Eva M. Goetz

MAP Kinase Pathway Alterations in BRAF-Mutant Melanoma Patients with Acquired Resistance to Combined RAF/MEK Inhibition

Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.

Reactivation of ERK Signaling Causes Resistance to EGFR Kinase Inhibitors

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors is limited by the development of drug resistance. The irreversible EGFR kinase inhibitor WZ4002 is effective against the most common mechanism of drug resistance mediated by the EGFR T790M mutation. Here, we show, in multiple complementary models, that resistance to WZ4002 develops through aberrant activation of extracellular signal-regulated kinase (ERK) signaling caused by either an amplification of mitogen-activated protein kinase 1 (MAPK1) or by downregulation of negative regulators of ERK signaling. Inhibition of MAP-ERK kinase (MEK) or ERK restores sensitivity to WZ4002 and prevents the emergence of drug resistance. We further identify MAPK1 amplification in an erlotinib-resistant EGFR-mutant non-small cell lung carcinoma patient. In addition, the WZ4002-resistant MAPK1-amplified cells also show an increase both in EGFR internalization and a decrease in sensitivity to cytotoxic chemotherapy. Our findings provide insights into mechanisms of drug resistance to EGFR kinase inhibitors and highlight rational combination therapies that should be evaluated in clinical trials.

ERK Mutations Confer Resistance to Mitogen-Activated Protein Kinase Pathway Inhibitors

The use of targeted therapeutics directed against BRAF(V600)-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAF(V600)-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor-resistant alleles were sensitive to RAF/MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents.

Phenotypic Characterization of a Comprehensive Set of MAPK1/ERK2 Missense Mutants

Tumor-specific genomic information has the potential to guide therapeutic strategies and revolutionize patient treatment. Currently, this approach is limited by an abundance of disease-associated mutants whose biological functions and impacts on therapeutic response are uncharacterized. To begin to address this limitation, we functionally characterized nearly all (99.84%) missense mutants of MAPK1/ERK2, an essential effector of oncogenic RAS and RAF. Using this approach, we discovered rare gain- and loss-of-function ERK2 mutants found in human tumors, revealing that, in the context of this assay, mutational frequency alone cannot identify all functionally impactful mutants. Gain-of-function ERK2 mutants induced variable responses to RAF-, MEK-, and ERK-directed therapies, providing a reference for future treatment decisions. Tumor-associated mutations spatially clustered in two ERK2 effector-recruitment domains yet produced mutants with opposite phenotypes. This approach articulates an allele-characterization framework that can be scaled to meet the goals of genome-guided oncology.

Genomic Correlate of Exceptional Erlotinib Response in Head and Neck Squamous Cell Carcinoma

IMPORTANCE Randomized clinical trials demonstrate no benefit for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in unselected patients with head and neck squamous cell carcinoma (HNSCC). However, a patient with stage IVA HNSCC received 13 days of neoadjuvant erlotinib and experienced a near-complete histologic response. OBJECTIVE To determine a mechanism of exceptional response to erlotinib therapy in HNSCC. DESIGN, SETTING, AND PARTICIPANTS Single patient with locally advanced HNSCC who received erlotinib monotherapy in a window-of-opportunity clinical trial (patients scheduled to undergo primary cancer surgery are treated briefly with an investigational agent). Whole-exome sequencing of pretreatment tumor and germline patient samples was performed at a quaternary care academic medical center, and a candidate somatic variant was experimentally investigated for mediating erlotinib response. INTERVENTION A brief course of erlotinib monotherapy followed by surgical resection. MAIN OUTCOMES AND MEASURES Identification of pretreatment tumor somatic alterations that may contribute to the exceptional response to erlotinib. Hypotheses were formulated regarding enhanced erlotinib response in preclinical models harboring the patient tumor somatic variant MAPK1 E322K following the identification of tumor somatic variants. RESULTS No EGFR alterations were observed in the pretreatment tumor DNA. Paradoxically, the tumor harbored an activating MAPK1 E322K mutation (allelic fraction 0.13), which predicts ERK activation and erlotinib resistance in EGFR-mutant lung cancer. The HNSCC cells with MAPK1 E322K exhibited enhanced EGFR phosphorylation and erlotinib sensitivity compared with wild-type MAPK1 cells. CONCLUSIONS AND RELEVANCE Selective erlotinib use in HNSCC may be informed by precision oncology approaches.

