Multi-organ landscape of therapy-resistant melanoma.

Metastasis and failure of present-day therapies represent the most common causes of mortality in patients with cutaneous melanoma. To identify the underlying genetic and transcriptomic landscapes, in this study we analyzed multi-organ metastases and tumor-adjacent tissues from 11 rapid autopsies after treatment with MAPK inhibitor (MAPKi) and/or immune checkpoint blockade (ICB) and death due to acquired resistance. Either treatment elicits shared genetic alterations that suggest immune-evasive, cross-therapy resistance mechanisms. Large, non-clustered deletions, inversions and inter-chromosomal translocations dominate rearrangements. Analyzing data from separate melanoma cohorts including 345 therapy-naive patients and 35 patients with patient-matched pre-treatment and post-acquired resistance tumor samples, we performed cross-cohort analyses to identify MAPKi and ICB as respective contributors to gene amplifications and deletions enriched in autopsy versus therapy-naive tumors. In the autopsy cohort, private/late mutations and structural variants display shifted mutational and rearrangement signatures, with MAPKi specifically selecting for signatures of defective homologous-recombination, mismatch and base-excision repair. Transcriptomic signatures and crosstalks with tumor-adjacent macroenvironments nominated organ-specific adaptive pathways. An immune-desert, CD8+-macrophage-biased archetype, T-cell exhaustion and type-2 immunity characterized the immune contexture. This multi-organ analysis of therapy-resistant melanoma presents preliminary insights with potential to improve therapeutic strategies.

Blocking Genomic Instability Prevents Acquired Resistance to MAPK Inhibitor Therapy in Melanoma.

Blocking cancer genomic instability may prevent tumor diversification and escape from therapies. We show that, after MAPK inhibitor (MAPKi) therapy in patients and mice bearing patient-derived xenografts (PDX), acquired resistant genomes of metastatic cutaneous melanoma specifically amplify resistance-driver, nonhomologous end-joining (NHEJ), and homologous recombination repair (HRR) genes via complex genomic rearrangements (CGR) and extrachromosomal DNAs (ecDNA). Almost all sensitive and acquired-resistant genomes harbor pervasive chromothriptic regions with disproportionately high mutational burdens and significant overlaps with ecDNA and CGR spans. Recurrently, somatic mutations within ecDNA and CGR amplicons enrich for HRR signatures, particularly within acquired resistant tumors. Regardless of sensitivity or resistance, breakpoint-junctional sequence analysis suggests NHEJ as critical to double-stranded DNA break repair underlying CGR and ecDNA formation. In human melanoma cell lines and PDXs, NHEJ targeting by a DNA-PKCS inhibitor prevents/delays acquired MAPKi resistance by reducing the size of ecDNAs and CGRs early on combination treatment. Thus, targeting the causes of genomic instability prevents acquired resistance. SIGNIFICANCE: Acquired resistance often results in heterogeneous, redundant survival mechanisms, which challenge strategies aimed at reversing resistance. Acquired-resistant melanomas recurrently evolve resistance-driving and resistance-specific amplicons via ecDNAs and CGRs, thereby nominating chromothripsis-ecDNA-CGR biogenesis as a resistance-preventive target. Specifically, targeting DNA-PKCS/NHEJ prevents resistance by suppressing ecDNA/CGR rearrangements in MAPKi-treated melanomas. This article is highlighted in the In This Issue feature, p. 799.

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

Enhancing PD-L1 Degradation by ITCH during MAPK Inhibitor Therapy Suppresses Acquired Resistance.

MAPK inhibitor (MAPKi) therapy in melanoma leads to the accumulation of tumor-surface PD-L1/L2, which may evade antitumor immunity and accelerate acquired resistance. Here, we discover that the E3 ligase ITCH binds, ubiquitinates, and downregulates tumor-surface PD-L1/L2 in MAPKi-treated human melanoma cells, thereby promoting T-cell activation. During MAPKi therapy in vivo, melanoma cell-intrinsic ITCH knockdown induced tumor-surface PD-L1, reduced intratumoral cytolytic CD8+ T cells, and accelerated acquired resistance only in immune-competent mice. Conversely, tumor cell-intrinsic ITCH overexpression reduced MAPKi-elicited PD-L1 accumulation, augmented intratumoral cytolytic CD8+ T cells, and suppressed acquired resistance in BrafV600MUT, NrasMUT, or Nf1MUT melanoma and KrasMUT-driven cancers. CD8+ T-cell depletion and tumor cell-intrinsic PD-L1 overexpression nullified the phenotype of ITCH overexpression, thereby supporting an in vivo ITCH-PD-L1-T-cell regulatory axis. Moreover, we identify a small-molecular ITCH activator that suppresses acquired MAPKi resistance in vivo. Thus, MAPKi-induced PD-L1 accelerates resistance, and a PD-L1-degrading ITCH activator prolongs antitumor response. SIGNIFICANCE: MAPKi induces tumor cell-surface PD-L1 accumulation, which promotes immune evasion and therapy resistance. ITCH degrades PD-L1, optimizing antitumor T-cell immunity. We propose degrading tumor cell-surface PD-L1 and/or activating tumor-intrinsic ITCH as strategies to overcome MAPKi resistance. This article is highlighted in the In This Issue feature, p. 1825.

