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Clin Cancer Res. Author manuscript; available in PMC 2016 December 15. Published in final edited form as: Clin Cancer Res. 2015 December 15; 21(24): 5412–5414. doi:10.1158/1078-0432.CCR-14-3132.
CCR 20th Anniversary Commentary: MAPK/ERK Pathway Inhibition in Melanoma–Kinase Inhibition Redux Diwakar Davar and John M. Kirkwood Division of Hematology-Oncology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
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Summary In the January 15, 2012, issue of Clinical Cancer Research, Kirkwood and colleagues published a study comparing the MEK inhibitor selumetinib to temozolomide in unselected metastatic melanoma. Although selumetinib did not improve survival or response, most responders had BRAF activating mutations, and selumetinib has since demonstrated efficacy in BRAF-mutant melanoma. This study laid the groundwork for the evaluation of BRAF/MEK inhibitors in BRAFmutant melanoma.
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Major advances in the treatment of advanced melanoma over the past three years are directly linked to advances in our understanding of how activating mutations in canonical signaling and tumor-mediated T-cell dysfunction moderate melanoma tumorigenesis and progression. Deep sequencing approaches established that activating mutations in mitogen activated protein kinase (MAPK) pathway constituents (BRAF/NRAS) are common, mutually exclusive and carry oncogenic potential in human cancers (1). RAS mutations (typically at G12, G13, or Q61 residues) impair GTP hydrolysis resulting in constitutive activation of GTP-bound RAS. BRAF is a serine/threonine protein kinase and mutations (most commonly a glutamic acid substitution for valine at codon 600 - V600E) occur within the kinase domain’s activation segment. Oncogenic BRAF and NRAS mutations result in constitutive activation of downstream targets including MAPK/ERK kinases 1 and 2 (MEK1/MEK2) and unrestrained proliferation. Seminal work by Bastian and colleagues identified distinct patterns of mutations in chronically sun damaged skin (higher mutation load, lower BRAF/ NRAS mutant incidence) and intermittent sun exposed skin (lower mutation load, BRAF/ NRAS mutant) – underscoring the concept of melanomas a heterogeneous disease with multiple genetically distinct subtypes (2). Concurrently, Allison and Honjo identified CTLA-4 and PD-1 to be key negative regulatory immune checkpoints hijacked by tumors to
Corresponding Author: John M. Kirkwood, Division of Hematology-Oncology, University of Pittsburgh Medical Center, 5117 Centre Avenue, Pittsburgh, PA 15232. Phone: 412-623-7707; Fax: 412-623-7704; [email protected]. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed by the other author. Authors’ Contributions Conception and design: D. Davar, J.M. Kirkwood Writing, review, and/or revision of the manuscript: D. Davar, J.M. Kirkwood Disclaimer The content is solely the responsibility of the authors and does not necessarily represent the official views of the NCI or the NIH.
Davar and Kirkwood
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subvert antitumor immunity and mediate tolerogenic tumor microenvironments (3, 4). Subsequent work has since identified multiple other positive and negative checkpoints and their characterization in murine and human models of various cancers is ongoing.
Author Manuscript
Consequently, agents targeting the MAPK pathway and inhibitory immune checkpoints were developed and tested. In melanoma, 6 new agents have been approved including two targeting BRAF (vemurafenib and dabrafenib), one targeting MEK (trametinib), and CTLA-4 (ipilimumab) as well as two targeting PD-1 (pembrolizumab and nivolumab). Several other agents in each class are under evaluation including BRAF inhibitor encorafenib (LGX818, Array BioPharma); MEK inhibitors cobimetinib (GDC-0973/XL518, Exelixis) and binimetinib (MEK162, Array BioPharma); PD-1 inhibitors AMP-224 and AMP-514 (Amplimmune and GlaxoSmithKlein), pidilizumab (CT-011, Cure Tech); and PD-L1 inhibitors BMS-936559 (Bristol-Meyers Squibb), MEDI4736 (MedImmune and AstraZeneca), Atezolizumab (MPDL3280A, Roche), and Avelumab (MSB0010718C, Merck).
