Introduction

Cancer therapy has arguably entered a transformation fueled by the success of targeted agents such as kinase inhibitors deployed against tumors harboring “druggable” oncogene mutations. Unfortunately, resistance to these therapies remains a formidable challenge (1). In some cases of therapeutic resistance, cancer patients fail to respond to treatment at the outset (termed ‘de novo’ or innate resistance). Alternatively, resistance may emerge after several weeks or months of clinical response (termed ‘acquired’ resistance).

The identification of B-RAF mutations as key driver events in malignant melanoma spurred the development of small molecule inhibitors of the mitogen-activated protein kinase (MAP kinase) pathway in an effort to block dysregulated signal transduction engendered by the mutant B-RAF oncoprotein. As a result, B-RAF inhibitors such as vemurafenib or dabrafenib, or MEK inhibitors such as trametinib, elicited striking clinical response rates when administered as single agents in patients with B-RAF^V600E-mutant melanomas (2–4). The use of B-RAF and MEK inhibitors in combination further extends the magnitude and duration of clinical benefit (5). However, intrinsic or acquired resistance to these regimens remains a major clinical problem. Systematic characterization of resistance to these agents is therefore needed in order to further the development of combined therapeutic strategies that either complement existing therapies or provide alternative treatment avenues.

Several mechanisms of resistance to vemurafenib have been described, most of which involve reactivation of downstream MEK/ERK signaling. Interestingly, secondary mutations involving the B-RAF gatekeeper residue (a threonine at codon 529) – common in drug-resistant CML and EGFR-mutant lung cancers – have not been observed, although preclinical data may support such a mechanism (6–9). Multiple laboratories have generated resistant cell line subclones by chronic exposure to RAF inhibitors in vitro, which may facilitate identification of resistance mechanisms by functional or genomic characterization of these cells. Such efforts have implicated amplification or mutation of RAS isoforms (10), enhanced C-RAF expression (11), activation of receptor tyrosine kinases (12) and a splice variant of B-RAF that constitutively dimerizes in the presence of inhibitor, producing sustained MEK/ERK signaling (13). Additionally, systematic gain of function screens identified COT (MAP3K8) as a resistance effector (14). Moreover, secretion of HGF by stromal cells may activate MET to promote resistance to B-RAF inhibition (15, 16). In the aggregate, these studies provided a rationale for the combined use of RAF and MEK inhibitors, and potentially additional therapeutic avenues that may overcome specific resistance mechanisms.

Genome-scale RNAi interference screens may offer a systematic and unbiased genetic approach to study tumor biology and therapeutic resistance. In order to elucidate potential loss-of-function mechanisms of resistance to B-RAF or MEK inhibition, we performed a pooled, lentiviral short hairpin RNA (shRNA) screen in drug-sensitive B-RAF-mutant melanoma cells exposed to high RAF inhibitor concentrations. Characterization of surviving cells identified inactivation of NF1 as a novel mechanism of resistance to both B-RAF and MEK inhibition.

Results

To identify genes capable of suppressing resistance to B-RAF inhibition, we utilized a library of 90,000 shRNAs targeting approximately 16,600 genes expressed in A375 cells, which harbor the B-RAF^V600E mutation and are sensitive to small-molecule RAF and MEK inhibitors (17). Following infection with the lentiviral shRNA library, these cells were cultured in the presence of either DMSO or 3 μM of the RAF inhibitor PLX4720 for 14 and 7 population doublings respectively (Figure 1A and 1B). A drug-resistant population began to emerge within two weeks following shRNA infection (Figure 1B), whereas cells expressing a control shRNA targeting red fluorescent protein (shRFP) remained fully inhibited during this time frame (data not shown). The relative abundance of each shRNA was determined by PCR amplification and massively parallel sequencing of lentiviral library DNA (see Methods). The log fold-enrichment for shRNAs in the PLX4720-treated arm compared to the DMSO-treated arm was assessed. Using RNAi gene enrichment (RIGER) analysis (18), a ranked gene list was generated based upon the degree of enrichment of shRNAs targeting a given gene in the PLX4720-treated arm (Figure 1C). Only 31 genes had 2 or more shRNAs ranked in the top 1000 enriched subgroup (a criterion for candidacy as a “hit” from this screen)(Supplemental table 1). The top-ranking gene was NF1, which encodes the RAS-GTPase activating protein (RAS-GAP) neurofibromin (19). In particular, enrichment of 2 shRNAs targeting NF1 was clearly observed across six PLX4720-treated replicates (Figure 1D). This data raised the possibility that suppression of NF1 activity might permit proliferation of B-RAF-mutant melanoma cells in the presence of inhibitory RAF inhibitor concentrations.

