BRAF-mutant Cutaneous Melanoma Research Articles

Combined therapy of dabrafenib and an anti-HER2 antibody–drug conjugate for advanced BRAF-mutant melanoma

BackgroundMelanoma is the most lethal skin cancer characterized by its high metastatic potential. In the past decade, targeted and immunotherapy have brought revolutionary survival benefits to patients with advanced and metastatic melanoma, but these treatment responses are also heterogeneous and/or do not achieve durable responses. Therefore, novel therapeutic strategies for improving outcomes remain an unmet clinical need. The aim of this study was to evaluate the therapeutic potential and underlying molecular mechanisms of RC48, a novel HER2-target antibody drug conjugate, either alone or in combination with dabrafenib, a V600-mutant BRAF inhibitor, for the treatment of advanced BRAF-mutant cutaneous melanoma.MethodsWe evaluated the therapeutic efficacy of RC48, alone or in combination with dabrafenib, in BRAF-mutant cutaneous melanoma cell lines and cell-derived xenograft (CDX) models. We also conducted signaling pathways analysis and global mRNA sequencing to explore mechanisms underlying the synergistic effect of the combination therapy.ResultsOur results revealed the expression of membrane-localized HER2 in melanoma cells. RC48 effectively targeted and inhibited the growth of HER2-positive human melanoma cell lines and corresponding CDX models. When used RC48 and dabrafenib synergically induced tumor regression together in human BRAF-mutant melanoma cell lines and CDX models. Mechanically, our results demonstrated that the combination therapy induced apoptosis and cell cycle arrest while suppressing cell motility in vitro. Furthermore, global RNA sequencing analysis demonstrated that the combination treatment led to the downregulation of several key signaling pathways, including the PI3K-AKT pathway, MAPK pathway, AMPK pathway, and FOXO pathway.ConclusionThese findings establish a preclinical foundation for the combined use of an anti-HER2 drug conjugate and a BRAF inhibitor in the treatment of BRAF-mutant cutaneous melanoma.

Abstract 202: Spry1 targeting enhances E-cadherin expression in cutaneous melanoma

Abstract Background: Cutaneous melanoma (CM) is a very aggressive malignancy that still represents the deadliest form of skin cancer. About 50% of CM harbors the activating BRAFV600 mutation which exerts most of the oncogenic effects through the MAPK signaling pathway. In the last years, a number of MAPK modulators have been identified, including Spry1. In this context, we have recently reported that Spry1 knockdown (Spry1KO) reduced the expression of several markers of epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Loss of E-cadherin is a hallmark of EMT in CM, and the presence of aberrant E-cadherin expression in CM is usually associated with a significantly worse overall survival. Based on these premises, in this study we wondered to explore whether Spry1 influences EMT through the modulation of E-cadherin expression. Material and Methods: SPRY1 gene was knocked-out using the CRISPR strategy in BRAF wild-type (wt) and in BRAF-mutant CM cell lines. By using in vitro and in vivo models, the effects of Spry1KO on E-cadherin expression and localization was investigated through RNA-sequencing (RNA-seq), quantitative real-time PCR, western blot, and immunofluorescence analyses. To gain insight into Spry1 interactome, immunoprecipitation coupled to mass spectrometry (IP-MS) was performed. Results and Discussion: E-cadherin mRNA and protein levels were significantly increased both in vitro and in vivo in Spry1KO clones. In addition, immunofluorescence analysis revealed a sustained redistribution of E-cadherin to the plasma membrane following Spry1 loss, thus confirming that Spry1 plays a role in EMT in CM. E-cadherin requires p120-catenin (p120) to maintain cell adhesion and functional adherens junctions. Indeed, p120 dysregulation results in the rapid turnover of E-cadherin complexes. Intriguingly, IP-MS analyses identified several Spry1 protein partners, including p120. Although a number of functional studies are still ongoing, preliminary western blot analysis of cytosolic and mitochondrial proteins indicated that Spry1 and p120 were mostly located in mitochondria. Conclusion: Spry1 reduced E-cadherin expression in vitro and in vivo. Starting from preliminary data, we hypothesized that Spry1-p120 interaction might influence E-cadherin distribution over the plasma membrane. Citation Format: Barbara Montico, Giorgio Giurato, Roberto Guerrieri, Annamaria Salvati, Francesca Colizzi, Lorena Baboci, Luca Sigalotti, Alessia Covre, Michele Maio, Agostino Steffan, Tuula Anneli Nyman, Alessandro Weisz, Maurizio Mongiat, Eva Andreuzzi, Elisabetta Fratta. Spry1 targeting enhances E-cadherin expression in cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 202.