Author Manuscript
The use of monoclonal antibodies targeting inhibitory immune checkpoints in melanoma is marked by relatively low response rates that however, tend to be durable. Pooled analyses from multiple phase II/III trials reveal that ipilimumab has a lower response rate (11%–15%) compared to nivolumab (30%–35%) and pembrolizumab (45%–50%); with commensurately doubled 2-/3- year survival rates for PD-1 inhibitors compared to ipilimumab (5). Combined inhibition of both CTLA-4 and PD-1 increases response rates (to ~50%) and median progression-free survival, albeit with significant attendant toxicity (55% grade 3/4 adverse events) (5). Conversely, BRAF inhibitor use is marked by higher initial response rates (45– 53%) with inevitable progression at a median of 6–9 months typically though the acquisition of resistance mutations that reactivate the MAPK pathway. Addition of MEK inhibitors improves response rate (64–76%) and response duration (9–11 months) while reducing the incidence of cutaneous toxicities – though progression is similarly inevitable (5). Initial development of MAPK pathway inhibitors proceeded concurrently with inhibitors of MEK (AZD8330/ARRY-704, PD0325901, XL518, RDEA119, AS703026, CH5126766, CL-1040, GSK1120212, AZD6244) and RAF (GDC-0879, RAF265, ARQ 680, XL281/ BMS-908662, GSK2118436, PLX4032, sorafenib) (6). Importantly, initial clinical development (CL-1040) occurred prior to the identification of BRAF mutations in human cancer; and initial trials were not selectively enriched for patients with NRAS/BRAF mutated tumors or cancers in which these oncogenic mutations were most commonly detected such as melanoma (6).
Author Manuscript
Smalley et al first reported the antitumor activity of AZD6244 (ARRY-142886) in melanoma in a cell culture model where BRAF V600E mutant (WM35, WM793, 1205Lu, and 451Lu), NRAS Q61R mutant (SbCl2, WM2032, WM1361a, and WM852) and wild type (C8161) lines revealed that AZD6244 inhibited melanoma growth additively with docetaxel in vitro and in vivo (7). Further studies characterized selumetinib (AZD6244/ ARRY-142886) as an inhibitor of both MEK1 (IC50 14nM) and ERK1/2 phosphorylation (IC50 10nM) and led to evaluation of selumetinib in phase I trials against multiple tumor types (6).
Clin Cancer Res. Author manuscript; available in PMC 2016 December 15.
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In the phase II study we reported, 200 patients with advanced stage III/IV melanoma were assigned at random to receive either temozolomide or selumetinib (8). Most patients had cutaneous primaries (73%), but uveal (10%), mucosal (3%) and unknown primary tumors (14%) were also included. BRAF/NRAS and GNAQ mutation status were retrospectively analyzed in the cutaneous and uveal primaries although enrolment did not stratify patients on mutational status. Of the 158 samples that had adequate archival tissue for evaluation, 73 tumors (46% of tested samples or 50% of cutaneous primaries) were BRAF mutant; 28 tumors (18% of tested samples or 19% of cutaneous primaries) were NRAS mutant; only 4 tumors (3% of tested samples, or 20% of uveal primaries) were GNAQ mutant. The primary end-point of this trial was progression-free survival (PFS). Sample size of 182 (91 per arm) was calculated to provide 80% power to detect improvement in PFS with a hazard ratio of 0.74 (overall) at a one-sided 20% significance level, which would represent a HR 0.67 among BRAF mutant. PFS comparisons assumed that BRAF and NRAS mutant melanoma comprised 60% and 30% of study participants respectively; and that selumetinib would have equal effect in NRAS mutant and BRAF wild type patients, a reasonable assumption given prior reports of selumetinib efficacy in the BRAF/NRAS wild type C8161 cell line. We reported no significant difference in response or PFS between the treatment arms among unselected patients and in patients with tumors displaying BRAF mutation. Response rates in both arms were low (6% vs. 9%) – and surprisingly lower in the selumetinib arm. Further, patients assigned to selumetinib therapy had lower overall survival even after adjusting for imbalances in adverse prognostic factors between the two arms. Notably, 5 of 6 selumetinib responders and 1 of the 2 responding patients who had been crossed over to selumetinib from temozolomide post-progression were identified to be BRAF mutants.