To validate this observation, A375 cells were re-infected with two distinct shRNAs targeting NF1. Reduced expression of NF1 protein was then confirmed by Western blotting (Figure 1E), and cell proliferation was assessed in the presence or absence of 3 μM PLX4720 for 2 weeks. Both NF1 shRNAs permitted robust cell proliferation (as measured by a colony formation assay in vitro) in the presence of PLX4720 (Figure 1F). These results suggested that loss of NF1 in A375 cells may reduce cellular dependency on oncogenic B-RAF for proliferation.

To determine whether suppression of NF1 could provide a generalizable loss-of-function mechanism of resistance to RAF inhibition, the B-RAF mutant melanoma cell lines SKMEL28 and UACC62 were treated with PLX4720 alongside A375 cells following infection with the shNF1 constructs. In each case, sustained cell proliferation in the presence of the RAF inhibitor was observed following NF1 silencing, compared to cells expressing a control shRNA (Figure 2A). Indeed, suppression of NF1 conferred a 5- to 31-fold shift in the GI₅₀ values observed for PLX4720 (e.g. up to 2.2–3.5 μM compared to 0.112 μM in control A375 cells). By comparison, ectopic expression of oncogenic KRAS^G12V, a positive control for resistance to PLX4720 (and which is functionally equivalent to oncogenic forms of NRAS) (14) produced a GI₅₀ of 26.3 μM. In parallel, cells were treated with 1 μM PLX4720 for 16 h and cell lysates were analyzed by Western blotting for ERK phosphorylation (Figure 2B). Loss of NF1 enabled sustained ERK phosphorylation in the presence of PLX4720 compared to mock-treated or shLuc-treated cells, although to a lesser extent than the KRAS^G12V control (Figure 2B). Thus, NF1 silencing was sufficient to confer resistance to RAF inhibition in multiple B-RAF^V600E melanoma cell contexts.

Recent whole-exome sequencing studies of melanoma tumors suggest that mutant NF1 might provide a “driver” genetic event that dysregulates MAP kinase signaling in some melanoma cells that lack B-RAF and NRAS mutations. B-RAF/NRAS “wild-type” melanoma cells are typically unresponsive to RAF inhibition (20–22). We therefore sought to test the ability of NF1 silencing to compensate for mutated B-RAF and modulate sensitivity to RAF inhibitors in an immortalized melanocyte model system (23). Knockdown of NF1 in primary human melanocytes expressing oncogenic B-RAF caused a 10-fold shift in the PLX4720 GI₅₀, permitted robust proliferation in the presence of 3 μM PLX4720, and allowed sustained ERK phosphorylation in the presence of 0.2 and 1 μM PLX4720 (Supplemental Figure 1A–C). These experiments provided independent evidence that silencing of NF1 could confer resistance to RAF inhibition in a MAP kinase pathway-dependent manner.

