Mitogen‐activated protein kinase dependency in BRAF/RAS wild‐type melanoma: A rationale for combination inhibitors

Inhibitors targeting the mitogen‐activated protein kinase (MAPK) pathway and immune checkpoint molecules have dramatically improved the survival of patients with BRAF^V600‐mutant melanoma. For BRAF/RAS wild‐type (WT) melanoma patients, however, immune checkpoint inhibitors remain the only effective therapeutic option with 40% of patients responding to PD‐1 inhibition. In the present study, a large panel of 10 BRAF^V600‐mutant and 13 BRAF/RAS WT melanoma cell lines was analyzed to examine MAPK dependency and explore the potential utility of MAPK inhibitors in this melanoma subtype. We now show that the majority of BRAF/RAS WT melanoma cell lines (8/13) display some degree of sensitivity to trametinib treatment and resistance to trametinib in this melanoma subtype is associated with, but not mediated by NF1 suppression. Although knockdown of NF1 stimulates RAS and CRAF activity, the activation of CRAF by NF1 knockdown is limited by ERK‐dependent feedback in BRAF‐mutant cells, but not in BRAF/RAS WT melanoma cells. Thus, NF1 is not a dominant regulator of MAPK signaling in BRAF/RAS WT melanoma, and co‐targeting multiple MAP kinase nodes provides a therapeutic opportunity for this melanoma subtype.     

PTEN regulates IGF‐1R‐mediated therapy resistance in melanoma

Inhibition of the mitogen‐activated protein kinase (MAPK) pathway is a major advance in the treatment of metastatic melanoma. However, its therapeutic success is limited by the rapid emergence of drug resistance. The insulin‐like growth factor‐1 receptor (IGF‐1R) is overexpressed in melanomas developing resistance toward the BRAF^V600 inhibitor vemurafenib. Here, we show that hyperactivation of BRAF enhances IGF‐1R expression. In addition, the phosphatase activity of PTEN as well as heterocellular contact to stromal cells increases IGF‐1R expression in melanoma cells and enhances resistance to vemurafenib. Interestingly, PTEN‐negative melanoma cells escape IGF‐1R blockade by decreased expression of the receptor, implicating that only in melanoma patients with PTEN‐positive tumors treatment with IGF‐1R inhibitors would be a suitable strategy to combat therapy resistance. Our data emphasize the crosstalk and therapeutic relevance of microenvironmental and tumor cell‐autonomous mechanisms in regulating IGF‐1R expression and by this sensitivity toward targeted therapies.     

Inhibition of mutant BRAF splice variant signaling by next‐generation,selective RAF inhibitors

Vemurafenib and dabrafenib block MEK‐ERK1/2 signaling and cause tumor regression in the majority of advanced‐stage BRAF^V600E melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next‐generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF^V600E melanoma cells without paradoxical effects in wild‐type cells. Furthermore, PLX7904 blocks the growth of vemurafenib‐resistant BRAF^V600E cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF^V600E splice variant‐mediated vemurafenib‐resistant cells. We show that paradox‐breaker RAF inhibitors potently block MEK‐ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720‐resistant cells harboring distinct BRAF^V600E splice variants. These data support the further investigation of paradox‐breaker RAF inhibitors as a second‐line treatment option for patients failing on vemurafenib or dabrafenib.     

Modification,Biological Evaluation and SAR Studies of Novel 1H‐Pyrazol Derivatives Containing N,N′‐Disubstituted Urea Moiety as Potential Anti‐melanoma Agents

Malignant melanomas are amongst the most aggressive cancers. BRAF Inhibitors have exhibited therapeutic effects against BRAF‐mutant melanoma. In continuation of our earlier studies on anti‐melanoma agents based on 1H‐pyrazole skeleton, two sets of novel compounds that include 1H‐pyrazole‐4‐amines FA 1 – FA13 and corresponding urea derivatives FN 1 – FN13 have been synthesized and evaluated for their BRAF^V600E inhibitory and antiproliferation activities. Compound FN 10 displayed the most potent biological activity against BRAF^V600E (IC₅₀ = 0.066 μm ) and the A375 human melanoma cell line (GI₅₀ = 0.81 μm ), which was comparable to the positive control vemurafenib, and more potent than our previously reported 1H‐pyrazole‐3‐amines and their urea derivatives. The results of SAR studies and molecular docking can guide further optimization and may help to improve potency of these pyrazole‐based anti‐melanoma agents.     