High-throughput chemogenetic drug screening reveals PKC-RhoA/PKN as a targetable signaling vulnerability in GNAQ-driven uveal melanoma

Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death invitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

Understanding cancer drug resistance with Sleeping Beauty functional genomic screens: Application to MAPK inhibition in cutaneous melanoma

Combined BRAF and MEK inhibition is an effective treatment for BRAF-mutant cutaneous melanoma. However, most patients progress on this treatment due to drug resistance. Here, we applied the Sleeping Beauty transposon system to understand how melanoma evades MAPK inhibition. We found that the specific drug resistance mechanisms differed across melanomas in our genetic screens of five cutaneous melanoma cell lines. While drivers that reactivated MAPK were highly conserved, many others were cell-line specific. One such driver, VAV1, activated a de-differentiated transcriptional program like that of hyperactive RAC1, RAC1P29S. To target this mechanism, we showed that an inhibitor of SRC, saracatinib, blunts the VAV1-induced transcriptional reprogramming. Overall, we highlighted the importance of accounting for melanoma heterogeneity in treating cutaneous melanoma with MAPK inhibitors. Moreover, we demonstrated the utility of the Sleeping Beauty transposon system in understanding cancer drug resistance.

Abstract A005: The aged tumor microenvironment influences tolerance to targeted therapy via NR2F1 overexpression in BRAF-mutant melanoma

Abstract Despite the clinical success of targeted inhibitors, tumor responses to these agents are transient, and drug-tolerant residual cells seed resistance. Understanding the role of tumor-intrinsic mechanisms and effects of the tumor microenvironment in mediating drug tolerance will guide and optimize targeted therapies. Given similarities between drug tolerance and cellular dormancy, we studied the role of nuclear receptor subfamily 2 group F member 1 (NR2F1) in response to targeted therapy. We used BRAF-mutant cutaneous melanoma models treated with BRAF and MEK inhibitors (BRAFi + MEKi) since patients treated with this combination typically develop resistance. The aged tumor microenvironment has been shown to increase therapy resistance, and we find that melanoma cells in aged mice express higher levels of NR2F1 than when the same cells are injected into young animals. Transcriptomic analysis of melanoma patient samples treated with BRAFi + MEKi showed increased expression of NR2F1 post-treatment. Similarly, NR2F1 was highly expressed in minimal residual disease collected on BRAFi + MEKi treatment in patient- and xenograft-derived tumors. High expression of NR2F1 promotes tumor survival and invasion in the presence of BRAFi + MEKi in vitro leading to tolerance to BRAFi + MEKi efficacy in vivo. Depletion of NR2F1 in YUMM1.7 allografts grown in aged mice improved response to the combination therapy. Altogether, our findings suggest that NR2F1 promotes drug tolerance leading to minimal residual disease in melanoma and that NR2F1-high cells may be targeted with CDK4/6 inhibitors to improve targeted therapy outcomes in melanoma patients. Citation Format: Manoela Tiago, Timothy J. Purwin, Mitchell E. Fane, Yash Chhabra, Jessica L. F. Teh, Rama Kadamb, Weijia Cai, Inna Chervoneva, Sheera Rosenbaum, Vivian Chua, Nir Hacohen, Michael A. Davies, Jessie Villanieva, Ashani T. Weeraratna, Claudia Capparelli, Julio A. Aguirre-Ghiso, Andrew E. Aplin. The aged tumor microenvironment influences tolerance to targeted therapy via NR2F1 overexpression in BRAF-mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A005.

Evaluation of treatment patterns in patients with BRAF-mutant cutaneous melanoma in adjuvant setting in real practice.