Given that NF1 is a known negative regulator of RAS activity (24), we queried the activation state of RAS in A375 cells following NF1 knockdown using a RAS-GTP pull down assay. As expected, NF1 suppression caused a substantial increase in the level of active GTP-bound RAS (Figure 3A). Associated with the increased RAS-GTP, we observed a concomitant increase in C-RAF activation, as measured by phosphorylation of Ser338 (Figure 3B). C-RAF (Ser338) phosphorylation was further enhanced in the presence of PLX4720, suggesting that the enhanced RAS-GTP produced by NF1 silencing was competent to potentiate so-called “paradoxical” RAF activation (25–27).

Based on these observations, we next tested the requirement for C-RAF to mediate NF1-driven resistance to RAF inhibition by performing combined shRNA-mediated knockdown of both C-RAF and NF1, followed by assessment of MAPK signaling and sensitivity to RAF inhibition. Silencing of C-RAF alone had little effect on the responsiveness of MAPK signaling to PLX4720—this was expected given prior observations that C-RAF is inactive in B-RAF^V600E melanoma under steady state conditions (28). However, under conditions of combined C-RAF/NF1 knockdown, ERK phosphorylation was inhibited more effectively compared to knockdown of NF1 alone (Figure 3B). Moreover, whereas cyclin D1 levels were inhibited by PLX4720 in A375 cells (as expected given the known regulation of this protein by MAP kinase signaling in B-RAF^V600E melanoma), but NF1 silencing partially alleviated this effect (Figure 3B). Effects of PLX4720 on cyclin D1 protein expression were restored when both NF1 and C-RAF were depleted concomitantly. Quantitative analysis of the Western blots confirmed these observations (Figure 3C). Cellular sensitivity to PLX4720 followed a similar trend, whereby combinatorial knockdown of C-RAF and NF1 reversed the resistance to PLX4720 conferred by NF1 loss (Figure 3D).

Having established that loss of NF1 could promote resistance to RAF inhibition, we sought to determine if NF1 silencing could also affect sensitivity to pharmacologic MEK inhibition. In particular, we hypothesized that inhibition of MEK, a substrate of both B-RAF and C-RAF, might have equal potency regardless of NF1 expression (given that MEK is a limiting MAPK kinase effector downstream of C-RAF). Interestingly, cells exhibiting NF1 knockdown were partially resistant to AZD6244 (Figure 4A). Here, NF1 silencing increased theGI₅₀ to the MEK inhibitor AZD6244 by approximately 7-fold. ERK phosphorylation was also sustained in the presence of AZD6244 (Figure 4B). Combined inhibition of B-RAF and MEK by simultaneous treatment with PLX4720 and AZD6244 achieved greater efficacy than either agent alone; however, NF1 knockdown was still associated with residual resistance to combined RAF/MEK inhibition in vitro (Figure 4C). Moreover, a clear correlation was evident between residual ERK phosphorylation and inhibition of cell proliferation (Figure 4D). Nonetheless, a robust resistance phenotype remained apparent in the setting of NF1 knockdown, even under conditions of combined RAF/MEK inhibitor exposure.

In the presence of activated RAS, PLX4720 can activate C-RAF through loss of feedback inhibition and induction of RAF-dimerization (25–27). Conceivably then, the RAS activation engendered by NF1 loss might promote a biochemical state wherein the activated RAF/MEK complex is less vulnerable to MEK inhibition, particularly under conditions of concomitant RAF inhibitor exposure. To test this hypothesis, we used the irreversible RAF inhibitor AZ628. This compound prevents C-RAF activation due to persistent occupation of the ATP binding site (25). In contrast to the aforementioned drug conditions, only minimal resistance (2-fold) was observed to AZ628 (Figure 5A). Moreover, ERK phosphorylation was effectively suppressed (between 80–90%) by this irreversible inhibitor, even in the setting of NF1 knockdown but only by 65% in the presence of oncogenic KRAS (Figure 5B). Furthermore, combined NF1/C-RAF knockdown resensitized cells to treatment with the MEK inhibitor (Supplemental Figure 2). These results provided additional support for the notion that C-RAF signaling is required for the resistance phenotype induced by NF1 loss.

We also tested the effect of pharmacologic ERK inhibition in this setting using the compound VTX-11e (29). Interestingly, B-RAF^V600E melanoma cells harboring NF1 knockdown remained sensitive to ERK inhibition (Figure 5C). Consistent with this observation, phosphorylation of the ERK substrate FRA1 was inhibited equally in the presence or absence of NF1 knockdown (Figure 5D). This pattern of activity was confirmed in colony formation assays, whereby robust resistance was observed to the selective RAF inhibitors PLX4720, PLX4032, GDC0879 and the MEK inhibitor AZD6244. However, NF1 knockdown produced only modest resistance to the irreversible RAF inhibitor AZ628, and no resistance to the ERK inhibitor VTX-11e (Supplemental Figure 3A&B).

To understand the potential for endogenous NF1 inactivation to mediate resistance to RAF inhibition, we leveraged the Cancer Cell Line Encyclopedia (CCLE) (30) to identify cell line models that contained putative inactivating NF1 mutations in the context of oncogenic B-RAF^V600E mutation. Toward this end, over 500 CCLE lines have previously been profiled for pharmacologic sensitivity to RAF and MEK inhibition (30). We identified five B-RAF^V600E cell lines (3 melanomas and 2 colorectal tumors) with co-occurring mutations in NF1 (Supplemental Figure 4). Of these, three contained obvious “damaging” NF1 mutations (HS695T: NF1^Q959*, LOXIMVI: NF1^Q1174*and RKO: NF1^{L626fs,Q2340fs,V2205A}) whereas two contained point mutations (WM88: NF1^R1306Q, LS411N: NF1^T2805I), deemed unlikely to affect NF1 function according to the Mutation Assessor algorithm (data not shown)(31). HS695T and RKO cells also exhibited reduced NF1 mRNA expression. Firstly, we assessed NF1 protein expression by Western blotting and those cell lines with damaging mutations in NF1 expressed significantly less protein than the wild-type cells (Figure 6A). The sensitivity of HS695T, LOXIMVI and RKO cells to MAPK pathway inhibition by PLX4720 was assessed alongside A375, SKMEL28 and UACC62 B-RAF^V600E/NF1^WT melanoma cells. B-RAF^V600E cell lines that expressed no or undetectable levels of NF1 protein (LOXIMVI and RKO) were highly resistant to the inhibition of ERK phosphorylation by PLX4720, whereas HS695T cells which express low levels of NF1 protein were still relatively sensitive (Figure 6B). In accord with this finding, NF1 mutation correlated with resistance to PLX4720 in these cells (Figure 6C). The mean GI₅₀ was 0.247 μM in NF1-wild-type cells which increased greater than 50-fold in the NF1-mutant cell lines to 15.8 μM

Given that NF1 knockdown did not confer resistance to pharmacologic ERK inhibition in A375 cells (as described above), we tested the hypothesis that B-RAF^V600E/NF1-mutant cancer cell lines might remain sensitive to VTX-11e. Notably, the ERK inhibitor showed activity against NF1-mutant cell lines with a mean GI₅₀ of 0.781 μM compared to 0.219 μM in B-RAF-mutant cells with wild-type NF1 (Figure 6D). These results suggested that pharmacologic ERK inhibition might in principle provide a viable therapeutic strategy in B-RAF^V600E cancer cells harboring NF1 loss.