Inhibition of both BRAF and MEK in BRAFV600E mutant melanoma restores compromised dendritic cell (DC) function while having differential direct effects on DC properties

Purpose

Dendritic cells (DCs) can induce strong tumor-specific T-cell immune responses. Constitutive upregulation of the mitogen-activated protein kinase (MAPK) pathway by a BRAF^V600 mutation, which is present in about 50 % of metastatic melanomas, may be linked to compromised function of DCs in the tumor microenvironment. Targeting both MEK and BRAF has shown efficacy in BRAF^V600 mutant melanoma.

Methods

We co-cultured monocyte-derived human DCs with melanoma cell lines pretreated with the MEK inhibitor U0126 or the BRAF inhibitor vemurafenib. Cytokine production (IL-12 and TNF-α) and surface marker expression (CD80, CD83, and CD86) in DCs matured with the Toll-like receptor 3/Melanoma Differentiation-Associated protein 5 agonist polyI:C was examined. Additionally, DC function, viability, and T-cell priming capacity were assessed upon direct exposure to U0126 and vemurafenib.

Results

Cytokine production and co-stimulation marker expression were suppressed in polyI:C-matured DCs exposed to melanoma cells in co-cultures. This suppression was reversed by MAPK blockade with U0126 and/or vemurafenib only in melanoma cell lines carrying a BRAF^V600E mutation. Furthermore, when testing the effect of U0126 directly on DCs, marked inhibition of function, viability, and DC priming capacity was observed. In contrast, vemurafenib had no effect on DC function across a wide range of dose concentrations.

Conclusions

BRAF^V600E mutant melanoma cells modulate DC through the MAPK pathway as its blockade can reverse suppression of DC function. MEK inhibition negatively impacts DC function and viability if applied directly. In contrast, vemurafenib does not have detrimental effects on important functions of DCs and may therefore be a superior candidate for combination immunotherapy approaches in melanoma patients.     

The broad‐spectrum receptor tyrosine kinase inhibitor dovitinib suppresses growth of BRAF‐mutant melanoma cells in combination with other signaling pathway inhibitors

BRAF inhibitors have revolutionized treatment of mutant BRAF metastatic melanomas. However, resistance develops rapidly following BRAF inhibitor treatment. We have found that BRAF‐mutant melanoma cell lines are more sensitive than wild‐type BRAF cells to the small molecule tyrosine kinase inhibitor dovitinib. Sensitivity is associated with inhibition of a series of known dovitinib targets. Dovitinib in combination with several agents inhibits growth more effectively than either agent alone. These combinations inhibit BRAF‐mutant melanoma and colorectal carcinoma cell lines, including cell lines with intrinsic or selected BRAF inhibitor resistance. Hence, combinations of dovitinib with second agents are potentially effective therapies for BRAF‐mutant melanomas, regardless of their sensitivity to BRAF inhibitors.     

Metabolic stress regulates ERK activity by controlling KSR‐RAF heterodimerization

Altered cell metabolism is a hallmark of cancer, and targeting specific metabolic nodes is considered an attractive strategy for cancer therapy. In this study, we evaluate the effects of metabolic stressors on the deregulated ERK pathway in melanoma cells bearing activating mutations of the NRAS or BRAF oncogenes. We report that metabolic stressors promote the dimerization of KSR proteins with CRAF in NRAS‐mutant cells, and with oncogenic BRAF in BRAF^V600E‐mutant cells, thereby enhancing ERK pathway activation. Despite this similarity, the two genomic subtypes react differently when a higher level of metabolic stress is induced. In NRAS‐mutant cells, the ERK pathway is even more stimulated, while it is strongly downregulated in BRAF^V600E‐mutant cells. We demonstrate that this is caused by the dissociation of mutant BRAF from KSR and is mediated by activated AMPK. Both types of ERK regulation nevertheless lead to cell cycle arrest. Besides studying the effects of the metabolic stressors on ERK pathway activity, we also present data suggesting that for efficient therapies of both genomic melanoma subtypes, specific metabolic targeting is necessary.     

