Steven P. Angus

Molecular Pathways: Adaptive Kinome Reprogramming in Response to Targeted Inhibition of the BRAF-MEK-ERK Pathway in Cancer

The central role of the BRAF–MEK–ERK pathway in controlling cell fate has made this pathway a primary target for deregulated activation in cancer. BRaf is activated by Ras proteins allowing Ras oncogenes to constitutively activate the pathway. Activating BRaf mutations are also frequent in several cancers, being the most common oncogenic mutation in thyroid carcinoma and melanoma. There are currently two inhibitors, vemurafenib and dabrafenib, approved for treatment of malignant melanoma having activating BRaf mutations. Concurrent administration of BRAF and MAP–ERK kinase (MEK) inhibitor (trametinib) is significantly more active in patients with BRAF-mutant melanoma than either single agent alone, but progression to resistance ultimately occurs by different mechanisms that increase the activation of extracellular signal–regulated kinase (ERK). Such adaptive changes in tumor cell signaling networks allow bypass of targeted oncoprotein inhibition. This is true with targeted inhibitors for BRaf and MEK as well as specific inhibitors for AKT, mTOR, and many receptor tyrosine kinases such as EGF receptor (EGFR) and HER2. It is this adaptive response to targeted kinase inhibitors that contributes to the failure of single-agent kinase inhibitors to have durable responses. This failure is seen in virtually all cancers treated with single-agent kinase inhibitors, most of which are not as dependent on a single signaling pathway such as BRaf–MEK–ERK in melanoma. Thus, understanding the breadth of adaptive reprogramming responses to specific targeted kinase inhibition will be critical to develop appropriate combination therapies for durable clinical responses. Clin Cancer Res; 20(10); 2516–22. ©2014 AACR.

MAP3K1: Genomic Alterations in Cancer and Function in Promoting Cell Survival or Apoptosis

MAP3K1 is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of serine/threonine kinases. MAP3K1 regulates JNK activation and is unique among human kinases in that it also encodes an E3 ligase domain that ubiquitylates c-Jun and ERK1/2. Full length MAP3K1 regulates cell migration and contributes to pro-survival signaling while its caspase 3-mediated cleavage generates a C-terminal kinase domain that promotes apoptosis. The critical function of MAP3K1 in cell fate decisions suggests that it may be a target for deregulation in cancer. Recent large-scale genomic studies have revealed that MAP3K1 copy number loss and somatic missense or nonsense mutations are observed in a significant number of different cancers, being most prominent in luminal breast cancer. The alteration of MAP3K1 in diverse cancer types demonstrates the importance of defining phenotypes for possible therapeutic targeting of tumor cell vulnerabilities created when MAP3K1 function is lost or gained.

Enhancer Remodeling during Adaptive Bypass to MEK Inhibition Is Attenuated by Pharmacologic Targeting of the P-TEFb Complex

Targeting the dysregulated BRAF-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb-associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.Significance: Widespread transcriptional adaptation to pharmacologic MEK inhibition was observed in TNBC patient tumors. In preclinical models, MEK inhibition induces dramatic genome-wide modulation of chromatin, in the form of de novo enhancer formation and enhancer remodeling. Pharmacologic targeting of P-TEFb complex members at enhancers is an effective strategy to durably inhibit such adaptation. Cancer Discov; 7(3); 302-21. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Combination therapy with potent PI3K and MAPK inhibitors overcomes adaptive kinome resistance to single agents in preclinical models of glioblastoma

Background Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. Methods We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. Results PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. Conclusion Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.

New mechanisms of resistance to MEK inhibitors in melanoma revealed by intravital imaging

Targeted therapeutics that are initially effective in cancer patients nearly invariably engender resistance at some stage, an inherent challenge in the use of any molecular-targeted drug in cancer settings. In this study, we evaluated resistance mechanisms arising in metastatic melanoma to MAPK pathway kinase inhibitors as a strategy to identify candidate strategies to limit risks of resistance. To investigate longitudinal responses, we developed an intravital serial imaging approach that can directly visualize drug response in an inducible RAF-driven, autochthonous murine model of melanoma incorporating a fluorescent reporter allele (tdTomatoLSL). Using this system, we visualized formation and progression of tumors in situ, starting from the single-cell level longitudinally over time. Reliable reporting of the status of primary murine tumors treated with the selective MEK1/2 inhibitor (MEKi) trametinib illustrated a time-course of initial drug response and persistence, followed by the development of drug resistance. We found that tumor cells adjacent to bundled collagen had a preferential persistence in response to MEKi. Unbiased transcriptional and kinome reprogramming analyses from selected treatment time points suggested increased c-Kit and PI3K/AKT pathway activation in resistant tumors, along with enhanced expression of epithelial genes and epithelial-mesenchymal transition downregulation signatures with development of MEKi resistance. Similar trends were observed following simultaneous treatment with BRAF and MEK inhibitors aligned to standard-of-care combination therapy, suggesting these reprogramming events were not specific to MEKi alone. Overall, our results illuminate the integration of tumor-stroma dynamics with tissue plasticity in melanoma progression and provide new insights into the basis for drug response, persistence, and resistance.Significance: A longitudinal study tracks the course of MEKi treatment in an autochthonous imageable murine model of melanoma from initial response to therapeutic resistance, offering new insights into the basis for drug response, persistence, and resistance. Cancer Res; 78(2); 542-57. ©2017 AACR.