Plasticity of Extrachromosomal and Intrachromosomal BRAF Amplifications in Overcoming Targeted Therapy Dosage Challenges.

Focal amplifications (FA) can mediate targeted therapy resistance in cancer. Understanding the structure and dynamics of FAs is critical for designing treatments that overcome plasticity-mediated resistance. We developed a melanoma model of dual MAPK inhibitor (MAPKi) resistance that bears BRAFV600 amplifications through either extrachromosomal DNA (ecDNA)/double minutes (DM) or intrachromosomal homogenously staining regions (HSR). Cells harboring BRAFV600E FAs displayed mode switching between DMs and HSRs, from both de novo genetic changes and selection of preexisting subpopulations. Plasticity is not exclusive to ecDNAs, as cells harboring HSRs exhibit drug addiction-driven structural loss of BRAF amplicons upon dose reduction. FA mechanisms can couple with kinase domain duplications and alternative splicing to enhance resistance. Drug-responsive amplicon plasticity is observed in the clinic and can involve other MAPK pathway genes, such as RAF1 and NRAS. BRAF FA-mediated dual MAPKi-resistant cells are more sensitive to proferroptotic drugs, extending the spectrum of ferroptosis sensitivity in MAPKi resistance beyond cases of dedifferentiation. SIGNIFICANCE: Understanding the structure and dynamics of oncogene amplifications is critical for overcoming tumor relapse. BRAF amplifications are highly plastic under MAPKi dosage challenges in melanoma, through involvement of de novo genomic alterations, even in the HSR mode. Moreover, BRAF FA-driven, dual MAPKi-resistant cells extend the spectrum of resistance-linked ferroptosis sensitivity. This article is highlighted in the In This Issue feature, p. 873.

Anti-PD-1/L1 lead-in before MAPK inhibitor combination maximizes antitumor immunity and efficacy.

Rationally sequencing and combining PD-1/L1-and MAPK-targeted therapies may overcome innate and acquired resistance. Since increased clinical benefit of MAPK inhibitors (MAPKi) is associated with previous immune checkpoint therapy, we compare the efficacies of sequential and/or combinatorial regimens in subcutaneous murine models of melanoma driven by BrafV600, Nras, or Nf1 mutations as well as colorectal and pancreatic carcinoma driven by KrasG12C. Anti-PD-1/L1 lead-in preceding MAPKi combination optimizes response durability by promoting pro-inflammatory polarization of macrophages and clonal expansion of interferon-γhi, and CD8+ cytotoxic and proliferative (versus CD4+ regulatory) T cells that highly express activation genes. Since therapeutic resistance of melanoma brain metastasis (MBM) limits patient survival, we demonstrate that sequencing anti-PD-1/L1 therapy before MAPKi combination suppresses MBM and improves mouse survival with robust T cell clonal expansion in both intracranial and extracranial metastatic sites. We propose clinically testing brief anti-PD-1/L1 (± anti-CTLA-4) dosing before MAPKi co-treatment to suppress therapeutic resistance.

SPRED1 deletion confers resistance to MAPK inhibition in melanoma.

Functional evaluation of genetic lesions can discover a role in cancer initiation and progression and help develop novel therapeutic strategies. We previously identified the negative MAPK regulator SPRED1 as a novel tumor suppressor in KIT-driven melanoma. Here, we show that SPRED1 is also frequently deleted in human melanoma driven by mutant BRAF. We found that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma conferred resistance to BRAFV600E inhibition in vitro and in vivo. Mechanistically, SPRED1 loss promoted melanoma cell proliferation under mutant BRAF inhibition by reactivating MAPK activity. Consistently, biallelic deletion of SPRED1 was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy. These studies combining work in human cells and in vivo modeling in zebrafish demonstrate a new mechanism of resistance to BRAFV600E inhibition in melanoma.