Identification of PLX4032‐resistance mechanisms and implications for novel RAF inhibitors

BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole‐exome sequencing of drug‐resistant BRAF^V600K melanoma cells. We further describe a new screening approach, a genome‐wide piggyBac mutagenesis screen that revealed clinically relevant aberrations (N‐terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution (BRAF^L505H), is the first resistance‐conferring second‐site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF‐PLX4032 interface with a larger polar residue. Moreover, we show that BRAF^L505H, found in human prostate cancer, is itself a MAPK‐activating, PLX4032‐resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032‐resistant melanoma cells are sensitive to novel, next‐generation BRAF inhibitors, especially the ‘paradox‐blocker’ PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF‐mutations.     

NVP-LDE225, a Potent and Selective SMOOTHENED Antagonist Reduces Melanoma Growth In Vitro and In Vivo

Melanoma is one of the most aggressive cancers and its incidence is increasing worldwide. So far there are no curable therapies especially after metastasis. Due to frequent mutations in members of the mitogen-activated protein kinase (MAPK) signaling pathway, this pathway is constitutively active in melanoma. It has been shown that the SONIC HEDGEHOG (SHH)-GLI and MAPK signaling pathway regulate cell growth in many tumors including melanoma and interact with each other in the regulation of cell proliferation and survival.Here we show that the SHH-GLI pathway is active in human melanoma cell lines as they express downstream target of this pathway GLI1. Expression of GLI1 was significantly higher in human primary melanoma tissues harboring BRAF^V600E mutation than those with wild type BRAF. Pharmacologic inhibition of BRAF^V600E in human melanoma cell lines resulted in decreased expression of GLI1 thus demonstrating interaction of SHH-GLI and MAPK pathways. Inhibition of SHH-GLI pathway by the novel small molecule inhibitor of smoothened NVP-LDE225 was followed by inhibition of cell growth and induction of apoptosis in human melanoma cell lines, interestingly with both BRAF^V600E and BRAF^{Wild Type} status. NVP-LDE225 was potent in reducing cell proliferation and inducing tumor growth arrest in vitro and in vivo, respectively and these effects were superior to the natural compound cyclopamine.Finally, we conclude that inhibition of SHH-GLI signaling pathway in human melanoma by the specific smoothened inhibitor NVP-LDE225 could have potential therapeutic application in human melanoma even in the absence of BRAF^V600E mutation and warrants further investigations.     

SHOC2 and CRAF Mediate ERK1/2 Reactivation in Mutant NRAS-mediated Resistance to RAF Inhibitor

ERK1/2 signaling is frequently dysregulated in tumors through BRAF mutation. Targeting mutant BRAF with vemurafenib frequently elicits therapeutic responses; however, durable effects are often limited by ERK1/2 pathway reactivation via poorly defined mechanisms. We generated mutant BRAF^V600E melanoma cells that exhibit resistance to PLX4720, the tool compound for vemurafenib, that co-expressed mutant (Q61K) NRAS. In these BRAF^V600E/NRAS^Q61K co-expressing cells, re-activation of the ERK1/2 pathway during PLX4720 treatment was dependent on NRAS. Expression of mutant NRAS in parental BRAF^V600 cells was sufficient to by-pass PLX4720 effects on ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in a manner dependent on the RAF binding site in NRAS. ERK1/2 activation in BRAF^V600E/NRAS^Q61K cells required CRAF only in the presence of PLX4720, indicating a switch in RAF isoform requirement. Both ERK1/2 activation and resistance to apoptosis of BRAF^V600E/NRAS^Q61K cells in the presence of PLX4720 was modulated by SHOC-2/Sur-8 expression, a RAS-RAF scaffold protein. These data show that NRAS mutations confer resistance to RAF inhibitors in mutant BRAF cells and alter RAF isoform and scaffold molecule requirements to re-activate the ERK1/2 pathway.     

