Meghna Das Thakur

Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance

Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥50% of tumours expressing the BRAF(V600E) oncoprotein. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xenograft models in which drug resistance is selected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E)→MEK→ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations.

The Evolution of Melanoma Resistance Reveals Therapeutic Opportunities

The RAS-RAF-MEK-ERK pathway is a key driver of proliferation and survival signals in tumor cells and has been the focus of intense drug development efforts over the past 20 years. The recent regulatory approval of RAF inhibitors and a MAP-ERK kinase (MEK) inhibitor for metastatic melanoma provides clinical validation of tumor dependency on this pathway. Unfortunately, the therapeutic benefit of these agents is often short lived and resistance develops within a matter of months. Preclinical models of resistance to vemurafenib have provided critical insights into predicting, validating, and characterizing potential mechanisms. A key observation has been that vemurafenib-resistant tumor cells suffer a fitness deficit in the absence of drug treatment and this led to the predication that modulating the selective pressure of drug treatment through intermittent dosing could delay or prevent the emergence of resistant tumors. Most importantly, the preclinical data are supported by observations in vemurafenib-treated patients with melanoma providing a strong rationale for clinical testing of alternative dosing regimens.

Ligand independent EphA2 signaling drives the adoption of a targeted therapy-mediated metastatic melanoma phenotype

UNLABELLED Many patients with BRAF inhibitor resistance can develop disease at new sites, suggesting that drug-induced selection pressure drives metastasis. Here, we used mass spectrometry-based phosphoproteomic screening to uncover ligand-independent EPHA2 signaling as an adaptation to BRAF inhibitor therapy that led to the adoption of a metastatic phenotype. The EPHA2-mediated invasion was AKT-dependent and readily reversible upon removal of the drug as well as through PI3K and AKT inhibition. In xenograft models, BRAF inhibition led to the development of EPHA2-positive metastases. A retrospective analysis of patients with melanoma on BRAF inhibitor therapy showed that 68% of those failing therapy develop metastases at new disease sites, compared with 35% of patients on dacarbazine. Further IHC staining of melanoma specimens taken from patients on BRAF inhibitor therapy as well as metastatic samples taken from patients failing therapy showed increased EPHA2 staining. We suggest that inhibition of ligand-independent EPHA2 signaling may limit metastases associated with BRAF inhibitor therapy. SIGNIFICANCE This study provides evidence that BRAF inhibition promotes the adoption of a reversible, therapy-driven metastatic phenotype in melanoma. The cotargeting of ligand-independent EPHA2 signaling and BRAF may be one strategy to prevent the development of therapy-mediated disease at new sites.

Molecular Pathways: Response and Resistance to BRAF and MEK Inhibitors in BRAF V600E Tumors

The RAS–RAF–MEK (MAP–ERK kinase)–ERK (extracellular signal–regulated kinase) pathway plays a central role in driving proliferation, survival, and metastasis signals in tumor cells, and the prevalence of oncogenic mutations in RAS and BRAF and upstream nodes makes this pathway the focus of significant oncology drug development efforts. This focus has been justified by the recent success of BRAF and MEK inhibitors in prolonging the lives of patients with BRAFV600E/K-mutant melanoma. Although it is disappointing that cures are relatively rare, this should not detract from the value of these agents to patients with cancer and the opportunity they provide in allowing us to gain a deeper understanding of drug response and resistance. These insights have already provided the basis for the evaluation of alternative dosing regimens and combination therapies in patients with melanoma. Clin Cancer Res; 20(5); 1074–80. ©2013 AACR.

Mouse tumour models to guide drug development and identify resistance mechanisms

We need improved, translatable and predictive tumour models for the evaluation of response and the evolution of resistance to targeted therapeutics. We provide a review of the use of different types of preclinical tumour models to evaluate novel anticancer agents, and model the rapidly evolving landscape of resistance to targeted therapy. We focus on describing the various preclinical models available for candidate drug development and design considerations for preclinical experiments, depending on the aspect of drug action being interrogated. We discuss selected examples of how experimental findings have translated into clinical outcomes for targeted agents, predicted mechanisms that drive resistance and strategies to overcome the evolution thereof. We discuss challenges in preclinical experimental design and interpretation and possible improvements in animal models of therapeutic response and resistance, with an emphasis on improved translation of experimental research into clinical practice. Copyright

Abstract 1610: MAPK inhibitor resistance leads to ligand-independent Ephrin A2 receptor signaling and the formation of new melanoma metastases