Durable Suppression of Acquired MEK Inhibitor Resistance in Cancer by Sequestering MEK from ERK and Promoting Antitumor T-cell Immunity.

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAF non-V600, and BRAF V600 mutations. Tumor cell-intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8+ T cells, and durable tumor regression elicited by this combination requires CD8+ T cells, which can be reinvigorated by anti-PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy. SIGNIFICANCE: Type I RAFi + MEKi are indicated only in certain BRAF V600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8+ T cells are mechanisms that may be further exploited.This article is highlighted in the In This Issue feature, p. 521.

High-Speed Live-Cell Interferometry: A New Method for Quantifying Tumor Drug Resistance and Heterogeneity.

We report the development of high-speed live-cell interferometry (HSLCI), a new multisample, multidrug testing platform for directly measuring tumor therapy response via real-time optical cell biomass measurements. As a proof of concept, we show that HSLCI rapidly profiles changes in biomass in BRAF inhibitor (BRAFi)-sensitive parental melanoma cell lines and in their isogenic BRAFi-resistant sublines. We show reproducible results from two different HSLCI platforms at two institutions that generate biomass kinetic signatures capable of discriminating between BRAFi-sensitive and -resistant melanoma cells within 24 h. Like other quantitative phase imaging (QPI) modalities, HSLCI is well-suited to noninvasive measurements of single cells and cell clusters, requiring no fluorescence or dye labeling. HSLCI is substantially faster and more sensitive than field-standard growth inhibition assays, and in terms of the number of cells measured simultaneously, the number of drugs tested in parallel, and temporal measurement range, it exceeds the state of the art by more than 10-fold. The accuracy and speed of HSLCI in profiling tumor cell heterogeneity and therapy resistance are promising features of potential tools to guide patient therapeutic selections.

Exploiting Drug Addiction Mechanisms to Select against MAPKi-Resistant Melanoma.

Melanoma resistant to MAPK inhibitors (MAPKi) displays loss of fitness upon experimental MAPKi withdrawal and, clinically, may be resensitized to MAPKi therapy after a drug holiday. Here, we uncovered and therapeutically exploited the mechanisms of MAPKi addiction in MAPKi-resistant BRAFMUT or NRASMUT melanoma. MAPKi-addiction phenotypes evident upon drug withdrawal spanned transient cell-cycle slowdown to cell-death responses, the latter of which required a robust phosphorylated ERK (pERK) rebound. Generally, drug withdrawal-induced pERK rebound upregulated p38-FRA1-JUNB-CDKN1A and downregulated proliferation, but only a robust pERK rebound resulted in DNA damage and parthanatos-related cell death. Importantly, pharmacologically impairing DNA damage repair during MAPKi withdrawal augmented MAPKi addiction across the board by converting a cell-cycle deceleration to a caspase-dependent cell-death response or by furthering parthanatos-related cell death. Specifically in MEKi-resistant NRASMUT or atypical BRAFMUT melanoma, treatment with a type I RAF inhibitor intensified pERK rebound elicited by MEKi withdrawal, thereby promoting a cell death-predominant MAPKi-addiction phenotype. Thus, MAPKi discontinuation upon disease progression should be coupled with specific strategies that augment MAPKi addiction.Significance: Discontinuing targeted therapy may select against drug-resistant tumor clones, but drug-addiction mechanisms are ill-defined. Using melanoma resistant to but withdrawn from MAPKi, we defined a synthetic lethality between supraphysiologic levels of pERK and DNA damage. Actively promoting this synthetic lethality could rationalize sequential/rotational regimens that address evolving vulnerabilities. Cancer Discov; 8(1); 74-93. ©2017 AACR.See related commentary by Stern, p. 20This article is highlighted in the In This Issue feature, p. 1.

Recurrent Tumor Cell-Intrinsic and -Extrinsic Alterations during MAPKi-Induced Melanoma Regression and Early Adaptation.