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Several aspects of clinical trial design may have contributed to these findings. First, the study was likely underpowered to detect a true difference between treatment arms, given the discrepancy between the assumed (pre-study) and observed (on-study) proportions of BRAF/ NRAS mutations, despite the increase in enrollment (to 200) from an original accrual target (of 182). Second, subsequent reports of atypical BRAF/KRAS (G464E and G12D respectively) mutations in the C8161 cell line raises doubt as to selumetinib’s efficacy in true BRAF/NRAS wild type patients (9). Third, patients assigned to selumetinib had a higher incidence of known adverse prognostic features including worse WHO performance status and higher lactate dehydrogenase levels. Although these differences were adjusted for in the final analysis of overall survival, the non-significant p-values preclude definitive conclusions regarding the efficacy or non-efficacy of selumetinib, in either the overall or BRAF/NRAS mutant populations. Finally, given that the study allowed crossover on investigator assessed progression, selumetinib efficacy is difficult to ascertain as although overall survival and PFS statistics were reported separately, unequal crossover suggests an element of investigator bias. Concurrently, two other phase II monotherapy studies in nonsmall cell lung cancer and colorectal cancer reported that selumetinib did not offer a response and/or survival advantage over standard chemotherapy (10, 11). Subsequent evaluation in metastatic uveal, BRAF wild type (in combination with docetaxel) and BRAF mutant (in combination with dacarbazine) melanoma suggested that selumetinib improved PFS and response rates without significant improvements in overall survival (12–14).
Clin Cancer Res. Author manuscript; available in PMC 2016 December 15.
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Lessons learned from these studies have fundamentally altered studies the next generation MEK inhibitors. MEK 1/2 inhibitor tremetinib (GSK1120212) was evaluated in a tiered approach that is characteristic of the current paradigm of targeted therapy development: 1) highly specific and potent candidate (trametinib MEK 1/2 IC50 0.92 nM/1.8 nM vs. selumetinib MEK1 IC50 14nM) was defined through high-throughput screening; 2) maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) was defined in a non-selected cohort; 3) RP2D was evaluated in selected tumor types enriched for the target of interest; and 4) biologically active dose ranges were further characterized (15). Melanoma patients were included in the latter 3 phases; although enrollment was stratified by BRAF status, reflecting our growing understanding of the importance of pre-treatment stratification by mutational status. Given reports of disease stabilization and tumor regression among BRAF mutated (V600E/V600K) melanoma patients, subsequent phase II/III evaluation was restricted to BRAF V600E or V600K mutated melanoma (16).
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Evaluation of BRAF inhibitors in melanoma has paralleled that of MEK inhibitors. Initial forays utilized sorafenib (BAY43-9006) in unselected patients - with only minimal (singleagent) or modest (in combination with chemotherapy) clinical activity that did not correlate with BRAF mutational status (17). Subsequent analysis suggested that while initially considered a selective RAF inhibitor, sorafenib targeted VEGFR-2, VEGFR-3, PDGFR, Flt-3, c-KIT, and FGFR-1 (18). Subsequent evaluation centered on more potent agents with greater BRAF selectivity. Vemurafenib (PLX4032/RG7204) was developed as a more potent agent with greater selectivity for mutant BRAF compared to wild type BRAF (where it actually activates ERK). Significant activity in the phase I trial (69% in dose-escalation and 81% in dose-expansion phases) limited to BRAF V600E mutated patients prompted rapid further evaluation in phase II/III trials and led to regulatory approval by 2011. The development of dabrafenib (GSK2118436) has been similarly rapid.
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Underscoring the "One Companion Diagnostic - One Drug" paradigm, recent development of targeted therapies has been marked by concurrent development of in vitro companion diagnostic tests in concordance with guidelines from US and European regulatory agencies. Companion diagnostic tests must be rigorously validated prior to adoption as they are prone to many of the same vagaries of testing as the drugs they accompany. To wit, the Cobas® BRAF V600 assay was used in the pivotal BRIM studies to evaluate BRAF V600E mutation in formalin-fixed paraffin-embedded tissues and subsequently approved for this purpose concurrently with vemurafenib. Subsequently, vemurafenib was noted to have similar efficacy in patients with V600K BRAF mutations – missed in the prior report as the Cobas assay was poor at detecting these less common mutations. Dabrafenib’s approval in 2013 was accompanied by a companion diagnostic (THxID BRAF test) that detected both V600E and V600K mutations. Since selumetinib was first evaluated for melanoma in 2006, the field has advanced rapidly, with the approval of multiple more highly selective BRAF and MEK inhibitors between 2011 and the present. Multiple modes of resistance to BRAF inhibition have been identified, including MAPK pathway dependent (alternative splicing, increased gene amplification and copy number gains, development of oncogenic mutations in NRAS or MEK) and MAPK pathway independent [COT (MAP3K8) overexpression] mechanisms (19). Combining MEK Clin Cancer Res. Author manuscript; available in PMC 2016 December 15.