Cotargeting histone deacetylases and oncogenic BRAF synergistically kills human melanoma cells by necrosis independently of RIPK1 and RIPK3

Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAF^V600E melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAF^V600E melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAF^V600E melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.     

Immunoregulatory protein B7‐H3 promotes growth and decreases sensitivity to therapy in metastatic melanoma cells

B7‐H3 (CD276) belongs to the B7 family of immunoregulatory proteins and has been implicated in cancer progression and metastasis. In this study, we found that metastatic melanoma cells with knockdown expression of B7‐H3 showed modest decrease in proliferation and glycolytic capacity and were more sensitive to dacarbazine (DTIC) chemotherapy and small‐molecule inhibitors targeting MAP kinase (MAPK) and AKT/mTOR pathways: vemurafenib (PLX4032; BRAF inhibitor), binimetinib (MEK‐162; MEK inhibitor), everolimus (RAD001; mTOR inhibitor), and triciribidine (API‐2; AKT inhibitor). Similar effects were observed in melanoma cells in the presence of an inhibitory B7‐H3 monoclonal antibody, while the opposite was seen in B7‐H3‐overexpressing cells. Further, combining B7‐H3 inhibition with small‐molecule inhibitors resulted in significantly increased antiproliferative effect in melanoma cells, as well as in BRAF^V600E mutated cell lines derived from patient biopsies. Our findings indicate that targeting B7‐H3 may be a novel alternative to improve current therapy of metastatic melanoma.     

The BRAF mutation confers sensitivity of thyroid cancer cells to the BRAF inhibitor PLX4032 (RG7204)

Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF^V600E, as a result of the BRAF^T1799A mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF^V600E-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF^T1799A mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF^T1799A mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC₅₀ values (0.115–1.156 μM) in BRAF^V600E mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC₅₀ values (56.674–1349.788 μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF^T1799A mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF^T1799A mutation-selective therapeutic agent for thyroid cancer.     

Activities of multiple cancer-related pathways are associated with BRAF mutation and predict the resistance to BRAF/MEK inhibitors in melanoma cells

Drug resistance is a major obstacle in the targeted therapy of melanoma using BRAF/MEK inhibitors. This study was to identify BRAF V600E-associated oncogenic pathways that predict resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors. We took in silico approaches to analyze the activities of 24 cancer-related pathways in melanoma cells and identify those whose activation was associated with BRAF V600E and used the support vector machine (SVM) algorithm to predict the resistance of BRAF-mutated melanoma cells to BRAF/MEK inhibitors. We then experimentally confirmed the in silico findings. In a microarray gene expression dataset of 63 melanoma cell lines, we found that activation of multiple oncogenic pathways preferentially occurred in BRAF-mutated melanoma cells. This finding was reproduced in 5 additional independent melanoma datasets. Further analysis of 46 melanoma cell lines that harbored BRAF mutation showed that 7 pathways, including TNFα, EGFR, IFNα, hypoxia, IFNγ, STAT3, and MYC, were significantly differently expressed in AZD6244-resistant compared with responsive melanoma cells. A SVM classifier built on this 7-pathway activation pattern correctly predicted the response of 10 BRAF-mutated melanoma cell lines to the MEK inhibitor AZD6244 in our experiments. We experimentally showed that TNFα, EGFR, IFNα, and IFNγ pathway activities were also upregulated in melanoma cell A375 compared with its sub-line DRO, while DRO was much more sensitive to AZD6244 than A375. In conclusion, we have identified specific oncogenic pathways preferentially activated in BRAF-mutated melanoma cells and a pathway pattern that predicts resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors, providing novel clinical implications for melanoma therapy.     