Treatment of advanced BRAFV600-mutant melanoma using a BRAF inhibitor or its combination with a MEK inhibitor typically elicits partial responses. We compared the transcriptomes of patient-derived tumors regressing on MAPK inhibitor (MAPKi) therapy against MAPKi-induced temporal transcriptomic states in human melanoma cell lines or murine melanoma in immune-competent mice. Despite heterogeneous dynamics of clinical tumor regression, residual tumors displayed highly recurrent transcriptomic alterations and enriched processes, which were also observed in MAPKi-selected cell lines (implying tumor cell-intrinsic reprogramming) or in bulk mouse tumors (and the CD45-negative or CD45-positive fractions, implying tumor cell-intrinsic or stromal/immune alterations, respectively). Tumor cell-intrinsic reprogramming attenuated MAPK dependency, while enhancing mesenchymal, angiogenic, and IFN-inflammatory features and growth/survival dependence on multi-RTKs and PD-L2. In the immune compartment, PD-L2 upregulation in CD11c+ immunocytes drove the loss of T-cell inflammation and promoted BRAFi resistance. Thus, residual melanoma early on MAPKi therapy already displays potentially exploitable adaptive transcriptomic, epigenomic, immune-regulomic alterations.Significance: Incomplete MAPKi-induced melanoma regression results in transcriptome/methylome-wide reprogramming and MAPK-redundant escape. Although regressing/residual melanoma is highly T cell-inflamed, stromal adaptations, many of which are tumor cell-driven, could suppress/eliminate intratumoral T cells, reversing tumor regression. This catalog of recurrent alterations helps identify adaptations such as PD-L2 operative tumor cell intrinsically and/or extrinsically early on therapy. Cancer Discov; 7(11); 1248-65. ©2017 AACR.See related commentary by Haq, p. 1216This article is highlighted in the In This Issue feature, p. 1201.

JUN dependency in distinct early and late BRAF inhibition adaptation states of melanoma.

A prominent mechanism of acquired resistance to BRAF inhibitors in BRAF (V600) -mutant melanoma is associated with the upregulation of receptor tyrosine kinases. Evidences suggested that this resistance mechanism is part of a more complex cellular adaptation process. Using an integrative strategy, we found this mechanism to invoke extensive transcriptomic, (phospho-) proteomic and phenotypic alterations that accompany a cellular transition to a de-differentiated, mesenchymal and invasive state. Even short-term BRAF-inhibitor exposure leads to an early adaptive, differentiation state change-characterized by a slow-cycling, persistent state. The early persistent state is distinct from the late proliferative, resistant state. However, both differentiation states share common signaling alterations including JUN upregulation. Motivated by the similarities, we found that co-targeting of BRAF and JUN is synergistic in killing fully resistant cells; and when used up-front, co-targeting substantially impairs the formation of the persistent subpopulation. We confirmed that JUN upregulation is a common response to BRAF inhibitor treatment in clinically treated patient tumors. Our findings demonstrate that events shared between early- and late-adaptation states provide candidate up-front co-treatment targets.

Non-genomic and Immune Evolution of Melanoma Acquiring MAPKi Resistance.

Clinically acquired resistance to MAPK inhibitor (MAPKi) therapies for melanoma cannot be fully explained by genomic mechanisms and may be accompanied by co-evolution of intra-tumoral immunity. We sought to discover non-genomic mechanisms of acquired resistance and dynamic immune compositions by a comparative, transcriptomic-methylomic analysis of patient-matched melanoma tumors biopsied before therapy and during disease progression. Transcriptomic alterations across resistant tumors were highly recurrent, in contrast to mutations, and were frequently correlated with differential methylation of tumor cell-intrinsic CpG sites. We identified in the tumor cell compartment supra-physiologic c-MET up-expression, infra-physiologic LEF1 down-expression and YAP1 signature enrichment as drivers of acquired resistance. Importantly, high intra-tumoral cytolytic T cell inflammation prior to MAPKi therapy preceded CD8 T cell deficiency/exhaustion and loss of antigen presentation in half of disease-progressive melanomas, suggesting cross-resistance to salvage anti-PD-1/PD-L1 immunotherapy. Thus, melanoma acquires MAPKi resistance with highly dynamic and recurrent non-genomic alterations and co-evolving intra-tumoral immunity.

Vemurafenib resistance reprograms melanoma cells towards glutamine dependence.

BACKGROUND: (V600) BRAF mutations drive approximately 50% of metastatic melanoma which can be therapeutically targeted by BRAF inhibitors (BRAFi) and, based on resistance mechanisms, the combination of BRAF and MEK inhibitors (BRAFi + MEKi). Although the combination therapy has been shown to provide superior clinical benefits, acquired resistance is still prevalent and limits the overall survival benefits. Recent work has shown that oncogenic changes can lead to alterations in tumor cell metabolism rendering cells addicted to nutrients, such as the amino acid glutamine. Here, we evaluated whether melanoma cells with acquired resistance display glutamine dependence and whether glutamine metabolism can be a potential molecular target to treat resistant cells. METHODS: Isogenic BRAFi sensitive parental (V600) BRAF mutant melanoma cell lines and resistant (derived by chronic treatment with vemurafenib) sub-lines were used to assess differences in the glutamine uptake and sensitivity to glutamine deprivation. To evaluate a broader range of resistance mechanisms, isogenic pairs where the sub-lines were resistant to BRAFi + MEKi were also studied. Since resistant cells demonstrated increased sensitivity to glutamine deficiency, we used glutaminase inhibitors BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] and L-L-DON (6-Diazo-5-oxo-L-norleucine) to treat MAPK pathway inhibitor (MAPKi) resistant cell populations both in vitro and in vivo. RESULTS: We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts. In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro. We also showed that mutant NRAS was critical for glutamine addiction in mutant NRAS driven resistance. When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells. CONCLUSION: Our study is a proof-of-concept for the potential of targeting glutamine metabolism as an alternative strategy to suppress acquired MAPKi-resistance in melanoma.

