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

Novel Combination Therapy to Overcome Adaptive and Acquired MAPK-inhibitor Resistance in Melanoma

About 50% of metastatic melanomas harbor BRAF V600 mutations, most commonly a V600E substitution, which constitutively hyper-activate the MAPK pathway and result in oncogene addiction. Therefore, the emergence of BRAF inhibitors (BRAFi) into the clinical arena have represented a true therapeutic paradigm shift for advanced melanomas. However, the almost universal occurrence of acquired resistance to BRAFi and the overabundance of initial partial responses (innate resistance) are urgent clinical problems that limit prolonged patient survival. Our recent efforts have identified 2 core resistance pathways exhibited in disease-progressive melanoma, MAPK reactivation mechanisms and PI3K–PTEN–AKT–upregulating genetic alterations. We hypothesized that these essential survival signaling pathways play a significant role in acute or adaptive resistance in BRAF-mutant melanoma. We observed that BRAF inhibition leads to early and adaptive PI3K–AKT pathway signaling, where preexisting determinants of AKT activation (e.g., PTEN mutation/expression status) limited this BRAFi-elicited PI3K–AKT signaling. However, the presence of a gain-of-function AKT mutant, for instance, would lift this restriction via BRAFi-mediated signal amplification. This study provided a mechanistic link between early, adaptive and late, acquired BRAF inhibitor resistance in melanoma, rationalizing clinical studies to target both MAPK and PI(3)K/AKT/mTOR pathways as combination therapy to delay tumor relapse. Combining BRAF/MEK targeted therapy, to combat acquired BRAFi resistance mechanisms reactivating the MAPK pathway, has been tested to be clinically superior to BRAFi monotherapy but is still challenged by acquired resistance. We show that melanomas acquire resistance to combined BRAF/MEK inhibition by augmenting or combining mechanisms observed in single-agent BRAFi resistance. Hyper-activated MAPK signaling configurations strongly favor ERK phosphorylation, leading to growth/survival finely tuned to the levels of dual inhibitors present. We show that excessive ERK rebound from MAPKi withdrawal induces cell-cycle slowdown (mediated by p38-FRA1-JUNB induction) in weakly addicted, MAPKi-resistant melanoma cells but cell death (by DNA damage and AIF cleavage) in strongly addicted cells. Insights on mechanisms of drug addiction have provided translational potential of novel combination therapy utilizing the synthetic lethality between ERK super-activation and DNA damage. Taken together, our studies have provided rationale for rapid translation of novel combination therapy to overcome relapse

Novel Combination Therapy to Overcome Adaptive and Acquired MAPK-inhibitor Resistance in Melanoma

About 50% of metastatic melanomas harbor BRAF V600 mutations, most commonly a V600E substitution, which constitutively hyper-activate the MAPK pathway and result in oncogene addiction. Therefore, the emergence of BRAF inhibitors (BRAFi) into the clinical arena have represented a true therapeutic paradigm shift for advanced melanomas. However, the almost universal occurrence of acquired resistance to BRAFi and the overabundance of initial partial responses (innate resistance) are urgent clinical problems that limit prolonged patient survival. Our recent efforts have identified 2 core resistance pathways exhibited in disease-progressive melanoma, MAPK reactivation mechanisms and PI3K–PTEN–AKT–upregulating genetic alterations. We hypothesized that these essential survival signaling pathways play a significant role in acute or adaptive resistance in BRAF-mutant melanoma. We observed that BRAF inhibition leads to early and adaptive PI3K–AKT pathway signaling, where preexisting determinants of AKT activation (e.g., PTEN mutation/expression status) limited this BRAFi-elicited PI3K–AKT signaling. However, the presence of a gain-of-function AKT mutant, for instance, would lift this restriction via BRAFi-mediated signal amplification. This study provided a mechanistic link between early, adaptive and late, acquired BRAF inhibitor resistance in melanoma, rationalizing clinical studies to target both MAPK and PI(3)K/AKT/mTOR pathways as combination therapy to delay tumor relapse. Combining BRAF/MEK targeted therapy, to combat acquired BRAFi resistance mechanisms reactivating the MAPK pathway, has been tested to be clinically superior to BRAFi monotherapy but is still challenged by acquired resistance. We show that melanomas acquire resistance to combined BRAF/MEK inhibition by augmenting or combining mechanisms observed in single-agent BRAFi resistance. Hyper-activated MAPK signaling configurations strongly favor ERK phosphorylation, leading to growth/survival finely tuned to the levels of dual inhibitors present. We show that excessive ERK rebound from MAPKi withdrawal induces cell-cycle slowdown (mediated by p38-FRA1-JUNB induction) in weakly addicted, MAPKi-resistant melanoma cells but cell death (by DNA damage and AIF cleavage) in strongly addicted cells. Insights on mechanisms of drug addiction have provided translational potential of novel combination therapy utilizing the synthetic lethality between ERK super-activation and DNA damage. Taken together, our studies have provided rationale for rapid translation of novel combination therapy to overcome relapse

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

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

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

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.SignificanceAcquired 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

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 PDGFRβ), 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