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and BRAF inhibitors increases response rate and prolongs duration of response by subverting multiple escape mechanisms. BRAF/MEK inhibitors are being combined in BRAF mutant malignancies (primarily melanoma) with a myriad of other agents including: small molecule inhibitors of bcl-2 (navitoclax, NCT01989585), Akt (GSK2141795, NCT01902173); autophagy inducers hydroxychloroquine (NCT02257424) and metformin (NCT02143050); VEGF antagonist bevacizumab (melanoma, NCT01495988); and EGFR antagonist panitumumab in colorectal cancer (NCT01750918). Further, BRAF/MEK inhibitors are being combined with CTLA-4 blocking agents including ipilimumab (NCT01940809); tremelimumab (NCT02027961); and PD-1/PD-L1 blocking agents including nivolumab (NCT02357732), pembrolizumab (KEYNOTE-022, NCT02130466), MPDL3280A (NCT01656642); and in a BRAF/MEK inhibitor vs. CTLA-4/PD-1 inhibitor sequence study (NCT02224781). It is hoped that these combinatorial strategies will improve upon the outcomes achieved with MEK/BRAF inhibition alone.
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Acknowledgments J.M. Kirkwood is a consultant/advisory board member for Bristol-Myers Squibb, Merck, and Roche/Genentech. Grant Support This work was supported by the NCI of the NIH under award number P50CA121973 (J.M. Kirkwood).
References
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1. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature. 2002; 417:949–954. [PubMed: 12068308] 2. Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005; 353:2135–2147. [PubMed: 16291983] 3. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996; 271:1734–1736. [PubMed: 8596936] 4. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A. 2002; 99:12293–12297. [PubMed: 12218188] 5. Johnson DB, Sosman JA. Therapeutic Advances and Treatment Options in Metastatic Melanoma. JAMA Oncol. 2015; 1:380–386. [PubMed: 26181188] 6. Daud A, Bastian BC. Beyond BRAF in melanoma. Curr Top Microbiol Immunol. 2012; 355:99– 117. [PubMed: 21826607] 7. Haass NK, Sproesser K, Nguyen TK, Contractor R, Medina CA, Nathanson KL, et al. The mitogenactivated protein/extracellular signal-regulated kinase kinase inhibitor AZD6244 (ARRY-142886) induces growth arrest in melanoma cells and tumor regression when combined with docetaxel. Clin Cancer Res. 2008; 14:230–239. [PubMed: 18172275] 8. Kirkwood JM, Bastholt L, Robert C, Sosman J, Larkin J, Hersey P, et al. Phase II, open-label, randomized trial of the MEK1/2 inhibitor selumetinib as monotherapy versus temozolomide in patients with advanced melanoma. Clin Cancer Res. 2012; 18:555–567. [PubMed: 22048237] 9. Yu X, Ambrosini G, Roszik J, Eterovic AK, Stempke-Hale K, Seftor EA, et al. Genetic analysis of the 'uveal melanoma' C918 cell line reveals atypical BRAF and common KRAS mutations and single tandem repeat profile identical to the cutaneous melanoma C8161 cell line. Pigment Cell Melanoma Res. 2015; 28:357–359. [PubMed: 25515650] 10. Bennouna J, Lang I, Valladares-Ayerbes M, Boer K, Adenis A, Escudero P, et al. A Phase II, open-label, randomised study to assess the efficacy and safety of the MEK1/2 inhibitor AZD6244 (ARRY-142886) versus capecitabine monotherapy in patients with colorectal cancer who have