Tunable-combinatorial mechanisms of acquired resistance limit the efficacy of BRAF/MEK cotargeting but result in melanoma drug addiction.

Combined BRAF- and MEK-targeted therapy improves upon BRAF inhibitor (BRAFi) therapy but is still beset by acquired resistance. We show that melanomas acquire resistance to combined BRAF and MEK inhibition by augmenting or combining mechanisms of single-agent BRAFi resistance. These double-drug resistance-associated genetic configurations significantly altered molecular interactions underlying MAPK pathway reactivation. (V600E)BRAF, expressed at supraphysiological levels because of (V600E)BRAF ultra-amplification, dimerized with and activated CRAF. In addition, MEK mutants enhanced interaction with overexpressed (V600E)BRAF via a regulatory interface at R662 of (V600E)BRAF. Importantly, melanoma cell lines selected for resistance to BRAFi+MEKi, but not those to BRAFi alone, displayed robust drug addiction, providing a potentially exploitable therapeutic opportunity.

Imatinib mesylate exerts anti-proliferative effects on osteosarcoma cells and inhibits the tumour growth in immunocompetent murine models.

Osteosarcoma is the most common primary malignant bone tumour characterized by osteoid production and/or osteolytic lesions of bone. A lack of response to chemotherapeutic treatments shows the importance of exploring new therapeutic methods. Imatinib mesylate (Gleevec, Novartis Pharma), a tyrosine kinase inhibitor, was originally developed for the treatment of chronic myeloid leukemia. Several studies revealed that imatinib mesylate inhibits osteoclast differentiation through the M-CSFR pathway and activates osteoblast differentiation through PDGFR pathway, two key cells involved in the vicious cycle controlling the tumour development. The present study investigated the in vitro effects of imatinib mesylate on the proliferation, apoptosis, cell cycle, and migration ability of five osteosarcoma cell lines (human: MG-63, HOS; rat: OSRGA; mice: MOS-J, POS-1). Imatinib mesylate was also assessed as a curative and preventive treatment in two syngenic osteosarcoma models: MOS-J (mixed osteoblastic/osteolytic osteosarcoma) and POS-1 (undifferentiated osteosarcoma). Imatinib mesylate exhibited a dose-dependent anti-proliferative effect in all cell lines studied. The drug induced a G0/G1 cell cycle arrest in most cell lines, except for POS-1 and HOS cells that were blocked in the S phase. In addition, imatinib mesylate induced cell death and strongly inhibited osteosarcoma cell migration. In the MOS-J osteosarcoma model, oral administration of imatinib mesylate significantly inhibited the tumour development in both preventive and curative approaches. A phospho-receptor tyrosine kinase array kit revealed that PDGFRα, among 7 other receptors (PDFGFRß, Axl, RYK, EGFR, EphA2 and 10, IGF1R), appears as one of the main molecular targets for imatinib mesylate. In the light of the present study and the literature, it would be particularly interesting to revisit therapeutic evaluation of imatinib mesylate in osteosarcoma according to the tyrosine-kinase receptor status of patients.

A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition.

BRAF inhibitor (BRAFi) therapy leads to remarkable anti melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants, causing late, acquired resistance. We show here that BRAFi (or BRAFi + MEKi) therapy in patients frequently led to rebound phosphorylated AKT (p-AKT) levels in their melanomas early on-treatment. In cell lines, BRAFi treatment led to rebound levels of receptor tyrosine kinases (RTK; including PDGFRß), phosphatidyl (3,4,5)-triphosphate (PIP3), pleckstrin homology domain recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by the introduction of a mutant AKT1 (Q79K) known to confer acquired BRAFi resistance. Functionally, AKT1(Q79K) conferred BRAFi resistance via amplification of BRAFi-elicited PI3K-AKT signaling. In addition, mitogen-activated protein kinase pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy.