BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model

Abstract

BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼80% of BRAF V600 -mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf V618E (analogous to the human BRAF V600E mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf V618E transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes. melanoma | drug resistance | BRAF inhibitors | mouse models T he discovery that ∼50-60% of melanomas carry BRAF V600E point mutations (1) prompted the generation of compounds specifically targeting this hyperactive mutated kinase. One such compound, PLX4032, has shown unprecedented therapeutic efficacy in clinical trials and was therefore FDA-approved for clinical therapy under the name vemurafenib. Despite its remarkable efficacy, almost all patients receiving BRAF inhibitor treatment relapsed after weeks to months of therapy (2-5). Acquired resistance to BRAF inhibitors has since been a major focus of research and two major paths to resistance have emerged: MAPK-dependent and MAPK-independent mechanisms. MAPK-dependent mechanisms primarily involve reactivation of the MAPK pathway to substitute for the inhibition of BRAF V600E. This may be achieved through mechanisms including expression of alternative splicing forms of BRAF V600E

Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf −/− mouse model

“…Importantly, our findings were confirmed in patient-derived xenograft models, underlining the benefit of large-scale in vivo forward genetic screening to identify potential resistance mechanisms to targeted therapeutics. (20). Consequently, we conducted a large-scale transposon-based insertional mutagenesis screen to investigate the resistance to HDM201 in mice.…”

Identification of NRAS isoform 2 overexpression as a mechanism facilitating BRAF inhibitor resistance in malignant melanoma

“…Additionally, studies with the combination of vemurafenib and the MEK inhibitor selumetinib showed that combination with different PI3K pathway inhibitors led to synergistic reduction of cell viability and enhanced apoptosis in vitro (18). Perna et al (19) also observed that in a mouse model of BRAF mutant melanoma, hyperphosphorylation of AKT conferred resistance to vemurafenib and this resistance could be reversed by combinatorial treatment with both vemurafenib and an AKT inhibitor. However, a better understanding of the mechanism of how and when NRAS isoform 2 binds to RAF and PI3K could serve to elucidate additional treatment strategies.…”

Use of DNA transposons for functional genetic screens in mouse models of cancer

“…They treated Tyrosinase-Cre ERT2 ; Rosa26-LSL-SB13; T2/Onc; LSL-Braf V618E/+ mice displaying melanomas with the BRAF inhibitor PLX4720, and identified ERas as a novel candidate that mediates resistance to PLX4720 [22 ]. This study shows that insertional mutagenesis can be also used for the identification of molecular mechanisms involved in drug resistance.…”