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This supplementary data file contains 5 figures pertaining to the tumor distribution of MEK1 hotspot mutations, functional characterization of further MEK1/2 mutants with and without MEK or ERK inhibitor treatment, and the sequence paralogy alignment of MEK1 and MEK2. This file also contains 4 tables detailing hotspot analysis q values for MEK1/2 mutants, tumor incidence of MEK1 in-frame deletions, and a summary of MEK1/2 paralogous residues and their concordant activation status.

Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on MAP2K1 and MAP2K2 mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part in silico methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. In silico prediction accurately distinguished functional from benign MAP2K1 and MAP2K2 mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted in silico modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response.Significance:Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation.See related commentary by Whitehead and Sebolt-Leopold, p. 4042

Supplementary Figures S1-S8. Levels of RAS-GTP in melanoma cell lines (S1); Changes in pERK and cyclin D1 levels as function of time in cells treated with the MEK inhibitor PD0325901 (S2); Sk-Mel-113 (NF1 null) melanoma cells are resistant to AKT inhibition (S3); Variability in induction of pMEK levels following exposure to four allosteric MEK inhibitors (S4); Exon-capture deep sequencing via IMPACT of the NF1 gene in M308 shows a nonsense Q1070* mutation (S5); Expression of activated RAS is sufficient to confer resistance to vemurafenib in Sk-Mel-239 (BRAF V600E) cells (S6); Sensitivity of A375 (BRAF V600E) cells to vemurafenib (S7); The MAPK pathway and the inhibitors used in the study of NF1-null cells (S8).

Large scale clinical genomic sequencing efforts have revealed inactivating mutations in the RAS-GTPase Neurofibromin 1 (NF1) in a significant subset of melanomas. To date, immunotherapy and MAPK pathway-directed targeted therapies have been largely inactive in this molecularly defined cohort and immunogenic models that reflect the distinct co-mutation patterns found in NF1-mutant melanoma patients are lacking. Leveraging a population-scale tumor genomic profiling initiative, we identified TP53 as a gene significantly co-altered with NF1 in melanoma. We thus generated and molecularly characterized a cohort of genetically engineered mice with targeted deletion of NF1 in melanocytes. Melanocyte-specific, homozygous knockout of NF1 induced hyperpigmentation yet was insufficient for tumorigenesis. Addition of TP53 knockout and/or conditional activating mutation in BRAF (BRAFVE), resulted in melanoma formation with variable and high penetrance, respectively, along with histologic features consistent with human melanomas. Tumor latency and overall survival in NF1/TP53 double knockout mice was similar to NF1/BRAFVE double mutants. NF1 knockout did not shorten the latency to tumor formation in the setting of BRAFVE/TP53 mutation but did intensify melanocytic hyperpigmentation in all genetic backgrounds tested. To facilitate preclinical and functional studies, we derived 22 congenic cell lines from harvested mouse tumors from NF1 knockout mice, with and without BRAFVE mutation, and tested their sensitivity to targeted agents. As expected, loss of NF1 conditioned the response to BRAF inhibition, while NF1-mutant cells retained sensitivity to MEK inhibition. To abrogate the effects of adaptive RAS reactivation after MEK inhibitor therapy, combined MEK/SHP inhibition in NF1/TP53 knockout cells and BRAF/SHP inhibition in NF1/TP53/BRAFVE mutant cells strongly blunted ERK phosphorylation and cell proliferation better than single agent therapy. However, this response to the addition of SHP inhibition was transient and ERK rebound was driven by continued MEK activation and dependance. In syngeneic xenograft models of NF1/TP53/BRAFVE mutation, MEK inhibition alone, or in combination with RAF and/or SHP inhibition, induced tumor regression and delayed the onset of resistance and progression as compared to doublet RAF/SHP inhibitor therapy. Overall, we demonstrated the efficacy and feasibility of vertical MAPK pathway targeting in a novel cohort of genetically relevant mouse and cell line models of NF1-mutant melanoma and provide justification for future studies of vertical MAPK pathway targeting to achieve maximal ERK pathway inhibition in this molecularly defined patient cohort. Citation Format: Alexis M. Jones, Aphrothiti J. Hanrahan, Moriah H. Nissan, Sebastien Monette, Ziyu Chen, Wenhuo Hu, Sandra Misale, Isabell Schulze, Naresh Vasani, Cailian Liu, Xia Yang, Mohsen Abu-Akeel, Elisa de Stanchina, Nikolaus Schultz, Michael F. Berger, Neal Rosen, Taha Merghoub, David B. Solit. Vertical MAPK pathway targeting in novel genetically engineered mouse and cell line models of NF1-altered melanoma: the mSK-Mel murine cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2.

Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on MAP2K1 and MAP2K2 mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part in silico methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. In silico prediction accurately distinguished functional from benign MAP2K1 and MAP2K2 mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted in silico modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response. Significance: Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation. See related commentary by Whitehead and Sebolt-Leopold, p. 4042

Genomic sequencing of tumors is a routine part of cancer patient care. To address the need for a comprehensive resource that annotates the oncogenic effects and clinical actionability of somatic alterations in cancer, we developed OncoKB, a precision oncology knowledgebase. Since its introduction in 2016, OncoKB has grown to include annotations for >500 alterations in 682 genes. This includes 42 Level 1 genes (included in the FDA drug label), 12 Level 2 genes (included in the NCCN guidelines), and 29 Level 3A genes (predictive of drug response in well-powered clinical studies). We evaluated changes in the clinical actionability landscape and evolution of the OncoKB annotation rules and processes by comparing the AACR Project GENIE cohort (v8.1) annotated with the OncoKB version from February 2018 to that from November 2020. Even within a short window of time, this comparison reveals a significant shift of the proportion of samples that harbor a standard care alteration (Level 1 and 2), increasing from ~9% in 2018 to ~24% in 2020, and a Level 3A alteration, decreasing from ~11% to ~5%. This shift is partially attributable to the FDA approval of a PI3K inhibitor in PIK3CA-mutant ER+/HER2- breast cancer, approval of RAF inhibitors in BRAF V600E mutant anaplastic thyroid cancer and colorectal cancer, approval of NTRK-inhibitors in NTRK fusion-positive solid tumors, FGFR-inhibitor approval in FGFR2 fusion-positive bladder cancer and cholangiocarcinoma, RET-inhibitor approval in RET fusion-positive thyroid cancer and non-small cell lung cancer and expansion of indications for PARP inhibitors to include prostate cancer. The tumor agnostic approval of the checkpoint blockade inhibitor, pembrolizumab, in TMB-H solid tumors additionally gave rise to a ~4% increase in Level 1 samples. Analysis of the AACR Project GENIE cohort also revealed significant changes to the OncoKB process, including those reflected in the updated OncoKB Levels of Evidence v2.0. The refined levels system deprioritized the significance of standard care biomarkers when present in indications outside of the FDA-approved/NCCN listed indication. This change was based on clinical data demonstrating that patients with investigational predictive biomarkers for a specific tumor type based on compelling clinical evidence from phase 3 trials (currently Level 3A) are more likely to experience clinical benefit compared to patients with predictive biomarkers that are considered standard care in a different tumor type (previously Level 2B, currently Level 3B), and is consistent with guidelines published by ASCO/AMP/CAP and ESMO. Knowledgebases such as OncoKB have emerged as key informational resources that the clinical oncology and scientific communities have used to rapidly connect sequencing results to clinical actionability. Their utility depends on their ability to stay abreast of clinical data and seamlessly adapt their rules and processes to the evolving field of precision medicine. Citation Format: Sarah P. Suehnholz, Ritika Kundra, Hongxin Zhang, Shaleigh Smith, Moriah Nissan, Yifu Yao, Lindsay LaFave, Kinisha Gala, Linde Miles, Maria E. Arcila, Marc Ladanyi, Michael F. Berger, Ahmet Zehir, Aijaz Syed, Julia Rudolph, Paul Sabbatini, Ross Levine, Ahmet Dogan, Jianjiong Gao, David Solit, Nikolaus Schultz, Debyani Chakravarty. Evolution of OncoKB, a precision oncology knowledgebase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 99.

Purpose With prospective clinical sequencing of tumors emerging as a mainstay in cancer care, an urgent need exists for a clinical support tool that distills the clinical implications associated with specific mutation events into a standardized and easily interpretable format. To this end, we developed OncoKB, an expert-guided precision oncology knowledge base. Methods OncoKB annotates the biologic and oncogenic effects and prognostic and predictive significance of somatic molecular alterations. Potential treatment implications are stratified by the level of evidence that a specific molecular alteration is predictive of drug response on the basis of US Food and Drug Administration labeling, National Comprehensive Cancer Network guidelines, disease-focused expert group recommendations, and scientific literature. Results To date, > 3,000 unique mutations, fusions, and copy number alterations in 418 cancer-associated genes have been annotated. To test the utility of OncoKB, we annotated all genomic events in 5,983 primary tumor samples in 19 cancer types. Forty-one percent of samples harbored at least one potentially actionable alteration, of which 7.5% were predictive of clinical benefit from a standard treatment. OncoKB annotations are available through a public Web resource ( http://oncokb.org ) and are incorporated into the cBioPortal for Cancer Genomics to facilitate the interpretation of genomic alterations by physicians and researchers. Conclusion OncoKB, a comprehensive and curated precision oncology knowledge base, offers oncologists detailed, evidence-based information about individual somatic mutations and structural alterations present in patient tumors with the goal of supporting optimal treatment decisions.

OncoKB is a precision oncology knowledge base that annotates the oncogenic effects and clinical actionability of somatic alterations in cancer. Initially focused on solid tumors, OncoKB was introduced in 2016 with >200 genes and almost 3000 somatic alterations via a public website (oncokb.org) and through the cBioPortal for Cancer Genomics. OncoKB now contains annotations for &gt5000 alterations in 642 genes. This includes 30 Level 1 alterations (included in the FDA drug label; a growth of 114% since 2016), 15 Level 2 alterations (included in the NCCN guidelines; 50% growth), and 38 Level 3A alterations (predictive of drug response in well-powered clinical studies; 65% growth). OncoKB now also supports hematologic malignancies with two new levels of evidence systems that encompass diagnostic and prognostic implications (in addition to therapeutic implications) and 288 heme-specific alterations in 156 newly curated cancer-associated genes. At MSK, OncoKB is used for the annotation of 1000 molecular patient reports per month. To assess the clinical utility of OncoKB and changes in the frequency of actionable alterations, we performed a comparison between the AACR Project GENIE cohort from 2017 and the most recent one (Table 1). With an increased number of tumor types and greater inclusion of hematologic malignancies, the overall potential actionability rate increased by 3.6 percentage points. A shift in access to targeted cancer therapies is also observed, where Level 1 or 2 alterations increased over 5 percentage points and Level 3 alterations decreased by ~3 percentage points, perhaps reaping the benefits of recent successful phase III trials. August 2017 (v1.1)December 2019 (v7.2)AACR Project GENIE cohort size18,80480,248Tumor types with >100 samples3151Hematologic malignancies included29Level 1 or 2 annotation7.3%12.9%Level 3A annotation6.4%4.7%Level 3B annotation17.8%17.5%Total potential actionability31.5%35.1% While only a subset of patients with targetable alterations will benefit from treatments, there is ample evidence that targeted cancer therapies can have profound and durable clinical activity. Knowledgebases such as OncoKB have become a key component to support clinical decision making, and there is a continued need to expand their capabilities while maintaining a nuanced approach to annotation. Citation Format: Sarah Suehnholz, Hongxin Zhang, Moriah Nissan, Ritika Kundra, Jing Su, Lindsay LaFave, Kinisha Gala, Chad Vanderbilt, Maria Arcila, Marc Ladanyi, Michael Berger, Ahmet Zehir, Julia E. Rudolph, Paul Sabbatini, Ross Levine, Ahmet Dogan, Jianjiong Gao, David B. Solit, Nikolaus Schultz, Debyani Chakravarty. OncoKB, a precision oncology knowledgebase [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3208.