Detailed imaging and genetic analysis reveal a secondary BRAFL505H resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three‐dimensional imaging. In all patients, only a subset of lesions progressed. Next‐generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib‐resistant patient‐derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.     

Reversing melanoma cross-resistance to BRAF and MEK inhibitors by co-targeting the AKT/mTOR pathway

Background

The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF^V600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.

Methodology/Principal Findings

The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.

Conclusions/Significance

Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.     

Receptor tyrosine kinases in cancer escape from BRAF inhibitors

The BRAF inhibitors (BRAFi) induce anti-tumor responses in nearly 60% of patients with advanced ^V600BRAF-mutant melanomas but only 5% of patients with ^V600BRAF-mutant colorectal carcinomas. Earlier studies of how a subset of melanoma that initially responds to BRAFi but later acquires drug resistance pointed to the importance of receptor tyrosine kinases (RTKs) in drug escape. In a pair of recent reports, this RTK-mediated mechanism of acquired BRAFi resistance in melanoma is re-surfacing in the context of innate or primary BRAFi resistance in ^V600BRAF-mutant colorectal carcinomas, suggesting potential upfront therapeutic strategies to prevent BRAFi resistance.^V600BRAF mutations are found in >50% of melanomas, nearly 100% of hairy cell leukemias but smaller subsets of more common human malignancies (e.g., colorectal, thyroid)¹. The in-human “druggability” of mutant BRAF has been best demonstrated in metastatic BRAF mutant melanomas using the novel small-molecule BRAF inhibitor (BRAFi) PLX4032/vemurafenib, producing survival benefits². Early clinical results of BRAFi in colorectal carcinoma, however, were disappointing, with only 5% of patients (1 of 21 patients) experiencing a partial response and 19% of patients (4 of 21 patients) experiencing minor responses³. This difference in the clinical results (melanoma vs. colorectal carcinoma) may relate less to their ontological origins but more to alternative states of a dynamic and plastic survival signaling network.The majority of BRAF mutant melanomas responds to BRAFi rapidly but acquires drug resistance within a median time of 6-7 months. The specific mechanisms of acquired BRAFi resistance are variegated but fall under two core pathways: 1) reactivation of RAF-MEK-ERK MAPK signaling, and 2) activation of MAPK-redundant signaling via the receptor tyrosine kinase (RTK)-PI3K-AKT pathway, which is parallel but interconnected to the MAPK pathway. MAPK reactivation can occur via NRAS activating mutations⁴, COT overexpression⁵, ^V600BRAF alternative splicing⁶, ^V600BRAF amplification⁷, and MEK1 activating mutation^8,9. MAPK-redundant signaling via RTK overexpression has been shown to result in AKT activation and RAS-CRAF-MEK signaling, bypassing mutant BRAF^4,10,11. The repertoire of RTK overexpressed appears restricted but shares a common pattern of PDGFRβ and EGFR overexpression, at least in melanoma cell lines with acquired resistance to vemurafenib⁴. It is unclear at present how this overexpression of a select number of wild-type RTKs contributes to the molecular details of survival pathway redundancy and cooperativity. Nevertheless, understanding how melanomas acquire BRAFi resistance via core pathways may shed key insights into mechanisms of innate BRAFi resistance in multiple malignancies. Hence, it came as not a complete surprise that a pair of papers published recently implicated RTKs in innate BRAFi resistance in colorectal cancer cell lines^12,13. Both studies pointed to EGFR activation and downstream signaling as a key component to innate BRAFi resistance, at least in a majority of colorectal carcinoma (CRC) cell lines examined.Corcoran et al.¹² showed that BRAF mutant CRC cell lines, in contrast to BRAF mutant melanoma cell lines, displayed innate resistance to growth inhibition by vemurafenib. An important clue implicating RTK involvement in innate vemurafenib resistance of BRAF mutant CRC cell lines came from the observation that p-ERK recovery occurred soon (hours to days) after vemurafenib treatment, unlike the kinetics of p-ERK recovery in BRAF mutant melanoma cell lines. This relatively rapid recovery of p-ERK post vemurafenib treatment in CRC cell lines is akin to that in melanoma cell lines with acquired BRAFi resistance driven by RTK overexpresion¹⁰. Corcoran et al. then traced this propensity for early p-ERK recovery to vemurafenib treatment (24 h)-dependent enhancement of (activated) RAS-GTP levels and MEK activity, parallel to elevated RAS-GTP levels in melanoma cell lines with RTK-driven, acquired BRAFi resistance⁴. In phospho-RTK arrays, they determined that the p-EGFR level (among others such as p-c-MET and p-IGF1R levels) was elevated in CRC cell lines relative to those in melanoma cells. Vemurafenib treatment (24 h) did not significantly enhance the p-EGFR level (but did elevate the p-IGFR1 level). Elevated p-EGFR levels in BRAF mutant CRC cell lines were correlated with elevated total EGFR levels (i.e., overexpressed compared with BRAF mutant melanoma cell lines). Thus, several observations