Mechanisms of Resistance to Mitogen-Activated Protein Kinase Pathway Inhibition in BRAF-Mutant Melanoma.

Anticancer drug resistance remains a crucial impediment to the care of many patients with cancer. Although the exact mechanisms of resistance may differ for each therapy, common mechanisms of resistance predominate, including drug inactivation or modification, mutation of the target protein, reduced drug accumulation, or bypass of target inhibition. With the discovery and use of targeted therapies (such as small-molecule kinase inhibitors), resistance has received renewed attention-especially in light of the dramatic responses that may emerge from such therapeutics in particular genetic or molecular contexts. Recently, the mitogen-activated protein kinase (MAPK) pathway has become exemplary in this regard, since it is activated in many different cancers. Drugs targeting RAF and MAPK kinase (MEK) are currently in clinical trials for the treatment of several types of cancer. Vemurafenib, a selective RAF kinase inhibitor recently approved for the treatment of BRAF(V600E) melanoma, shows strong efficacy initially; however, the development of resistance is nearly ubiquitous. In vitro testing and analysis of patient samples have uncovered several mechanisms of resistance to RAF inhibition. Surprisingly, mutations in the drug-binding pocket have not thus far been observed; however, other alterations at the level of RAF, as well as downstream activation of MEK and bypass of MEK/extracellular signal-regulated kinase (ERK) signaling altogether, confer resistance to vemurafenib. Looking forward, combined RAF and MEK inhibitor treatments may improve efficacy-yet we must anticipate mechanisms of resistance to this combination as well. Therefore, understanding and/or determining the mechanism of resistance are paramount to effective cancer treatment.

Abstract 2150: Alternative treatment strategies to overcome MAPK inhibitor resistance in melanoma

Inhibitors of the BRAF/MEK/ERK pathway have promise in the treatment of BRAF mutant melanoma and other MAPK activated cancers. Treatment of metastatic melanoma patients with the combination of RAF + MEK inhibitor has extended progression free survival over single agent therapy. However, in a large subset of patients, resistance invariably develops. Most clinical models show reactivation of the MAPK pathway in resistant samples, suggesting further downregulation of MAPK signaling might be beneficial. Therefore, we took BRAF mutant melanoma grown to resistance by continuous culture in dabrafenib plus trametinib (RAF + MEK inhibitor) or SCH772984 (ERK inhibitor), and treated them with a triple combination of RAF+MEK+ERK inhibitors. We found cells resistant to RAF+MEK inhibitors were cross-resistant to ERK inhibitor, but sensitive to RAF+MEK+ERK inhibitors. Conversely, cells resistant to the ERK inhibitor were cross-resistant to RAF+MEK inhibitor, but sensitive to the triple combination. However, given the possible deleterious side effects of a triple combination in patients, we explored whether alternative dosing and timing strategies could delay development of resistance. We compared chronic dosing of RAF+MEK or ERK inhibitors to weekly exposures alternating between RAF+MEK and ERK inhibitors. This alternative dosing strategy delayed development of resistance in vitro over chronic dosing, while also resulting in a slower proliferation rate in resistant cells. In addition, cells exposed to a higher dose of inhibitor for a shorter period of time was equivalent to chronic exposure. These studies suggest that alternative dosing strategies (other than chronic exposure) may prevent development of resistance, and allow for treatment of patients with higher order combinations and less side effects. Citation Format: Eva Goetz, Bokang Rabasha, Levi Garraway. Alternative treatment strategies to overcome MAPK inhibitor resistance in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2150.

