Acquired BRAF Inhibitor Resistance Research Articles

The path of least resistance: enhancing the effectiveness of BRAF inhibitors.

The combination of genetic, epigenetic and microenvironment-driven phenotypic heterogeneity within tumours is so great, some argue that resistance to therapy is inevitable and prevention will be the only effective anticancer strategy. For most melanoma, changes in behaviour that reduce sun exposure would reduce its incidence substantially. However, despite raised public awareness of the dangers of excessive sun exposure, the increasing incidence of the disease means that devising an effective antimelanoma therapy remains a priority. Until the identification of activating mutations in BRAF (Davies et al., 2002), an upstream activator of mitogen-activated kinase (MAPK) signalling, as a major driver of melanoma progression in around 50% patients, antimetastatic melanoma strategies were notoriously ineffective. Now, the ability to genotype rapidly those patients with BRAF-mutant melanoma means that inhibitors of activated BRAF such as vemurafenib can be used to deliver ‘personalized targeted therapy’. Consistent with the key role of activated BRAF in melanoma, its inhibition leads to rapid tumour regression and in most cases improved survival. However, despite its initial promise, resistance to anti-BRAF therapy is observed in almost every patient (Sosman et al., 2012). Resistance can be acquired through a wide range of mechanisms including activation of upstream and downstream effectors of MAPK signalling, increased expression of various activated BRAF isoforms and activation of the AKT survival pathway. Thus, currently, there remains no effective cure for metastatic melanoma. As BRAF inhibitors are extremely effective before the emergence of resistance, it is not surprising that considerable effort is being applied to working out how best to use them. Now in two papers, Das Thakur et al. and Beck et al. highlight potential strategies to enhance the effectiveness of vemurafenib. “Discontinuous vemurafenib therapy may forestall the emergence of lethal resistance” Although discontinuous drug administration may yield short-term advantages and prolong survival, it seems likely that other resistance mechanisms will eventually emerge and that remission-free survival will remain elusive. Complementary strategies can be envisaged that either work on BRAF-inhibitor resistant cells via a completely different mechanism or by enhancing cell death by exploiting synergies with BRAF inhibitors. In the report from Beck et al., an examination of the molecular mechanism underlying apoptosis induced by vemurafenib revealed one such potential synergy. While it has been long known that vemurafenib induces apoptosis, mechanistically how this was achieved was not entirely clear. Beck et al. now reveal that vemurafenib induces endoplasmic reticulum (ER) stress in BRAFV600E melanoma cells. Endoplasmic reticulum stress can trigger apoptosis and occurs in response to disruption of normal ER function; nutrient deprivation, calcium imbalance, hypoxia or dysregulated protein glycosylation can lead to accumulation of misfolded proteins and activation of the unfolded protein response (UPR), increased transcription of UPR-associated factors, reduced protein synthesis and ER-associated protein degradation (Hetz, 2012). Together this adaptive response aims to restore normal ER functionality, or if this is not possible, trigger apoptosis. BRAF-mutant melanoma cells treated with BRAF inhibitors increase expression of the pro-apoptotic factor BCL2-interacting mediator of cell death, BIM (Paraiso et al., 2011), which is also elevated in response to ER stress. Beck et al. showed that BIM knockdown reduced vemurafenib-mediated cell death, providing a clue that ER stress could occur on BRAF-inhibition. Consistent with this, they also demonstrated that vemurafenib induces phosphorylation of the translation regulator eIF2a and up-regulated expression of ER-stress-related genes including the transcription factor ATF4, all hallmarks of an ER-stress response. Importantly, knockdown of ATF4 substantially reduced vemurafenib-mediated apoptosis. Significantly, treatment of rapidly proliferating melanoma tumours in a chick embryo model with thapsigargin, a well-characterized activator of ER stress, indicated that activation of ER stress could lead to substantially reduced proliferation and invasiveness. Moreover, although thapsigargin did not increase the effectiveness of BRAF-inhibition in melanoma cells that were highly vemurafenib sensitive, in less sensitive cells, thapsigargin was able to enhance the antitumour effectiveness of the anti-BRAF drug. The authors then went on to ask whether induction of ER stress might be effective in melanoma cells resistant to BRAF inhibitors. Significantly, thapsigargin was able to induce apoptosis in vemurafenib-resistant cell lines in which it inhibited phosphorylation of AKT, but not ERK. Taken together, the data presented by Beck et al. suggest that at least in some circumstances, ER-stress inducers may provide potential therapeutic benefit, especially in melanomas that have acquired BRAF-inhibitor resistance, and also highlight the possible synergy between AKT inhibition and ER-stress induction. The studies from Thakur et al. and Beck et al. suggest that while resistance to BRAF inhibitors is a major barrier to remission-free survival, there are potential strategies that can be adopted that may enhance the efficacy of BRAF-targeted therapies.

