EGFRi) are effective at inducing transient tumor shrinkage in EGFR- mutant lung cancers. The efficacy of these drugs however is mitigated by the outgrowth of resistant cells: this is most often manifested by a secondary mutation in EGFR, T790M , which leads to reactivation of key intracellular signaling despite continued drug treatment. We recently demonstrated that T790M can occur both at low frequencies prior to initiation of EGFR inhibitor therapy, or alternatively arise de novo during treatment (Hata et al., Nat Med 2016). Since some cancers form T790M mutations de novo, one potential therapeutic strategy to thwart resistance is to identify the cells surviving initial therapy (referred to as persister cells or drug-tolerant cells [DTCs]) that eventually acquire the T790M mutation, and eliminate them prior to T790M acquisition. To this end, we hypothesized that some cells were refractory to EGFR inhibitor-induced apoptosis, surviving initial therapy and forming a reservoir of cells that could then eventually acquire T790M . We demonstrate that Western blots of lysates from EGFR- mutant lung cancers surviving initial therapy to the EGFR inhibitor gefitinib detect quick ( EGFR- mutant lung cancer tumors, and the emergence of DTCs could be largely thwarted by co-incubation with MCL-1 specific inhibitors A-1210477 and S63845. Mechanistically, we report DTCs undergo a “translational switch” that manifests in increased cap-dependent mRNA translation of MCL-1, which corresponds to cellular upregulation of mTOR/eIF4 and downregulation of ribosomal proteins. These data reveal a novel mechanism in which lung cancer cells adapt to short-term pressures of apoptosis-inducing kinase inhibitors by shifting protein biosynthesis through cap-dependent translation of MCL-1 protein. Moreover, in EGFR- mutant lung cancer, MCL-1 is a key molecule governing the emergence of early DTCs to EGFR inhibitors and can be effectively co-targeted with clinically-emerging MCL-1 inhibitors, which may delay the acquisition of secondary mutations including T790M mutations, therefore prolonging therapy efficacy. Citation Format: Kyung-A Song, Timothy L. Lochmann, Neha U. Patel, Jungoh Ham, Brad E. Windle, Hisashi Harada, Joel D. Leverson, Andrew J. Souers, Aaron N. Hata, Hiromichi Ebi, Anthony C. Faber. A protein synthesis switch underlies initial survival of EGFR -mutant lung cancer to EGFR inhibitors [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B31.

Abstract 3082: Deficient NOXA in HER2-amplified breast cancer drives kinase inhibitor resistance

The purpose of this study is the development of a novel combination therapy that targets HER2-amplified breast cancer. About one quarter of breast cancers harbor amplification of HER2. HER2 is a transmembrane receptor tyrosine kinase (RTK) belonging to the ERBB family of receptors (ERBB1-4). Upon hetero- and homo-dimerization, HER2 activates several key intracellular pathways, regulating many cellular functions including proliferation and survival. HER2 inhibitors (HER2i) (e.g. the receptor tyrosine kinase (RTK) inhibitor, lapatinib) are now part of standard care for treating HER2-amplified breast cancers. However, despite their anti-cancer benefit, these drugs have limited efficacy as monotherapies, which contrasts to other RTK inhibitors in other RTK-driven cancers. To better understand this apparent dichotomy, in the present study, we evaluated potential modifiers of HER2i therapy. Here, we found that the pro-apoptotic NOXA, a member of the B-cell CLL/lymphoma 2 (BCL2) family which acts mainly via inhibitory binding of the pro-survival MCL-1, was markedly down-regulated in breast cancers compared to other cancers, and this was largely attributed to the HER2-amplified subset. Experimentally, overexpressing NOXA or silencing MCL-1 dramatically sensitizes HER2 amplified breast cancer cell lines to lapatinib via apoptosis. Consistently, pharmaceutical inhibition of MCL-1 sensitizes HER2-amplified breast cancers to lapatinib in vitro and in vivo. Mechanistically, disruption of MCL-1:BIM complexes and MCL-1:BAK underlie dual HER2i/MCL-1i therapy. Therefore, deficient NOXA expression constitutes a bonafide apoptotic block in HER2 amplified breast cancers, contributes to mitigated HER2i responses, and presents a rational combination therapy that may improve HER2i responses. Citation Format: Konstantinos V. Floros, Kyung-A Song, Timothy L. Lochmann, Mark T. Hughes, Daniel A. Heisey, Hisashi Harada, Bin Hu, Jennifer Koblinski, Andrew J. Souers, Joel D. Leverson, Anthony C. Faber. Deficient NOXA in HER2-amplified breast cancer drives kinase inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3082. doi:10.1158/1538-7445.AM2017-3082