Background: Over 300 somatic molecular variants in hematologic diseases are either specified as diagnostic criteria in the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues, recognized as potentially actionable biomarkers in the National Comprehensive Cancer Network (NCCN) compendia, or supported by published well-powered clinical studies. Moreover, new molecular alterations with potential clinical implications in hematologic disease are continuously emerging in the scientific literature. These have critical use for a wide spectrum of clinicians, including hematopathologists who diagnose patient-specific hematologic malignancies, heme-oncologists who direct patient care, and clinical trial nurses who assist patients in finding appropriate clinical trials. Importantly, the utility of this information critically depends on the clinician's ability to interpret the significance of variants in a point-of-care setting. Therefore, there is an urgent and unmet need for a clinical decision support system that 1) distills the clinical implications associated with molecular alterations into a standardized and easily interpretable format and 2) democratizes access of this information to all members of the heme-oncology community. Methods: OncoKB is an established expert-guided precision oncology knowledge base that annotates the oncogenic effect and therapeutic implications of somatic molecular alterations (Chakravarty, D. et al., JCOPO, 2017). Previously, OncoKB was focused primarily on solid tumor mutation annotation. Recently, we expanded OncoKB to include alterations in hematologic malignancies. The heme-specific annotation efforts were guided by heme-oncology and hematopathology physician scientists at Memorial Sloan Kettering (MSK). Supplementing the previously published therapeutic levels of evidence (Fig. 1a), we further added level of evidence systems for diagnostic and prognostic implications (Fig. 1b, c). These three sets of evidence levels are consistent with the criteria set forth by the joint consensus of the ASCO/CAP/AMP guidelines (Li, MM. et al., J Mol Diagn, 2017). We assigned the newly curated heme-specific molecular alterations with diagnostic, prognostic or therapeutic levels of evidence, when applicable. Finally, we annotated and analyzed 1569 hematologic tumor samples from the AACR Project GENIE (release 6.1) with these levels of evidence. Results: In addition to alterations with both solid and heme clinical implications already curated in OncoKB, we annotated 288 unique heme-specific mutations, fusions, and copy number alterations in 156 newly curated cancer-associated genes. Based on MSK-expert consensus, the WHO and NCCN guidelines, and the scientific literature, we identified a total of 192 alterations with unique diagnostic levels of evidence, 65 alterations with unique prognostic levels of evidence and 55 alterations with unique therapeutic levels of evidence across 13 major hematologic tumor types (Fig. 2). To test the utility of OncoKB, we annotated all genomic events in 1569 heme cancer samples in 89 hematologic malignancies in the AACR GENIE cohort (V6.1) (Fig. 3a). Thirty-eight percent of samples harbored at least one potentially actionable alteration, and 8% were predictive of clinical benefit from an FDA-approved drug (Fig. 3b). Conclusions: OncoKB heme data is publicly available both through the web resource http://oncokb.org and through incorporation into the cBioPortal for Cancer Genomics. Heme-specific molecular alterations are used to make an accurate diagnosis, inform prognosis, optimize the use of stem cell transplant, and to link patients with the optimal mechanism-based therapies in the clinical trial setting and in routine clinical practice. This is the first study to annotate and analyze actionability of heme samples. In this proof-of-principle study, we demonstrate the ability to annotate clinical samples with their diagnostic, prognostic and therapeutic implications in a point-of-care setting. Disclosures Roshal: Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Ho:Invivoscribe, Inc.: Honoraria. Knorr:Fate Therapeutics: Patents & Royalties. LaFave:Epizyme: Patents & Royalties. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Berger:Roche: Consultancy. Solit:Pfizer: Consultancy; Lilly Oncology: Honoraria; Vivideon Therapeutics: Consultancy; Loxo Oncology: Consultancy, Equity Ownership; Illumina: Consultancy. Dogan:Celgene: Consultancy; Seattle Genetics: Consultancy; Corvus Pharmaceuticals: Consultancy; Roche: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy. Levine:C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Gilead: Consultancy; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding.

Summary: Resistance to RAF inhibitors is generally accompanied by reactivation of extracellular signal-regulated kinase (ERK) signaling. SCH772984, a selective, ATP-competitive inhibitor of ERK1 and ERK2, is effective in BRAF-mutant models in which resistance is the result of ERK reactivation. SCH772984 may also have a role in the treatment of tumors in which ERK is dysregulated by mutant RAS, NF1, or activated receptor tyrosine kinases, settings in which current RAF inhibitors are ineffective. Cancer Discov; 3(7); 719–21. ©2013 AACR. See related article by Morris et al., p. 742