correlated with innate BRAFi resistance in CRC cell lines: RTK (mostly consistently EGFR) overexpression (at baseline); upregulation of activation-associated phosphorylation of RTKs (at baseline); and upregulation of RAS-GTP levels (in response to BRAFi treatment). Curiously, although EGFR is highly phosphorylated at baseline, the RAS-GTP levels only rose in response to vemurafenib treatment.Corcoran et al. further showed that small-molecule EGFR inhibitors (EGFRi) could downregulate, partially or completely, the RAS-GTP level induced by vemurafenib treatment. The combination of vemurafenib (BRAFi) and gefitnib (EGFRi) could synergistically reduce p-ERK levels and the net growth inhibition of most but not all CRC cell lines studied, suggesting that survival in some CRC cell lines may also depend on other RTKs and downstream signaling (e.g., AKT). Consistently, the combination of vemurafenib and erlotinib (EGFRi) stabilized the growth of, but did not cause significant regression of, CRC xenografts. Simultaneous inhibition or genetic knockdown of multiple RTKs was not explored, leaving unresolved the issue of how multiple RTKs may potentially play cooperative survival roles at baseline or in response to kinase inhibitor therapy.Prahallad et al.¹³ also compared CRC and melanoma cell lines and showed that EGFR expression is generally higher in CRC cell lines. Vemurafenib treatment (6 h) of the WiDr CRC cell line led to an induction in p-EGFR and p-AKT levels, concomitant with the expected suppression of p-MEK and p-ERK. MEK inhibition, by AZD6244 treatment, similarly led to the rebound phosphorylation of EGFR. Based on earlier literature showing that the ERK kinase phosphorylates Cdc25c, activating its phosphatase activity, and that Cdc25c can dephosphorylate EGFR, Prahallad et al. went on to show that Cdc25c knockdown mimicked vemurafenib treatment in inducing p-EGFR levels. As predicted, vemurafenib treatment of CRC cell line inhibited Cdc25c phosphorylation at a key threonine (Thr 48), which was previously demonstrated to be a key event for its phosphatase activity. Addition of an EGFRi (cetuximab or gefitnib) to the BRAFi vemurafenib treatment downregulated the baseline level of p-ERK and the BRAFi-induced p-AKT level (but not the baseline p-AKT level). Moreover, addition of an EGFRi sensitized CRC cell lines to growth inhibition by vemurafenib in vitro but did not induce tumor regression in vivo, again suggesting incomplete survival signaling blockade. Accordingly, it has been shown that the effect of vemurafenib in shrinking CRC tumor xenografts was enhanced by combining with an AKT inhibitor (MK-2206)¹⁴. Moreover, in this study, the addition of vemurafenib to erlotinib treatment also resulted in increased anti-tumor activity and improved survival in xenograft models. It should be pointed out that Prahallad et al. did not formally assess BRAFi and EGFRi synergy, nor did they examine the diversity of RTK overexpression/activity and its contribution to downstream survival signaling (e.g., AKT).These works, along with prior studies^4,10, highlight the importance of expression and activity level of RTKs as a key sensitivity determinant of BRAFi resistance in BRAF mutant cancer cell lines (Figure 1). An important question remains as to whether the diversity of RTK overexpression and/or upregulation participates in and contributes to the full BRAFi resistance phenotype. A recent study afforded us a systems-wide view of the RTKinome reprogramming in response to MEK inhibition in the so-called triple-negative breast cancer cell lines¹⁵. The balance of the MAPK vs. RTK network signaling may be dynamically influenced by kinase inhibitors targeting RAF or MEK. This daunting diversity of RTK expression/activity may corner us into abandoning a combination of RTK inhibitors (already approved for clinical usage) with a BRAF inhibitor. Instead, we might need to resort to downstream pathway inhibitors not yet approved for clinical usage (e.g., an inhibitor of MEK with an inhibitor of the PI3K-AKT-mTORC1/2 axis) before we have a chance to corner BRAF mutant cancers into death.Open in a separate windowFigure 1Upregulation of receptor tyrosine kinase(s) (RTKs) as a key sensitivity determinant of BRAFi resistance in BRAF mutant cancer cell lines. (A) In BRAF mutant melanoma cell lines, RTKs are generally expressed at very low levels and contribute minimally to survival signaling, resulting in a strong addiction to mutant BRAF signaling and sensitivity to BRAFi. When BRAF mutant melanoma cell lines acquire BRAFi resistance, they upregulate the expression and activity of PDGFRb and other RTKs, resulting in reactivation of MEK-ERK as well as MAPK-redundant PI3K-AKT survival signaling. (B) In BRAF mutant colorectal carcinoma (CRC) cell lines, EGFR and other RTKs are upregulated by overexpression and some level of activation, resulting in MAPK-redundant survival signaling and conferring innate or primary BRAFi resistance. Treatment of CRC cell lines wth a BRAF or a MEK inhibitor can further activate EGFR and potentially other RTKs and stimulate GTP-RAS levels, consolidating innate BRAFi resistance. Red denotes mutated protein (e.g., BRAF); gray symbols denote weak signaling or interactions; multiplicity of protein symbols denotes overexpression; P in blue denotes activation-associated phosphorylation.     