To confirm that NF1 alterations may confer resistance to B-RAF inhibition in human melanoma, we assessed the status of NF1 using whole exome sequencing of tumors from patients who had relapsed during vemurafenib treatment. We identified four patients whose tumors expressed mutant alleles of NF1 (Figure 7A). Of these, one was a nonsense mutation (patient 46, p.R2450*) that would likely result in reduced protein expression. This mutation was present in both pre-treatment and post-relapse biopsies, and interestingly the patient had a progression-free survival of only ~10 weeks. This short duration of response raised the possibility that the concomitant NF1 mutation conferred intrinsic resistance to vemurafenib (Figure 7B). The other NF1 mutations were silent events (patient 15, c.135T>C, patient 45 c.4023G>A and patient 50 c3018C>T) that are not predicted to alter NF1 protein expression. However, given that NF1 is known to be affected by splice-site mutations (32), we assessed the predicted impact of these mutations on both canonical splice sites and exon splice enhancer sites (ESEs) using the Human Splicing Finder (33). This analysis revealed that all three mutations were located within candidate splicing motifs. Conceivably, then, these mutations may produce aberrant NF1 exon splicing. Like patient 46, patients 15 and 50 exhibited response rates of relatively short duration (10 and 12 weeks, respectively). Moreover, these mutations were also observed in both the pre-treatment and post-relapse biopsies, consistent with intrinsic resistance to vemurafenib. In contrast, patient 45 experienced a progression-free survival of five months, and the NF1 mutation was only present in the post-relapse biopsy (Figure 7C), indicative of acquired resistance. Together, these observations provide preliminary support for the notion that genomic dysregulation of NF1 may influence both de novo and acquired resistance to RAF inhibition in the clinical arena.

Discussion

In malignant melanoma, activating B-RAF mutations confer exquisite dependency on RAF/MEK/ERK signaling, which has been successfully targeted using small molecule RAF and MEK inhibitors. However, therapeutic resistance inevitably develops following a period of disease stabilization or regression, and some B-RAF^V600E-mutant melanomas exhibit intrinsic resistance to these agents (2). Understanding the mechanistic basis of drug resistance is essential to the development of new therapeutic strategies that maximize clinical benefit. Here, we queried resistance to RAF inhibition using an unbiased, genome-scale RNA interference screen for modifiers of sensitivity to small molecule RAF inhibition. Our approach builds upon complementary studies using gain-of-function approaches, such as systematic open reading frame (ORF) overexpression screens, (14) or the generation of resistant cell lines by chronic exposure to inhibitors (10, 11, 13, 34).

The identification of NF1 loss as a resistance effector describes one of the first loss-of-function events capable of mediating resistance to RAF inhibitors identified by systematic functional approaches. The protein encoded by the NF1 gene, neurofibromin, is a known tumor suppressor gene and negative regulator of RAS proteins. This function is achieved through stimulation of the GTPase activity of RAS by neurofibromin, thus converting it from an active, GTP-bound form to its inactive GDP-bound form. Therefore, loss of NF1 activates RAS and provides an upstream stimulus to activate C-RAF, driving resistance to a RAF inhibitor by reactivation of the MAPK pathway. This is consistent with other reported mechanisms whereby RAS is mutated or amplified in vemurafenib-resistant cell lines, signaling through C-RAF to maintain MAPK signaling (10, 35). These findings are therefore consistent with several published reports indicating that the majority of resistance mechanisms reported for RAF inhibitors involve reactivation of the MAPK pathway. This underscores a fundamental dependency on RAS signaling potentiated by NF1 loss. This event also confers a C-RAF dependency in the setting of pharmacologic RAF inhibition, as evidenced by a near-complete reversal of the resistance phenotype by C-RAF knockdown. This observation raises the possibility that, in the future, saturating pharmacologic inhibition of both B- and C-RAF (and possibly A-RAF as well) could reduce the capacity for resistance to develop. Such inhibition might be achieved by an irreversible RAF inhibitor that both promotes an inactive conformation of RAF. Consistent with this notion is the observation that NF1 was unable to confer resistance to AZ628, a tool compound with these characteristics (25). Consequently, we hypothesized that either a single-agent MEK inhibitor or combined RAF and MEK inhibition might circumvent NF1-mediated resistance. Indeed, combined exposure displayed more potent activity in vitro than did either agent alone in the setting of NF1 knockdown. However, the drug combinations tested herein did not fully restore sensitivity, at least in vitro. Conceivably, this limitation may be overcome by more potent MEK inhibitors or clinical RAF/MEK regimens that enable more complete suppression of MEK/ERK signaling. Alternatively, C-RAF may have MEK-independent roles in tumor cell survival and proliferation (36–39). Therefore, whilst combined RAF/MEK inhibition certainly improves clinical responses in B-RAF-mutant melanoma (5), the potential for NF1 loss (and perhaps other mechanisms of upstream RAS-activation) to drive resistance may remain. On the other hand, melanoma cells harboring NF1 knockdown remained sensitive to a selective ERK inhibitor, suggesting that ERK may represent a pivotal integration point for upstream MAP kinase signaling, and may offer therapeutic potential for circumventing multiple resistance mechanisms.

To ascertain whether NF1 loss might provide an endogenous resistance mechanism, we analyzed cancer cell lines in which NF1 mutation co-occurred with B-RAF^V600E mutation. Using the Cancer Cell Line Encyclopedia, we identified 5 B-RAF^V600E cell lines that had NF1 mutations, 3 of which were nonsense mutations. Each of these cell lines was insensitive to PLX4720, consistent with the NF1 shRNA screening and validation results in RAF-inhibitor-sensitive cell lines. Furthermore, by integrating our findings with a published cohort of 121 primary melanoma tumors, metastatic tumors and short-term cultures (20) previously analyzed by whole exome sequencing, we gained additional insights into the potential clinical relevance of NF1 mutations. In this cohort, putatively damaging NF1 mutations were observed in 25% (5/21) of samples that lacked highly recurrent mutations in either B-RAF or NRAS. Genomic studies of an independent melanoma cohort have also observed this association (21). However, NF1 mutations were also observed in B-RAF- or NRAS-mutant tumors, including a splice site mutation (NF1^G1166) co-occurring with the activating mutant B-RAF^K601E,P731S, a nonsense mutation (NF1^R440*) co-occurring with NRAS^Q61K, and a GAP-related domain mutation (NF1^P1323L) in a B-RAF^V600E tumor. Given that both melanoma and colorectal tumors harbor coexisting B-RAF and NF1 mutations, it therefore seemed likely that clinical scenarios could arise where NF1 loss serves as a gating resistance effector in the setting of B-RAF/MEK inhibition. Toward this end, we assessed NF1 alterations by whole exome sequencing in the tumors of melanoma patients treated with vemurafenib who progressed whilst on treatment. We identified four patients whose tumors had NF1 alterations that could impact upon protein function or expression. One event resulted in a nonsense mutation in NF1 (p.R2450*), likely to result in nonfunctional protein. The other three mutations were silent events but importantly, they occurred in putative splice regulatory or exon splicing enhancer sites predicted to regulate proper mRNA processing. These data affirm the potential for NF1 alterations to drive both de novo and acquired resistance to vemurafenib in melanoma. As deep genomic/molecular characterization accrues for tumor specimens obtained prior to treatment, during treatment and following relapse, the relevance of NF1 loss to clinical resistance should become clearer.

Our data may also endorse the development of irreversible RAF inhibitors. Such agents may in principle show benefit in NRAS-mutant tumors because of their enhanced dependency upon C-RAF signaling for tumor cell growth. Alternatively, the use of ERK inhibitors may overcome numerous melanoma resistance mechanisms if such agents can be administered safely at doses that achieve robust target inhibition. Toward this end, both irreversible-RAF and ERK inhibitors are entering preclinical/clinical trials, thereby offering forthcoming opportunities to evaluate their efficacy in B-RAF-mutant cancers. Altogether, these results demonstrate the promise of systematic preclinical studies combined with knowledge of tumor genetic/molecular alterations to elucidate cancer drug resistance mechanisms that may inform novel therapeutic avenues for many types of cancer.

Materials and Methods

Supplementary Material