Melanoma is a disease characterized by lesions that activate ERK. Although 70% of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the remaining 30% are largely undefined. Vemurafenib, a selective inhibitor of RAF kinases, has clinical utility restricted to BRAF-mutant tumors. MEK inhibitors, which have shown clinical activity in NRAS-mutant melanoma, may be effective in other ERK pathway-dependent settings. Here, we investigated a panel of melanoma cell lines wild type for BRAF and NRAS to determine the genetic alteration driving their transformation and their dependence on ERK signaling in order to elucidate a candidate set for MEK inhibitor treatment. A cohort of the BRAF/RAS wild type cell lines with high levels of RAS-GTP had loss of NF1, a RAS GTPase activating protein. In these cell lines, the MEK inhibitor PD0325901 inhibited ERK phosphorylation, but also relieved feedback inhibition of RAS, resulting in induction of pMEK and a rapid rebound in ERK signaling. In contrast, the MEK inhibitor trametinib impaired the adaptive response of cells to ERK inhibition, leading to sustained suppression of ERK signaling and significant antitumor effects. Notably, alterations in NF1 frequently co-occurred with RAS and BRAF alterations in melanoma. In the setting of BRAF(V600E), NF1 loss abrogated negative feedback on RAS activation, resulting in elevated activation of RAS-GTP and resistance to RAF, but not MEK, inhibitors. We conclude that loss of NF1 is common in cutaneous melanoma and is associated with RAS activation, MEK-dependence, and resistance to RAF inhibition. Cancer Res; 74(8); 2340–50. ©2014 AACR.

11583Background: Sequencing of tumor DNA is becoming part of routine clinical care, with the goal of matching patients to approved treatments or clinical trials based on specific mutations. However...

Activating mutations in the BRAF gene are among the most prevalent kinase mutations in human cancer. BRAF mutations are most frequent in patients with melanoma where they occur in approximately 50% of patients with advanced disease. Remarkable clinical activity has recently been reported with highly selective RAF inhibitors in melanoma patients whose tumors harbor V600E BRAF mutations. The response rates of RAF inhibitors in patients with BRAF-mutant melanomas far exceed the activity level of any prior therapy studied in this disease. The results suggest that we have entered an era of personalized therapy for patients with metastatic melanoma in which treatment selection will be guided by BRAF mutational status. This review will discuss the strengths, weaknesses, opportunities and threats ("SWOT") of developing RAF and MEK selective inhibitors as anti-cancer therapies, recent insights into the mechanisms of intrinsic and acquired resistance to these agents, and current efforts to develop mechanism-based combination therapies.

Though two-thirds of cutaneous melanomas harbor activating mutations in the BRAF and NRAS genes, the alterations that drive tumor progression in the roughly 30% of melanoma tumors wild-type for BRAF and NRAS remain largely uncharacterized. The selective RAF inhibitors vemurafenib and dabrafenib inhibit MAPK pathway activity only in BRAF mutant cells, thus their clinical utility is restricted to patients with BRAF mutant tumors. MEK inhibitors, which have broader antitumor activity and have shown promising activity in NRAS mutant melanoma, may be effective in a broader range of MAPK pathway-dependent tumors. We performed a functional and genomic analysis of melanoma cell lines wild-type for BRAF and NRAS to determine whether occult mutations in RAS signaling were present. Elevated RAS-GTP was common in BRAF/NRAS wild-type melanoma cell lines, a subset of which exhibited total loss of NF1 protein expression. The proliferation of NF1-null melanoma cells was dependent upon MEK, though the cellular potency of several allosteric MEK inhibitors that are currently in clinical testing (PD0325901, AZD6244, MEK162 and trametinib) varied widely in NF1-null melanoma cells. The greatest antitumor activity was noted with trametinib, a MEK inhibitor that also blocks RAF mediated phosphorylation of MEK. Notably, alterations in NF1 also co-occurred with RAS and BRAF mutations in both cell lines and human melanomas. In the setting of BRAF co-mutation, loss of NF1 abrogated upstream negative feedback on RAS activation resulting in constitutive expression of RAS-GTP and resistance to RAF but not MEK inhibition. In summary, NF1 loss is a common event in cutaneous melanoma that is associated with RAS activation, MEK dependence and RAF inhibitor resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C138. Citation Format: Moriah H. Nissan, Christine Pratilas, Alexis Jones, Helen Won, Li Kong, Zhan Yao, Taha Merghoub, Antoni Ribas, Paul Chapman, Rona Yaeger, Barry Taylor, Nikolaus Shultz, Michael F. Berger, Neal Rosen, David B. Solit. Loss of NF1 in melanoma cell lines is associated with active Ras and dependence on MEK even in the absence of BRAF or NRAS mutation. [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 C138.