Abstract B13: Modeling BRAF inhibitor resistance in genetically engineered mouse models of melanoma

Abstract Resistance to BRAF inhibitors in melanoma poses a serious clinical challenge to this otherwise potent therapy. Acquired resistance has been shown to arise from a wide variety of genetic and epigenetic sources including NRAS or MEK mutations, elevated PDGFRB or IGF1R levels, and BRAF splice variants. Many of these mechanisms were discovered in vitro and validated in patients, demonstrating the faithfulness of cell culture data. Nevertheless, cell culture may not be able to capture all sources of resistance, particularly if they involve interaction with the stroma. We have therefore undertaken the generation of de novo BRAF inhibitor resistance in a novel mouse model of melanoma, the iBIP (inducible Braf, Ink/Arf, Pten) model. Similar to existing BRAF mouse models, topical tamoxifen restricts BRAFV600E expression and PTEN loss to targeted melanocytes. The added utility of this model is the ability to extinguish BRAF transgene expression via withdrawal of doxycycline from the diet. We show that the molecular profile and tumor regression phentoype of BRAF inhibitor treatment in this model faithfully mirrors genetic BRAF extinction. Treatment with the BRAF inhibitor caused stable and complete tumor regression. However, long-term treatment with the inhibitor failed to generate de novo resistant melanomas. We hypothesized that the relatively stable mouse genome was not generating potentially resistance-conferring mutations or alterations at a fast enough rate. We therefore generated genomic instability in the iBIP model by crossing it onto a telomerase-deficient background. Melanomas in this model acquired resistance at a rate of approximately 30% after a short interval of sensitivity. Currently, characterization of these tumors is being carried out, including whole-exon sequencing, RNA-sequencing, and array comparative genomic hybridization. Preliminary aCGH data show a recurrent, whole-chromosome gain in two resistant tumors that is not present in untreated or sensitive tumors. Other efforts are focused on generating a large bank of resistant tumors, checking cross-resistance among BRAF inhibitors, and identifying any stromal and immune changes. This in vivo model of acquired BRAF inhibitor resistance provides an important complement to known resistance mechanisms and may also serve as a preclinical platform to test anti-resistance countertherapies.

Making Sense of MEK1 Mutations in Intrinsic and Acquired BRAF Inhibitor Resistance

In this issue of Cancer Discovery, Shi and colleagues add further insight into the role of exon 3 MEK1 mutations in BRAF inhibitor resistance by demonstrating the presence of P124SMEK1 and I111SMEK1 mutations concurrently with V600E/KBRAF mutations at baseline in 16% of melanoma specimens. Although the presence of P124SMEK1 or I111SMEK1 mutations did not predict for resistance, and these alleles were not selected for upon BRAF inhibition, other exon 3 MEK1 mutations, such as C121S, did convey resistance, suggesting a role for defined exon 3 MEK1 mutations in acquired BRAF inhibitor resistance.

Targeting mutant BRAF in melanoma: current status and future development of combination therapy strategies.

The discovery of activating BRAF mutations in ∼50% of all melanomas has proved to be a turning point in the therapeutic management of the disseminated disease. In this commentary, we review the latest research delineating the role of mutant BRAF in melanoma initiation and progression and discuss the remarkable 10-year journey leading up to the recent U.S. Food and Drug Administration approval of the small-molecule BRAF inhibitor vemurafenib. We further outline the most recent findings on the mechanisms that underlie intrinsic and acquired BRAF inhibitor resistance and describe ongoing preclinical and clinical studies designed to delay or abrogate the onset of therapeutic escape. It is hoped that our evolving understanding of melanoma genetics and intracellular signaling coupled with a growing armamentarium of signal transduction inhibitors will lead to significant improvements in the level and durability of therapeutic response in metastatic melanoma.

Melanoma whole-exome sequencing identifies V600EB-RAF amplification-mediated acquired B-RAF inhibitor resistance

The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor (B-RAFi) therapy for melanoma patients. Here we show V600EB-RAF copy number gain as a mechanism of acquired B-RAFi resistance in four out of twenty (20%) patients treated with B-RAFi. In cell lines, V600EB-RAF over-expression and knockdown conferred B-RAFi resistance and sensitivity, respectively. In V600EB-RAF amplification-driven (vs. mutant N-RAS-driven) B-RAFi resistance, ERK reactivation is saturable, with higher doses of vemurafenib down-regulating pERK and re-sensitizing melanoma cells to B-RAFi. These two mechanisms of ERK reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAFi vemurafenib. In contrast to mutant N-RAS-mediated V600EB-RAF bypass, which is sensitive to C-RAF knockdown, V600EB-RAF amplification-mediated resistance functions largely independently of C-RAF. Thus, alternative clinical strategies may potentially overcome distinct modes of ERK reactivation underlying acquired B-RAFi resistance in melanoma.