Melanoma is the deadliest form of skin cancer with the worst prognosis in patients with metastasis to distant sites such as brain, liver and bone. Studies have shown that MAPK reactivation is a key signaling event leading to BRAF inhibitor resistance. As such, clinical investigations are now underway to evaluate the efficacy of combining frontline BRAF plus MEK inhibitors. Though this approach appears to have meaningful clinical benefit, there are a number of patients who do not respond to therapy, or who through unknown mechanisms, succumb to refractory disease. In order to identify the dynamic changes that drive MAPK inhibitor resistance, we have developed a systems level approach combining mass spectrometry based phosphoproteomic and bioinformatics methodologies. Based on significant changes in tyrosine, threonine and serine phosphorylation events between naive and vemurafenib resistant melanoma cell lines, we have mapped a resistance interactome of ∼550 nodes. This resistance network was significantly enriched for pathways associated with metastatic disease where changes in network connectivity resulted in the appearance of new signaling hubs such as EGFR, EphA2, EphB4, STAT3, FAK1 and HDAC1. The clinical relevance of these findings was demonstrated in a retrospective study showing that 65% of BRAF V600E patients on vemurafenib therapy developed metastases at new sites, with 25% of these new metastases involving the brain. Consistent with our bioinformatics prediction, resistant lines had greater metastatic potential as seen by increased migration and invasion across matrigel and endothelial cell barriers. Extending our findings to a panel of BRAF and BRAF plus MEK inhibitor resistant cell lines, we found compelling evidence that EphA2 is essential for maintaining a resistance phenotype. This phenotype was associated with AKT activation and uncoupled Eph-ephrin signaling resulting in S897 phosphorylation and overexpression of EphA2. In vivo analysis of matched primary and metastatic tumors from vemurafenib resistant xenografts showed that EphA2 expression is increased in metastatic but not primary lesions. Importantly, analysis of clinical specimens from melanoma patients undergoing or failing vemurafenib therapy confirmed that EphA2 receptor expression is significantly upregulated in metastatic but not primary tumors. Functionally, EphA2 S897 phosphorylation was increased at the tumor leading edge of metastatic lesions while absent in primary lesions. Our results show that MAPK resistant melanomas can adaptively rewire their signaling leading to a more malignant phenotype and that through proteomics based approaches we can reliably identify resistance pathways that will translate into rational therapeutic strategies for disseminated disease. Citation Format: Kim H. T. Paraiso, Meghna Das Thakur, Jobin K. John, Bin Fang, John M. Koomen, Inna V. Fedorenko, Hensin Tsao, Keith T. Flaherty, Jane L. Messina, Elena M. Pasquale, Alejandro Villagra, John M. Kirkwood, Friedegund Meier, Sarah Sloot, Geoffrey T. Gibney, Darrin Stuart, Hussein Tawbi, Keiran S.M. Smalley. MAPK inhibitor resistance leads to ligand-independent Ephrin A2 receptor signaling and the formation of new melanoma metastases. [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 1610. doi:10.1158/1538-7445.AM2014-1610

Abstract LB-144: Modeling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥50% of tumors expressing the BRAF (V600E) oncoprotein. Moreover, the impressive tumor regression and increased survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance. Although vemurafenib therapy has transformed the treatment of late-stage melanoma patients, the durability of patient response to this drug is limited by the emergence of lethal drug resistant disease. Thus, understanding mechanisms and designing approaches to prevent the emergence of resistance are critical to providing improved therapy. Here we model the emergence of vemurafenib resistance using two early passage primary human-patient-derived xenograft (PDX) BRAF (T1799A)-mutated melanoma models, which were continuously treated with vemurafinib in immunocompromised mice. In one of these models vemurafenib resistance is due to selection of cells with elevated expression of the BRAF (V600E) oncoprotein. Remarkably, vemurafenib resistant tumors are not only drug resistant but also develop drug dependency such that tumors require exposure to vemurafenib for their continued proliferation. Indeed, cessation of drug administration in these PDX models leads to inhibition of tumor growth and, in many cases, tumor regression. This fitness deficit upon cessation of drug administration is based on an adaptive response to levels of MAPK pathway signaling output. Melanoma cells appear to proliferate best at a defined intermediate level of RAF/MEK/ERK signaling output. Hence, when drug resistant tumor cells are deprived of drug, the level of signaling output is increased leading to decreased proliferation. We sought to determine whether vemurafenib-resistant tumors in melanoma patients displayed similar evidence of drug dependency. In a small cohort of patients (n=42) with vemurafenib-resistant tumors for which CT scans were available following cessation of treatment (n=19), 14 out of 19 patients demonstrated evidence of reduced tumor growth velocity. Taken together, these data support the hypothesis that continuous treatment with vemurafenib can select for drug-resistant tumor cells that show a fitness deficit in the absence of the drug. Furthermore, we demonstrate in the preclinical setting that these observations can be leveraged therapeutically by using a discontinuous treatment regimen to forestall the emergence of resistance in two different PDX models. Consequently, these data may have important implications for the treatment of BRAF mutated melanoma patients with BRAF (V600E) inhibitors and suggest that altered dosing might delay or prevent the emergence of lethal drug resistant disease. Citation Format: Meghna Das Thakur, Rosalie Fisher, Fernando Salangsang, Allison Landman, William Sellers, Nancy Pryer, Mitchell Paul Levesque, Reinhard Dummer, Martin Gore, James Larkin, Martin McMahon, Darrin Stuart. Modeling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance. [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 LB-144. doi:10.1158/1538-7445.AM2013-LB-144

Abstract 5592: Modeling the evolution of resistance to RAF inhibitors in clinically relevant melanoma models

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Small molecule RAF inhibitors such as Vemurafenib have provided proof-of-concept that BRAFV600E is a key driver of proliferation and survival in melanoma, as evidenced by tumor responses in this patient population. Unfortunately, in many patients the tumor response is short-lived, as resistance to the inhibitor rapidly develops. Understanding the mechanisms that underlie drug resistance will be critical to providing improved treatment options for these patients. Here we model the emergence of resistance to selective RAF inhibitors, utilizing a limited passage BRAFV600E primary human melanoma xenograft model. This model, is expected to retain genetic diversity in a physiologically relevant microenvironment. Mice bearing HMEX1906 tumors were treated with one of two selective RAF inhibitors, Vemurafenib or the novel RAF inhibitor NVP-LGX818, at clinically-relevant doses, providing an opportunity to model pharmacokinetics, and replicate the selective pressure observed in patients. Long-term continuous treatment of BRAFV600E primary melanoma xenogafts with selective RAF inhibitors resulted in the appearance of resistant tumors over the course of 4 to 6 weeks. Pharmacodynamic (PD) analysis of individual tumors indicated that the MAPK pathway is suppressed in resistant tumors, although the degree and duration of suppression is less than in tumors that remain sensitive to RAF inhibitors. Furthermore, there is heterogeneity in the PD response, with greater extent and duration of P-Erk inhibition in sensitive tumors than in those resistant to RAF inhibitors. Biochemical analyses indicate that multiple receptor tyrosine kinases are activated and modulation of negative feedback loops may contribute to resistance. In addition increased expression of BRAFV600E, was detected in a subset of resistant tumors. Increasing the dose of RAF inhibitor administered to mice bearing resistant tumors leads to a significant yet transient tumor response, followed by tumor progression. Taken together with the PD data, this supports tumor cell heterogeneity, and rapid adaptation of tumors to the selective pressure being applied. Both in vivo drug withdrawal studies as well as in vitro analyses indicate that the resistant cells have become dependant on the presence of the RAF inhibitor for their survival. Based on these observations it appears that resistant tumor cells are less fit than sensitive tumor cells in the absence of drug. To test this hypothesis, mice were dosed on an intermittent regimen(4 weeks on / 2 weeks off) of RAF inhibitors. This regimen prevented the evolution of resistance for the duration of the 4 month experiment. These data could have implications in optimizing dose schedules to prevent the emergence of clinical resistance to targeted therapy. 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 5592. doi:1538-7445.AM2012-5592

Abstract B131: Modeling the evolution of resistance to RAF inhibitors in clinically relevant melanoma models.

Activating mutations in BRAF kinase are one of the most common genetic alterations in human melanoma, with over 50% of tumors expressing BRAFV600E. Small molecule RAF inhibitors such as vemurafenib have demonstrated proof-of-concept that BRAFV600E is a key driver of proliferation and survival in melanoma, as evidenced by tumor regression and prolonged survival in patients in late stage clinical trials. Unfortunately, in many patients the tumor response can be quite short-lived as resistance to the inhibitor rapidly develops. Understanding the mechanisms that underlie drug resistance will be critical to providing improved treatment options for these patients. To model the emergence of resistance to vemurafenib, we have established an early passage primary human melanoma xenograft model. In contrast to using tumor cell lines, this in vivo system recapitulates a more appropriate microenvironment, genetic diversity, and selective pressure observed in the clinical setting. The selection of resistance in vivo also reflects the clinical setting by modeling drug pharmacokinetics in patients. Treatment of these primary melanoma xenogafts with vemurafinib on a long term continuous dosing regimen at clinically relevant doses resulted in the appearance of resistant tumors over the course of 4 to 6 weeks. Pharmacodynamic (PD) analysis within individual tumors indicated that the RAF-MEK-ERK pathway is still suppressed by vemurafinib in resistant tumors, although the degree and duration of suppression is less than in sensitive tumors. Furthermore, the kinetics of pathway inhibition and recovery are different between each resistant tumor. While genetic analysis of resistant tumors is ongoing, biochemical analyses indicate that receptor tyrosine kinases (RTK) and modulation of negative feedback loops to RTK may be involved in resistance, as well as up-regulation of BRAFV600E expression in this model. Pharmacological evaluation of tumor response has provided insight into tumor cell populations and the evolution of resistance. We find that increasing the dose of drug administered to mice bearing resistant tumors leads to a significant yet transient tumor response, followed by tumor progression. Taken together with the PD data, this suggests there is a great deal of tumor cell heterogeneity, and that tumors are able to rapidly adapt to the selective pressure being applied. Further support for this was obtained by suspending drug treatment from mice with resistant tumors. Upon drug withdrawal, tumors initially regressed for several days to weeks, followed by re-growth. These data indicate that the adaptation which occurs within a tumor cell population under selective pressure make the cells less fit in the absence of drug and could have significant implications in optimizing dose schedules to prevent the emergence of resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B131.