Epigenetic Mechanisms Regulating Adaptive Responses to Targeted Kinase Inhibitors in Cancer

Although targeted inhibition of oncogenic kinase drivers has achieved remarkable patient responses in many cancers, the development of resistance has remained a significant challenge. Numerous mechanisms have been identified, including the acquisition of gatekeeper mutations, activating pathway mutations, and copy number loss or gain of the driver or alternate nodes. These changes have prompted the development of kinase inhibitors with increased selectivity, use of second-line therapeutics to overcome primary resistance, and combination treatment to forestall resistance. In addition to genomic resistance mechanisms, adaptive transcriptional and signaling responses seen in tumors are gaining appreciation as alterations that lead to a phenotypic state change-often observed as an epithelial-to-mesenchymal shift or reversion to a cancer stem cell-like phenotype underpinned by remodeling of the epigenetic landscape. This epigenomic modulation driving cell state change is multifaceted and includes modulation of repressive and activating histone modifications, DNA methylation, enhancer remodeling, and noncoding RNA species. Consequently, the combination of kinase inhibitors with drugs targeting components of the transcriptional machinery and histone-modifying enzymes has shown promise in preclinical and clinical studies. Here, we review mechanisms of resistance to kinase inhibition in cancer, with special emphasis on the rewired kinome and transcriptional signaling networks and the potential vulnerabilities that may be exploited to overcome these adaptive signaling changes.

Proteomic analysis defines kinase taxonomies specific for subtypes of breast cancer

Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative (basal-like and claudin-low) breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Understudied kinases, whose disease associations and pharmacology are generally unexplored, were highly represented in MIB-binding taxonomies and are integrated into signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.

EPH receptor signaling as a novel therapeutic target in NF2-deficient meningioma

Background Meningiomas are the most common primary brain tumor in adults, and somatic loss of the neurofibromatosis 2 (NF2) tumor suppressor gene is a frequent genetic event. There is no effective treatment for tumors that recur or continue to grow despite surgery and/or radiation. Therefore, targeted therapies that either delay tumor progression or cause tumor shrinkage are much needed. Our earlier work established mammalian target of rapamycin complex mTORC1/mTORC2 activation in NF2-deficient meningiomas. Methods High-throughput kinome analyses were performed in NF2-null human arachnoidal and meningioma cell lines to identify functional kinome changes upon NF2 loss. Immunoblotting confirmed the activation of kinases and demonstrated effectiveness of drugs to block the activation. Drugs, singly and in combination, were screened in cells for their growth inhibitory activity. Antitumor drug efficacy was tested in an orthotopic meningioma model. Results Erythropoietin-producing hepatocellular receptor tyrosine kinases (EPH RTKs), c-KIT, and Src family kinase (SFK) members, which are biological targets of dasatinib, were among the top candidates activated in NF2-null cells. Dasatinib significantly inhibited phospho-EPH receptor A2 (pEPHA2), pEPHB1, c-KIT, and Src/SFK in NF2-null cells, showing no cross-talk with mTORC1/2 signaling. Posttreatment kinome analyses showed minimal adaptive changes. While dasatinib treatment showed some activity, dual mTORC1/2 inhibitor and its combination with dasatinib elicited stronger growth inhibition in meningiomas. Conclusion Co-targeting mTORC1/2 and EPH RTK/SFK pathways could be a novel effective treatment strategy for NF2-deficient meningiomas.

A proteasome-resistant fragment of NIK mediates oncogenic NF-κB signaling in schwannomas

Schwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.

Kinome and transcriptome profiling reveal broad and distinct activities of erlotinib, sunitinib, and sorafenib in the mouse heart and suggest cardiotoxicity from combined signal transducer and activator of transcription and epidermal growth factor receptor inhibition

Background Most novel cancer therapeutics target kinases that are essential to tumor survival. Some of these kinase inhibitors are associated with cardiotoxicity, whereas others appear to be cardiosafe. The basis for this distinction is unclear, as are the molecular effects of kinase inhibitors in the heart. Methods and Results We administered clinically relevant doses of sorafenib, sunitinib (cardiotoxic multitargeted kinase inhibitors), or erlotinib (a cardiosafe epidermal growth factor receptor inhibitor) to mice daily for 2 weeks. We then compared the effects of these 3 kinase inhibitors on the cardiac transcriptome using RNAseq and the cardiac kinome using multiplexed inhibitor beads coupled with mass spectrometry. We found unexpectedly broad molecular effects of all 3 kinase inhibitors, suggesting that target kinase selectivity does not define either the molecular response or the potential for cardiotoxicity. Using in vivo drug administration and primary cardiomyocyte culture, we also show that the cardiosafety of erlotinib treatment may result from upregulation of the cardioprotective signal transducer and activator of transcription 3 pathway, as co‐treatment with erlotinib and a signal transducer and activator of transcription inhibitor decreases cardiac contractile function and cardiomyocyte fatty acid oxidation. Conclusions Collectively our findings indicate that preclinical kinome and transcriptome profiling may predict the cardiotoxicity of novel kinase inhibitors, and suggest caution for the proposed therapeutic strategy of combined signal transducer and activator of transcription/epidermal growth factor receptor inhibition for cancer treatment.