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failed one or two prior chemotherapeutic regimens. Invest New Drugs. 2011; 29:1021–1028. [PubMed: 20127139] 11. Hainsworth JD, Cebotaru CL, Kanarev V, Ciuleanu TE, Damyanov D, Stella P, et al. A phase II, open-label, randomized study to assess the efficacy and safety of AZD6244 (ARRY-142886) versus pemetrexed in patients with non-small cell lung cancer who have failed one or two prior chemotherapeutic regimens. J Thorac Oncol. 2010; 5:1630–1636. [PubMed: 20802351] 12. Carvajal RD, Sosman JA, Quevedo JF, Milhem MM, Joshua AM, Kudchadkar RR, et al. Effect of selumetinib vs chemotherapy on progression-free survival in uveal melanoma: a randomized clinical trial. JAMA. 2014; 311:2397–2405. [PubMed: 24938562] 13. Gupta A, Love S, Schuh A, Shanyinde M, Larkin JM, Plummer R, et al. DOC-MEK: A double blind randomized phase 2 trial of docetaxel with or without selumetinib in wild-type BRAF advanced melanoma. Ann Oncol. 2014; 25:968–974. [PubMed: 24567366] 14. Robert C, Dummer R, Gutzmer R, Lorigan P, Kim KB, Nyakas M, et al. Selumetinib plus dacarbazine versus placebo plus dacarbazine as first-line treatment for BRAF-mutant metastatic melanoma: a phase 2 double-blind randomised study. Lancet Oncol. 2013; 14:733–740. [PubMed: 23735514] 15. Infante JR, Fecher LA, Falchook GS, Nallapareddy S, Gordon MS, Becerra C, et al. Safety, pharmacokinetic, pharmacodynamic, and efficacy data for the oral MEK inhibitor trametinib: a phase 1 dose-escalation trial. Lancet Oncol. 2012; 13:773–781. [PubMed: 22805291] 16. Flaherty KT, Robert C, Hersey P, Nathan P, Garbe C, Milhem M, et al. Improved survival with MEK inhibition in BRAF-mutated melanoma. N Engl J Med. 2012; 367:107–114. [PubMed: 22663011] 17. Eisen T, Ahmad T, Flaherty KT, Gore M, Kaye S, Marais R, et al. Sorafenib in advanced melanoma: a phase II randomised discontinuation trial analysis. Br J Cancer. 2006; 95:581–586. [PubMed: 16880785] 18. Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, et al. BAY 43–9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 2004; 64:7099– 7109. [PubMed: 15466206] 19. Perna D, Karreth FA, Rust AG, Perez-Mancera PA, Rashid M, Iorio F, et al. BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model. Proc Natl Acad Sci U S A. 2015; 112:E536–E545. [PubMed: 25624498]
Author Manuscript Clin Cancer Res. Author manuscript; available in PMC 2016 December 15.
Clin Cancer Res. Author manuscript; available in PMC 2016 December 15. Published in final edited form as: Clin Cancer Res. 2015 December 15; 21(24): 5412–5414. doi:10.1158/1078-0432.CCR-14-3132.
CCR 20th Anniversary Commentary: MAPK/ERK Pathway Inhibition in Melanoma–Kinase Inhibition Redux Diwakar Davar and John M. Kirkwood Division of Hematology-Oncology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
Author Manuscript
Summary In the January 15, 2012, issue of Clinical Cancer Research, Kirkwood and colleagues published a study comparing the MEK inhibitor selumetinib to temozolomide in unselected metastatic melanoma. Although selumetinib did not improve survival or response, most responders had BRAF activating mutations, and selumetinib has since demonstrated efficacy in BRAF-mutant melanoma. This study laid the groundwork for the evaluation of BRAF/MEK inhibitors in BRAFmutant melanoma.