e21573 Background: Mutation in BRAF is a crucial biomarker for treatment choice in patient with locally advanced or metastatic melanoma. In patient with BRAF wild-type (WT) melanoma aPD1 is the preferred treatment regimen. In patients with BRAF V600 mutant melanoma combined targeted therapy (cTT) could also be chosen as adjuvant treatment, which significantly improves OS. Methods: We conducted a retrospective continuous study of all melanoma patients, who were tested for BRAF mutation by RT-PCR in 2020 (COIVD-19 pandemic year) in our institution and analyzed adjuvant therapy options they received. Patient demographics, mutational status, disease stage, treatment regimens and treatment outcomes were collected from electronic medical records. Results: Between January 2020 and December 2020, tumor samples from 297 pts were tested for BRAF mutations. The mean pts age was 58.2 years (95% CI 51.2 to 60.3) and 130 (43.7%) were male. BRAF mutations were detected in 147 (49.5%) patients. Most common mutation was V600E (140 out of 147 samples, 88.4%) and V600K (13 out of 147, 8.84%). We also found K601N, K601E, L597R and V600M mutations (in one sample each). Among 150 BRAF wild type samples 44 were also tested for NRAS mutations and in 13 (29.54%) pts NRAS mutations were detected: Q61R – in 6 (46.15%) pts, Q61L – in 2 (15.38%) pts, Q61K – in 3 (23.08%) pts, G12E – in 1 (7.69%) pt, and G12D - in 1 (7.69%) pt . Also among 150 BRAF wild type samples 56 were tested for KIT mutations and only one sample were found to harbour D186V mutation . Among BRAF-positive pts at time of the genetic test performed stage I CM had 11 (7.48%) out of 147 pts, stage II - 13 (8.84%) pts, stage III - 67 (45.57%) pts, and stage IV - 43 (29.25%) pts. In 13 (8.84%) pts stage could not be clearly identified. In most cases (32 out of 67, 47.76%) monotherapy with aPD1 was given as adjuvant treatment in stage III pts where as only 6 (8.95%) patients received cTT, 7 pts (10.41%) received IFN-alfa and 22 pts (32.84%) received no or were given with other treatment (Table). Median DFS did not differ significantly between treatment cohorts. Conclusions: In our retrospective study we found that among stage III BRAF-mutant patients monotherapy with aPD1 was the most common adjuvant treatment regimen. In some patients with stage III disease IFN-alfa is still given. More studies are needed to define best adjuvant treatment for BRAF mutant stage III melanoma pts.[Table: see text]

Economic evaluation of adverse events of dabrafenib plus trametinib versus nivolumab in patients with advanced BRAF-mutant cutaneous melanoma for adjuvant therapy in Germany

ABSTRACT Background: Adjuvant treatment options have become the standard therapy for stage III and IV resectable cutaneous melanoma. Two recent studies led to the registration of dabrafenib and trametinib as targeted therapies for BRAF-mutated melanoma, and of immunotherapy with nivolumab irrespective of BRAF-mutation status. Both therapies have different spectrums of adverse events. Objective: To estimate the financial impact of side effects from the perspective of the German statutory sick funds to compare both therapeutic options and to relate the burden to the overall costs of the treatment. Study design and setting: Thirty-six adverse event categories for the combination of dabrafenib and trametinib (‘combi treatment’) and for nivolumab were extracted from the original publications of the studies named COMBI-AD and CheckMate 238. Patients and intervention: For all event categories a diagnosis and therapy recommendation were determined according to current national or international guidelines or from leading German textbooks. Main outcome measure: The resulting diagnostic steps, treatments, and therapies were evaluated with unit costs based on the German fee schedule for ambulatory physicians, the German G-DRG scheme, and the German drug price list. Results: The number of events with nivolumab per one hundred treatments amounted to 3.8 mandatory hospitalizations, 3.5 emergency care events and 0.8 life-threatening events. For the combi treatment, the respective number of events per one hundred treatments was 2.7, 1.8, and 0.5. The overall cost burden was calculated as €899 for nivolumab and €861 for combi-treatment. Conclusion: The treatment of adverse events resulting from adjuvant melanoma therapy showed comparable costs for both therapies.

Abstract B06: Loss of SPRY1 expression in BRAF-mutant cutaneous melanoma inhibits cell proliferation and improves response to targeted therapy

