Timothy J. Stuhlmiller

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.

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.

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.

Epigenetic inhibition of adaptive bypass responses to lapatinib by targeting BET Bromodomains

ABSTRACT The characterization of kinases as oncogenic drivers has led to more than 30 FDA-approved targeted kinase inhibitors for cancer treatment. Unfortunately, these therapeutics fail to have clinical durability because of adaptive responses from the kinome and transcriptome that bypass inhibition of the targeted pathway. In our recent work, we describe a method to prevent these adaptive responses at an epigenetic level, generating a durable response to kinase inhibition.

Abstract B21: Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC

The RAF-MEK-ERK signaling pathway regulates the proliferation of Triple Negative Breast Cancer (TNBC). Although MEK inhibition by trametinib in TNBC cell lines and mouse models inhibits growth, the response is not durable and the tumor develops resistance. The lack of durability of targeted kinase inhibitors is a common problem in many tumor types due to induction of alternative bypass signaling pathways that lead to resistance. We have profiled the response of TNBC to MEK inhibition using trametinib in order to identify adaptive signaling pathways which drive resistance. We used a chemical proteomics method [multiplexed inhibitor beads coupled to mass spectrometry (MIB/MS)], to quantify global changes in kinase activity coupled with transcriptional changes in response to trametinib using RNAseq. Our studies revealed that adaptive resistance to trametinib occurs through increased expression and activation of multiple receptor tyrosine kinases, leading to reactivation of ERK. These adaptive kinases are heterogeneous across TNBC including PDGFRβ, VEGFR2, DDR1, AXL, KIT and FGFR2. There is no single targeted kinase inhibitor able to block all the adaptive kinases induced and activated, making the design of combination targeted kinase inhibitor therapies impractical. We therefore hypothesized an alternative therapeutic approach was required to inhibit adaptive reprogramming. Our goal was to block the initial transcriptional response to trametinib which upregulates adaptive kinase expression. Several studies in the lab determined that we could block adaptive kinase expression by inhibiting BET family bromodomain proteins that interact with acetylated histones and facilitate the formation of transcriptional regulatory complexes. Our work demonstrated that BET family bromodomain inhibitors (JQ1 and iBET151), synergize with trametinib to prevent TNBC growth in short-term (4 days) and long-term (4 weeks) growth assays. Using MIB/MS we found that combination trametinib/BET bromodomain inhibitor treatment blocks upregulation of adaptive kinase expression and activation. RNAseq demonstrated that BET bromodomain inhibitors synergize with trametinib to block the induction of kinases expressed as part of the adaptive bypass response. ChIP-seq of the BET bromodomain protein BRD4 and H3K27ac, a marker of active enhancers, showed BRD4 is recruited to super-enhancer regions proximal to adaptive response kinases as early as 4hr following trametinib treatment. Furthermore, the recruitment of BRD4 to enhancer regions is maintained in cells made resistant to trametinib by constant culture in low-dose drug. In sum, these results suggest that BRD4 recruitment to enhancer regions, following trametinib treatment, leads to the formation of transcriptional complexes which maintain adaptive gene expression. Ongoing studies seek to understand the mechanism by which BRD4 is recruited to super-enhancers following trametinib treatment in TNBC. Mouse xenograft studies also show promising evidence of enhanced tumor growth suppression in vivo using combination trametinib/iBET151 treatment. The results presented provide a novel treatment strategy to block adaptive resistance and have the potential to lead to durable clinical responses of TNBC. Citation Format: Samantha M. Miller, Jon S. Zawistowski, Timothy J. Stuhlmiller, Noah Sciaky, Charlene M. Santos, David B. Darr, Gary L. Johnson. Suppression of adaptive resistance mechanisms to trametinib by inhibition of BET bromodomains in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B21.

Abstract B25: BET bromodomain inhibition targets adaptive responses to lapatinib in HER2-positive breast cancer

Small molecule kinase inhibitors that target oncogene-driven cancers often elicit dramatic initial clinical responses, but adaptive responses from the kinome and transcriptome limit their efficacy and generate resistance. Such adaptive bypass responses are driven by the disruption of regulatory feedback and feedforward loops that govern many signaling networks. To understand these responses in the context of HER2-addicted cancer, we used a chemical proteomics method to assay whole-kinome activation dynamics in cells treated with the small molecule HER2/EGFR tyrosine kinase inhibitor lapatinib. We observed a rapid reactivation of oncogenic signaling and a global re-wiring of kinase signaling networks including activation of receptor tyrosine kinases (RTKs) HER3, DDR1, FGFRs, IGF1R, INSR, and multiple Ephrin receptors, as well as intracellular nodes PKC, SRC family kinases and FAK. Targeting compensatory kinases with a series of kinase inhibitors provided variable and incremental enhancement of growth inhibition but could not prevent resistant colony formation. Cell line heterogeneity and functional redundancy among compensatory kinases added further difficulty to effectively predict the best combination therapy. This presents a dilemma where combinations of two or even three kinase inhibitors would be insufficient to suppress all the bypass tracks that were rapidly induced by HER2 inhibition. RNAseq revealed a 2-fold dysregulation of approximately 20% of expressed genes within 2 days of lapatinib treatment, with many responsive kinases being transcriptionally upregulated. We argued that in order for lapatinib to have a durable effect, the adaptive response itself must be inhibited. We found that by targeting the BET bromodomain family of chromatin readers, we could suppress the transcriptional upregulation of RTKs that reactivate MAPK/AKT and drive the bypass survival tracks through SRC family kinase and FAK signaling. Treatment of HER2-positive cells with three different small molecule BET bromodomain inhibitors (JQ1, I-BET151, and I-BE762) had little effect on HER2 and downstream signaling pathways in the absence of lapatinib and accordingly allowed resistant colony formation in long-term growth assays. Combining BET bromodomain inhibitors with lapatinib caused a loss of signature RTK expression, prevented MAPK/AKT reactivation, and suppressed resistant colonies from forming. BET bromodomain inhibitors also downregulated responsive RTKs in cells made resistant to lapatinib and thus re-sensitized the cells to lapatinib. Transcriptional profiling defined the effects of BET bromodomain inhibition to be less pronounced than lapatinib, with only 8% of expressed genes being downregulated 2-fold or greater. However, more than 25% of lapatinib-induced genes were downregulated 2-fold, indicating BET bromodomains preferentially modulated the induced gene expression. Current literature describes a crucial role for the BET bromodomain protein BRD4 in the regulation and stability of large enhancer regions (super-enhancers) responsible for oncogene regulation and defining cell fate. ChIPseq analysis of enhancer dynamics in response to lapatinib and JQ1 identified a reorganization of the chromatin landscape within 24 hours of drug treatment, with multiple super-enhancers formed and decommissioned. Importantly, the combination of lapatinib and JQ1 caused a loss of the majority of super-enhancers observed in untreated cells. Together, these results demonstrate epigenetic targeting of adaptive response mechanisms can prevent resistance to small molecule kinase inhibitors and provide a more durable effect for oncogene targeting in the treatment of cancer. Citation Format: Timothy J. Stuhlmiller, Samantha M. Miller, Jon S. Zawistowski, James S. Duncan, Steven P. Angus, Deborah A. Granger, Rachel A. Reuther, Kyla A.L. Collins, Gomez M. Shawn, Pei-Fen Kuan, Xin Chen, Noah Sciaky, Gary L. Johnson. BET bromodomain inhibition targets adaptive responses to lapatinib in HER2-positive breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B25.