Author Manuscript Author Manuscript
Major advances in the treatment of advanced melanoma over the past three years are directly linked to advances in our understanding of how activating mutations in canonical signaling and tumor-mediated T-cell dysfunction moderate melanoma tumorigenesis and progression. Deep sequencing approaches established that activating mutations in mitogen activated protein kinase (MAPK) pathway constituents (BRAF/NRAS) are common, mutually exclusive and carry oncogenic potential in human cancers (1). RAS mutations (typically at G12, G13, or Q61 residues) impair GTP hydrolysis resulting in constitutive activation of GTP-bound RAS. BRAF is a serine/threonine protein kinase and mutations (most commonly a glutamic acid substitution for valine at codon 600 - V600E) occur within the kinase domain’s activation segment. Oncogenic BRAF and NRAS mutations result in constitutive activation of downstream targets including MAPK/ERK kinases 1 and 2 (MEK1/MEK2) and unrestrained proliferation. Seminal work by Bastian and colleagues identified distinct patterns of mutations in chronically sun damaged skin (higher mutation load, lower BRAF/ NRAS mutant incidence) and intermittent sun exposed skin (lower mutation load, BRAF/ NRAS mutant) – underscoring the concept of melanomas a heterogeneous disease with multiple genetically distinct subtypes (2). Concurrently, Allison and Honjo identified CTLA-4 and PD-1 to be key negative regulatory immune checkpoints hijacked by tumors to
Corresponding Author: John M. Kirkwood, Division of Hematology-Oncology, University of Pittsburgh Medical Center, 5117 Centre Avenue, Pittsburgh, PA 15232. Phone: 412-623-7707; Fax: 412-623-7704; [email protected]. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed by the other author. Authors’ Contributions Conception and design: D. Davar, J.M. Kirkwood Writing, review, and/or revision of the manuscript: D. Davar, J.M. Kirkwood Disclaimer The content is solely the responsibility of the authors and does not necessarily represent the official views of the NCI or the NIH.
Davar and Kirkwood
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subvert antitumor immunity and mediate tolerogenic tumor microenvironments (3, 4). Subsequent work has since identified multiple other positive and negative checkpoints and their characterization in murine and human models of various cancers is ongoing.
Author Manuscript
Consequently, agents targeting the MAPK pathway and inhibitory immune checkpoints were developed and tested. In melanoma, 6 new agents have been approved including two targeting BRAF (vemurafenib and dabrafenib), one targeting MEK (trametinib), and CTLA-4 (ipilimumab) as well as two targeting PD-1 (pembrolizumab and nivolumab). Several other agents in each class are under evaluation including BRAF inhibitor encorafenib (LGX818, Array BioPharma); MEK inhibitors cobimetinib (GDC-0973/XL518, Exelixis) and binimetinib (MEK162, Array BioPharma); PD-1 inhibitors AMP-224 and AMP-514 (Amplimmune and GlaxoSmithKlein), pidilizumab (CT-011, Cure Tech); and PD-L1 inhibitors BMS-936559 (Bristol-Meyers Squibb), MEDI4736 (MedImmune and AstraZeneca), Atezolizumab (MPDL3280A, Roche), and Avelumab (MSB0010718C, Merck).
Author Manuscript
The use of monoclonal antibodies targeting inhibitory immune checkpoints in melanoma is marked by relatively low response rates that however, tend to be durable. Pooled analyses from multiple phase II/III trials reveal that ipilimumab has a lower response rate (11%–15%) compared to nivolumab (30%–35%) and pembrolizumab (45%–50%); with commensurately doubled 2-/3- year survival rates for PD-1 inhibitors compared to ipilimumab (5). Combined inhibition of both CTLA-4 and PD-1 increases response rates (to ~50%) and median progression-free survival, albeit with significant attendant toxicity (55% grade 3/4 adverse events) (5). Conversely, BRAF inhibitor use is marked by higher initial response rates (45– 53%) with inevitable progression at a median of 6–9 months typically though the acquisition of resistance mutations that reactivate the MAPK pathway. Addition of MEK inhibitors improves response rate (64–76%) and response duration (9–11 months) while reducing the incidence of cutaneous toxicities – though progression is similarly inevitable (5). Initial development of MAPK pathway inhibitors proceeded concurrently with inhibitors of MEK (AZD8330/ARRY-704, PD0325901, XL518, RDEA119, AS703026, CH5126766, CL-1040, GSK1120212, AZD6244) and RAF (GDC-0879, RAF265, ARQ 680, XL281/ BMS-908662, GSK2118436, PLX4032, sorafenib) (6). Importantly, initial clinical development (CL-1040) occurred prior to the identification of BRAF mutations in human cancer; and initial trials were not selectively enriched for patients with NRAS/BRAF mutated tumors or cancers in which these oncogenic mutations were most commonly detected such as melanoma (6).
Author Manuscript
Smalley et al first reported the antitumor activity of AZD6244 (ARRY-142886) in melanoma in a cell culture model where BRAF V600E mutant (WM35, WM793, 1205Lu, and 451Lu), NRAS Q61R mutant (SbCl2, WM2032, WM1361a, and WM852) and wild type (C8161) lines revealed that AZD6244 inhibited melanoma growth additively with docetaxel in vitro and in vivo (7). Further studies characterized selumetinib (AZD6244/ ARRY-142886) as an inhibitor of both MEK1 (IC50 14nM) and ERK1/2 phosphorylation (IC50 10nM) and led to evaluation of selumetinib in phase I trials against multiple tumor types (6).