The tumor suppressor BAP1 cooperates with BRAFV600E to promote tumor formation in cutaneous melanoma

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAF^V600E) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1‐null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild‐type counterparts. Molecularly, Bap1‐null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1‐null tumors are completely responsive to BRAF‐ and MEK‐targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.     

Lkb1 Loss Promotes Tumor Progression of BRAFV600E-Induced Lung Adenomas

Aberrant activation of MAP kinase signaling pathway and loss of tumor suppressor LKB1 have been implicated in lung cancer development and progression. Although oncogenic KRAS mutations are frequent, BRAF mutations (BRAF^V600E) are found in 3% of human non-small cell lung cancers. Contrary to KRAS mutant tumors, BRAF^V600E-induced tumors are benign adenomas that fail to progess. Interestingly, loss of tumor supressor LKB1 coexists with KRAS oncogenic mutations and synergizes in tumor formation and progression, however, its cooperation with BRAF^V600E oncogene is unknown. Our results describe a lung cell population in neonates mice where expression of BRAF^V600E leads to lung adenoma development. Importantly, expression of BRAF^V600E concomitant with the loss of only a single-copy of Lkb1, overcomes senencence–like features of BRAF^V600E-mutant adenomas leading malignization to carcinomas. These results posit LKB1 haploinsufficiency as a risk factor for tumor progression of BRAF^V600E mutated lung adenomas in human cancer patients.