Abstract Background: Targeted therapy has proven efficacy in BRAF-mutant metastatic cutaneous melanoma (CM), but patients almost invariantly develop an early resistance. Although the underlying mechanisms are various and heterogeneous, resistance usually associates with an altered signaling. Sprouty 1 (SPRY1) has been described as an upstream mediator of MAPK signaling pathway, and although it has been found to be implicated in different biologic processes, its specific role in BRAF-mutant CM has been only partially addressed. Material and Methods: SPRY1 expression was assessed by quantitative real-time RT-PCR (qRT-PCR) and Western blotting (WB) analyses in BRAF-mutant CM cell lines and tissues. SPRY1 gene was knocked out using CRISPR-strategy. WB analyses evaluated the modulation of MAPK signaling pathways in SPRY1 knockout (SPRY1KO) clones. RNA-seq identified mRNAs that were differentially expressed in SPRY1KO clones respect to parental cells, and their expression was then validated by qRT-PCR. To further compare SPRY1KO clones to parental cells, cell proliferation was measured using xCELLigence instrument, whereas cell cycle progression, ROS induction, and apoptosis were assessed by flow cytometry. In vivo effects of SPRY1 silencing were investigated in xenografted mice treated with or without BRAF inhibitor (BRAFi). Results: SPRY1 was found highly expressed in a panel of BRAF-mutant CM cell lines and tissues, both at mRNA and protein level. To investigate the biologic role of SPRY1 in BRAF-mutant CM, SPRY1KO clones were obtained from 3 CM cell lines. RNA-seq identified 38 mRNAs that were significantly commonly modulated in SPRY1KO clones respect to parental cells. Ingenuity Pathway Analysis indicated that the top of the neighbor coding gene function of differentially expressed mRNAs was involved in cell proliferation and cell death. In addition, WB assays showed SPRY1 silencing was associated with i) decreased activation of p38 MAPK and beta-catenin signaling, ii) reduced expression of metalloproteinase 2, and iii) increased levels of p53. Consistent with these data, SPRY1 silencing markedly induced cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and impaired tumor growth in vivo. In addition, SPRY1 knockdown associated with improved response to BRAFi both in vitro and in vivo, which could be partly contributed by the higher levels of ROS observed in SPRY1KO clones. Interestingly, BRAFi treatment reduced mRNA and protein SPRY1 levels in parental BRAF-mutant CM cell lines, indicating that SPRY1 expression is downstream of the MEK-ERK signaling. Of note, some of the effects induced by BRAFi in parental cells were similar to those observed in SPRY1KO clones, suggesting that BRAFi activity may at least in part rely on SPRY1 downregulation. Conclusions: All together, our findings reveal important insights into SPRY1 role in BRAF-mutant CM and suggest its potential involvement in targeted therapy response. Citation Format: Barbara Montico, Giorgio Giurato, Francesca Colizzi, Alessia Covre, Elisa Comaro, Eliana Pivetta, Dania Benedetti, Alessandro Weisz, Agostino Steffan, Michele Maio, Luca Sigalotti, Elisabetta Fratta. Loss of SPRY1 expression in BRAF-mutant cutaneous melanoma inhibits cell proliferation and improves response to targeted therapy [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B06.

OCULAR ADVERSE EVENTS ASSOCIATED WITH MEK INHIBITORS.

Mitogen-activates protein kinase (MAPK) inhibitors, particularly MEK inhibitors, have shifted the treatment paradigm for metastatic BRAF-mutant cutaneous melanoma; however, oncologists, ophthalmologists, and patients have noticed different toxicities of variable importance. This review aims to provide an update of the ocular adverse events (OAEs), especially retinal toxicity, associated with the use of MEK inhibitors. We conducted a scientific literature search using the PubMed database up to July 2018 with the terms "MEK inhibitors" with a "review" filter and "MEK inhibitors" with a "clinical trials" filter. Phase I-III experimental studies and reviews were selected. Current principles and techniques for diagnosing and managing MEK inhibitor retinopathy and other OAEs are discussed. In patients treated with MEK inhibitors, including asymptomatic patients, OAEs occur with an incidence of up to 90%. Mild to severe ophthalmic toxicities are described, including visual disturbances, a 2-line decrease in Snellen visual acuity, dry eye symptoms, ocular adnexal abnormalities, visual field defects, panuveitis, and retinal toxicities, such as different degrees of MEK-associated retinopathy, vascular injury, and retinal vein occlusion. MEK inhibitors can lead to different degrees of retinal, uveal, and adnexal OAE, causing visual disturbances or discomfort. One of the most relevant OAE of MEK therapy is MEK inhibitor-associated retinopathy (MEKAR), which is usually mild, self-limited, and may subside after continuous use of the drug for weeks or months, or discontinuation, thereby restoring the normal visual function of the retina, with some exceptions. Ocular adverse events are often associated with other systemic adverse effects that can modify the dosage of treatment, so the communication with the oncologist is fundamental.

Encorafenib plus binimetinib: an embarrassment of riches.

Treatment of advanced BRAF-mutant cutaneous melanoma has undergone several breakthroughs, not only in terms of new therapeutic strategies but also in the number of drugs available. The BRAF–MEK inhibitor combinations vemurafenib plus cobimetinib and dabrafenib plus trametinib have been shown to improve overall survival along with pembrolizumab and nivolumab in monotherapy and when combined with ipilimumab. 1 Luke JJ Flaherty KT Ribas A Long GV Targeted agents and immunotherapies: optimizing outcomes in melanoma. Nat Rev Clin Oncol. 2017; 14: 463-482 Crossref PubMed Scopus (737) Google Scholar Now, another treatment option can be added to the list: encorafenib plus binimetinib. Overall survival in patients with BRAF-mutant melanoma receiving encorafenib plus binimetinib versus vemurafenib or encorafenib (COLUMBUS): a multicentre, open-label, randomised, phase 3 trialThe combination of encorafenib plus binimetinib provided clinically meaningful efficacy with good tolerability as shown by improvements in both progression-free survival and overall survival compared with vemurafenib. These data suggest that the combination of encorafenib plus binimetinib is likely to become an important therapeutic option in patients with BRAFV600-mutant melanoma. Full-Text PDF