Clin Cancer Res. Author manuscript; available in PMC 2016 December 15.
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In the phase II study we reported, 200 patients with advanced stage III/IV melanoma were assigned at random to receive either temozolomide or selumetinib (8). Most patients had cutaneous primaries (73%), but uveal (10%), mucosal (3%) and unknown primary tumors (14%) were also included. BRAF/NRAS and GNAQ mutation status were retrospectively analyzed in the cutaneous and uveal primaries although enrolment did not stratify patients on mutational status. Of the 158 samples that had adequate archival tissue for evaluation, 73 tumors (46% of tested samples or 50% of cutaneous primaries) were BRAF mutant; 28 tumors (18% of tested samples or 19% of cutaneous primaries) were NRAS mutant; only 4 tumors (3% of tested samples, or 20% of uveal primaries) were GNAQ mutant. The primary end-point of this trial was progression-free survival (PFS). Sample size of 182 (91 per arm) was calculated to provide 80% power to detect improvement in PFS with a hazard ratio of 0.74 (overall) at a one-sided 20% significance level, which would represent a HR 0.67 among BRAF mutant. PFS comparisons assumed that BRAF and NRAS mutant melanoma comprised 60% and 30% of study participants respectively; and that selumetinib would have equal effect in NRAS mutant and BRAF wild type patients, a reasonable assumption given prior reports of selumetinib efficacy in the BRAF/NRAS wild type C8161 cell line. We reported no significant difference in response or PFS between the treatment arms among unselected patients and in patients with tumors displaying BRAF mutation. Response rates in both arms were low (6% vs. 9%) – and surprisingly lower in the selumetinib arm. Further, patients assigned to selumetinib therapy had lower overall survival even after adjusting for imbalances in adverse prognostic factors between the two arms. Notably, 5 of 6 selumetinib responders and 1 of the 2 responding patients who had been crossed over to selumetinib from temozolomide post-progression were identified to be BRAF mutants.
Author Manuscript Author Manuscript
Several aspects of clinical trial design may have contributed to these findings. First, the study was likely underpowered to detect a true difference between treatment arms, given the discrepancy between the assumed (pre-study) and observed (on-study) proportions of BRAF/ NRAS mutations, despite the increase in enrollment (to 200) from an original accrual target (of 182). Second, subsequent reports of atypical BRAF/KRAS (G464E and G12D respectively) mutations in the C8161 cell line raises doubt as to selumetinib’s efficacy in true BRAF/NRAS wild type patients (9). Third, patients assigned to selumetinib had a higher incidence of known adverse prognostic features including worse WHO performance status and higher lactate dehydrogenase levels. Although these differences were adjusted for in the final analysis of overall survival, the non-significant p-values preclude definitive conclusions regarding the efficacy or non-efficacy of selumetinib, in either the overall or BRAF/NRAS mutant populations. Finally, given that the study allowed crossover on investigator assessed progression, selumetinib efficacy is difficult to ascertain as although overall survival and PFS statistics were reported separately, unequal crossover suggests an element of investigator bias. Concurrently, two other phase II monotherapy studies in nonsmall cell lung cancer and colorectal cancer reported that selumetinib did not offer a response and/or survival advantage over standard chemotherapy (10, 11). Subsequent evaluation in metastatic uveal, BRAF wild type (in combination with docetaxel) and BRAF mutant (in combination with dacarbazine) melanoma suggested that selumetinib improved PFS and response rates without significant improvements in overall survival (12–14).
Clin Cancer Res. Author manuscript; available in PMC 2016 December 15.
Davar and Kirkwood
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Lessons learned from these studies have fundamentally altered studies the next generation MEK inhibitors. MEK 1/2 inhibitor tremetinib (GSK1120212) was evaluated in a tiered approach that is characteristic of the current paradigm of targeted therapy development: 1) highly specific and potent candidate (trametinib MEK 1/2 IC50 0.92 nM/1.8 nM vs. selumetinib MEK1 IC50 14nM) was defined through high-throughput screening; 2) maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) was defined in a non-selected cohort; 3) RP2D was evaluated in selected tumor types enriched for the target of interest; and 4) biologically active dose ranges were further characterized (15). Melanoma patients were included in the latter 3 phases; although enrollment was stratified by BRAF status, reflecting our growing understanding of the importance of pre-treatment stratification by mutational status. Given reports of disease stabilization and tumor regression among BRAF mutated (V600E/V600K) melanoma patients, subsequent phase II/III evaluation was restricted to BRAF V600E or V600K mutated melanoma (16).
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Evaluation of BRAF inhibitors in melanoma has paralleled that of MEK inhibitors. Initial forays utilized sorafenib (BAY43-9006) in unselected patients - with only minimal (singleagent) or modest (in combination with chemotherapy) clinical activity that did not correlate with BRAF mutational status (17). Subsequent analysis suggested that while initially considered a selective RAF inhibitor, sorafenib targeted VEGFR-2, VEGFR-3, PDGFR, Flt-3, c-KIT, and FGFR-1 (18). Subsequent evaluation centered on more potent agents with greater BRAF selectivity. Vemurafenib (PLX4032/RG7204) was developed as a more potent agent with greater selectivity for mutant BRAF compared to wild type BRAF (where it actually activates ERK). Significant activity in the phase I trial (69% in dose-escalation and 81% in dose-expansion phases) limited to BRAF V600E mutated patients prompted rapid further evaluation in phase II/III trials and led to regulatory approval by 2011. The development of dabrafenib (GSK2118436) has been similarly rapid.
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Underscoring the "One Companion Diagnostic - One Drug" paradigm, recent development of targeted therapies has been marked by concurrent development of in vitro companion diagnostic tests in concordance with guidelines from US and European regulatory agencies. Companion diagnostic tests must be rigorously validated prior to adoption as they are prone to many of the same vagaries of testing as the drugs they accompany. To wit, the Cobas® BRAF V600 assay was used in the pivotal BRIM studies to evaluate BRAF V600E mutation in formalin-fixed paraffin-embedded tissues and subsequently approved for this purpose concurrently with vemurafenib. Subsequently, vemurafenib was noted to have similar efficacy in patients with V600K BRAF mutations – missed in the prior report as the Cobas assay was poor at detecting these less common mutations. Dabrafenib’s approval in 2013 was accompanied by a companion diagnostic (THxID BRAF test) that detected both V600E and V600K mutations. Since selumetinib was first evaluated for melanoma in 2006, the field has advanced rapidly, with the approval of multiple more highly selective BRAF and MEK inhibitors between 2011 and the present. Multiple modes of resistance to BRAF inhibition have been identified, including MAPK pathway dependent (alternative splicing, increased gene amplification and copy number gains, development of oncogenic mutations in NRAS or MEK) and MAPK pathway independent [COT (MAP3K8) overexpression] mechanisms (19). Combining MEK Clin Cancer Res. Author manuscript; available in PMC 2016 December 15.
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and BRAF inhibitors increases response rate and prolongs duration of response by subverting multiple escape mechanisms. BRAF/MEK inhibitors are being combined in BRAF mutant malignancies (primarily melanoma) with a myriad of other agents including: small molecule inhibitors of bcl-2 (navitoclax, NCT01989585), Akt (GSK2141795, NCT01902173); autophagy inducers hydroxychloroquine (NCT02257424) and metformin (NCT02143050); VEGF antagonist bevacizumab (melanoma, NCT01495988); and EGFR antagonist panitumumab in colorectal cancer (NCT01750918). Further, BRAF/MEK inhibitors are being combined with CTLA-4 blocking agents including ipilimumab (NCT01940809); tremelimumab (NCT02027961); and PD-1/PD-L1 blocking agents including nivolumab (NCT02357732), pembrolizumab (KEYNOTE-022, NCT02130466), MPDL3280A (NCT01656642); and in a BRAF/MEK inhibitor vs. CTLA-4/PD-1 inhibitor sequence study (NCT02224781). It is hoped that these combinatorial strategies will improve upon the outcomes achieved with MEK/BRAF inhibition alone.
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Acknowledgments J.M. Kirkwood is a consultant/advisory board member for Bristol-Myers Squibb, Merck, and Roche/Genentech. Grant Support This work was supported by the NCI of the NIH under award number P50CA121973 (J.M. Kirkwood).
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