Abstract PR02: Identification of ERK1/2 mutations that confer resistance to MAPK pathway inhibitors

Recent treatment advances in metastatic BRAF mutant melanoma using targeted therapy have increased progression free survival by many months. However, resistance to these targeted agents is a common occurrence, with relapse occurring in almost every case. Many of the alterations that confer clinical resistance to RAF and MEK inhibitors in BRAF mutant melanoma reactivate the MAPK pathway, but remain sensitive to ERK inhibition in vitro. Prospectively determining mechanisms of resistance to MAPK pathway inhibitors, such as an ERK inhibitor, before clinical trials begin may inform which patients will respond to therapy, potential markers of resistance, or best therapy combinations. Previous studies have shown that in vitro random mutagenesis screens can select specific alleles seen in resistant patient populations. Since ERK inhibitors are entering clinical trials, we wanted to determine mutations in ERK1 and ERK2 that could confer resistance to ERK or RAF/MEK inhibition. Therefore, we expressed dox-inducible ERK1 or ERK2 mutant libraries in A375 cells, which were then treated with MAPK inhibitor(s) and doxycycline until resistant cells emerged. Exogenous ERK1 or ERK2 was analyzed in resistant cells by massively parallel DNA sequencing. We validated 15 alleles that conferred resistance to the ERK inhibitor, VX-11e, and 11 alleles that conferred resistance to RAF, MEK, or RAF+MEK inhibition (dabrafenib and trametinib). Some resistance alleles were analogous between ERK1 and ERK2, while others were specific to ERK1 or ERK2. ERK inhibitor resistant alleles were localized to areas surrounding the ATP/drug binding site, including the glycine-rich loop, c-helix, and activation loop. These alleles were also cross-resistant to another ERK inhibitor, SCH-772984, albeit some only at lower concentrations. The RAF/MEK inhibitor resistant residues were located in the c-helix, activation loop, and common docking domain. Only a single ERK1 allele was found to confer resistance to RAF + MEK and ERK inhibition, suggesting that resistance mutations in ERK might be suppressed if RAF, MEK, and ERK inhibitors are given in combination. In addition, an ERK mutation arising in a patient treated with an ERK inhibitor would still be sensitive to RAF/MEK inhibition and vice versa. Screening for these alleles before administering therapy or after relapse may help inform patient treatment. Citation Format: Eva M. Goetz, Mahmoud Ghandi, Daniel Treacy, Nikhil Wagle, Levi A. Garraway. Identification of ERK1/2 mutations that confer resistance to MAPK pathway inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR02.

Abstract C221: A genome-wide RNA interference screen identifies synthetic lethal interactions with the BRAF oncogene.

Oncogenic mutations in BRAF are found in 50-70% of malignant melanomas and in ∼8% of all cancers. Activating BRAF mutations confer exquisite dependency on RAF/MEK/ERK signaling and RAF and MEK inhibitors achieved striking clinical response in the majority of patients with BRAFV600E-mutant melanoma. However, nearly all patients progress to therapeutic resistance and there are limited treatment options for patients with relapsed tumors. Identification of genes that are essential only in the presence of BRAFV600E mutation may inform alternative as well as combinatorial therapeutic approaches. Here, we used systematic RNA interference to discover synthetic lethal interactions in melanoma cells carrying BRAFV600E mutation. In addition, Project Achilles at the Broad Institute reported systematic loss-of-function studies in more than 100 human cancer cell lines. We intersected the list of genes that are synthetic lethal with BRAFV600E mutation in our melanoma study and that of Project Achilles. As a result, we have identified novel “druggable” targets for cells driven by BRAFV600E mutation. Our extensive in vitro and in vivo validation has confirmed and established key synthetic lethal interactions with BRAFV600E mutation. One major mechanism of acquired resistance to RAF and MEK inhibition is reactivation of MEK/ERK pathway. Importantly, we demonstrated that these synthetic lethal genes are also essential in the context of acquired resistance to RAF and MEK inhibition. Together, our study has identified novel targets for the treatment of oncogenic BRAF-driven melanoma and suggests targeting these genes as novel therapeutic strategies to overcome acquired resistance to RAF and MEK inhibition. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C221. Citation Format: Chengyin Min, David J. Konieczkowski, Christine Kwon, Marika Linja, Krishna Vasudevan, Barbara Weir, Eva Goetz, Levi Garraway. A genome-wide RNA interference screen identifies synthetic lethal interactions with the BRAF oncogene. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C221.