Abstract B157: YAP is a promising target in cutaneous melanoma

Abstract The transcriptional regulator YAP (Yes-associated protein) has been implicated in tissue development and homeostasis and in cancer growth (Harvey et al. 2013). In uveal melanoma, YAP mediates oncogenic signaling of mutated G-proteins, driving tumor growth in a therapeutically exploitable manner (Yu et al. & Feng et al. 2014). The role of YAP in cutaneous melanoma (CM) is less well defined, although previous studies implicating YAP in metastasis and disease maintenance/progression (Nallet-Staub et al. 2014; Menzel et al. 2014), and encouraging data suggesting synergism between combination targeting of YAP and the Mitogen-Activated Protein Kinases (MAPK) pathway (Lin et al. 2015). Here we show that YAP is a critical mediator of disease maintenance and progression in some but not all CMs. In a primary CM we identified novel YAP mutations, which are rare in human cancer but are predicted to relieve YAP from inhibition by the Hippo pathway. By quantifying YAP expression by immunofluorescence, we found YAP was increased early in human melanocytic transformation, being elevated in nevi and in over 50% of primary CMs. Gene targeting of YAP with inducible shRNAs induced profound proliferative arrest and death in cultured CM cells (measured by MTT assay, EdU incorporation and propidium iodide staining respectively). The dependency on YAP of melanoma maintenance was validated in vivo using xenograft models of cell lines and of early passage patient-derived melanomas. Further, efficacy of YAP targeting synergized with MAPK inhibition in BRAF mutant CMs. However, some CMs were resistant to YAP targeting independent of BRAF/NRAS genotype and of levels of YAP protein expression or activation. These data compel the development of anti-YAP therapies in CM and the identification of biomarkers that predict response to YAP targeting. Citation Format: Jian zhong Tang, Xiaomeng Zhang, Ismael Vergara, Anthony Papenfuss, Kieran Harvey, Mark Shackleton. YAP is a promising target in cutaneous melanoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B157.

Russian study of morphological prognostic factors characterization in BRAF-mutant cutaneous melanoma

Melanoma is one of the aggressive cancer types causing the majority of deaths in skin cancer patients. Mutational screening of the tumor revealed a number of driver mutations in oncogenes which enabled melanoma classification into a few molecular subtypes. BRAF is a key component of mitogen-activated kinase pathway; its activating mutation leads to accelerated melanoma cells proliferation, invasion and survival. Somatic mutations in BRAF were reported in various malignancies, including thyroid cancer, colorectal cancer and melanoma. Specific features of BRAF-positive tumors could have clinical implications as mutational alterations may have an impact on the biological behavior of the tumor and prognosis of the disease.In the present study, the frequency of BRAF V600E mutation was evaluated in Russian patients with melanocytic lesions, of which 41.25% were primary melanoma and 60% were melanocytic nevi. Melanoma patients with trunk localization were of younger age in the BRAF-positive group as compared with BRAF-negative patients. Immunohistochemical evaluations of Ki-67 expression, as well as matrix metalloproteinase-2, -9, were found to be equal in BRAF-positive and BRAF-negative tumors. MMP-2/MMP-9 immunoreactivity was observed in stromal and/or melanocytic cells both in melanoma and nevi patients. Besides tumor cells, MMP-9 expression was observed in lymphocytes in 27.2% of BRAF-positive and in 19.1% of BRAF-negative patients. Histopathological prognostic markers (Breslow thickness, mitotic index, ulceration, tumor infiltrating lymphocytes pattern) did not show any differences depending on BRAF V600E mutational status.The frequency of BRAF-positive melanomas in Russian cohort is similar to other Caucasian population rates. BRAF V600E mutation harboring tumors are more often observed in younger patients without specific features of morphological prognostic factors.

Basal and Treatment-Induced Activation of AKT Mediates Resistance to Cell Death by AZD6244 (ARRY-142886) in Braf-Mutant Human Cutaneous Melanoma Cells

The majority of melanomas show constitutive activation of the RAS-RAF-MAP/ERK kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway. AZD6244 is a selective MEK1/2 inhibitor that markedly reduces tumor P-MAPK levels, but it produces few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the phosphoinositide 3-kinase (PI3K)-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines showed AZD6244-induced activation of AKT. In contrast, sensitive cell lines showed AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or insulin-like growth factor I receptor blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells and the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients.