L. Boussemart

eIF4F is a nexus of resistance to anti-BRAF and anti-MEK cancer therapies

In BRAF(V600)-mutant tumours, most mechanisms of resistance to drugs that target the BRAF and/or MEK kinases rely on reactivation of the RAS–RAF–MEK–ERK mitogen-activated protein kinase (MAPK) signal transduction pathway, on activation of the alternative, PI(3)K–AKT–mTOR, pathway (which is ERK independent) or on modulation of the caspase-dependent apoptotic cascade. All three pathways converge to regulate the formation of the eIF4F eukaryotic translation initiation complex, which binds to the 7-methylguanylate cap (m7G) at the 5′ end of messenger RNA, thereby modulating the translation of specific mRNAs. Here we show that the persistent formation of the eIF4F complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, is associated with resistance to anti-BRAF, anti-MEK and anti-BRAF plus anti-MEK drug combinations in BRAF(V600)-mutant melanoma, colon and thyroid cancer cell lines. Resistance to treatment and maintenance of eIF4F complex formation is associated with one of three mechanisms: reactivation of MAPK signalling, persistent ERK-independent phosphorylation of the inhibitory eIF4E-binding protein 4EBP1 or increased pro-apoptotic BCL-2-modifying factor (BMF)-dependent degradation of eIF4G. The development of an in situ method to detect the eIF4E–eIF4G interactions shows that eIF4F complex formation is decreased in tumours that respond to anti-BRAF therapy and increased in resistant metastases compared to tumours before treatment. Strikingly, inhibiting the eIF4F complex, either by blocking the eIF4E–eIF4G interaction or by targeting eIF4A, synergizes with inhibiting BRAF(V600) to kill the cancer cells. eIF4F not only appears to be an indicator of both innate and acquired resistance but also is a promising therapeutic target. Combinations of drugs targeting BRAF (and/or MEK) and eIF4F may overcome most of the resistance mechanisms arising in BRAF(V600)-mutant cancers.

Skin Tumors Induced by Sorafenib; Paradoxic RAS–RAF Pathway Activation and Oncogenic Mutations of HRAS, TP53, and TGFBR1

Purpose: The emergence of skin tumors in patients treated with sorafenib or with more recent BRAF inhibitors is an intriguing and potentially serious event. We carried out a clinical, pathologic, and molecular study of skin lesions occurring in patients receiving sorafenib. Experimental Design: Thirty-one skin lesions from patients receiving sorafenib were characterized clinically and pathologically. DNA extracted from the lesions was screened for mutation hot spots of HRAS, NRAS, KiRAS, TP53, EGFR, BRAF, AKT1, PI3KCA, TGFBR1, and PTEN. Biological effect of sorafenib was studied in vivo in normal skin specimen and in vitro on cultured keratinocytes. Results: We observed a continuous spectrum of lesions: from benign to more inflammatory and proliferative lesions, all seemingly initiated in the hair follicles. Eight oncogenic HRAS, TGFBR1, and TP53 mutations were found in 2 benign lesions, 3 keratoacanthomas (KA) and 3 KA-like squamous cell carcinoma (SCC). Six of them correspond to the typical UV signature. Treatment with sorafenib led to an increased keratinocyte proliferation and a tendency toward increased mitogen-activated protein kinase (MAPK) pathway activation in normal skin. Sorafenib induced BRAF–CRAF dimerization in cultured keratinocytes and activated CRAF with a dose-dependent effect on MAP-kinase pathway activation and on keratinocyte proliferation. Conclusion: Sorafenib induces keratinocyte proliferation in vivo and a time- and dose-dependent activation of the MAP kinase pathway in vitro. It is associated with a spectrum of lesions ranging from benign follicular cystic lesions to KA-like SCC. Additional and potentially preexisting somatic genetic events, like UV-induced mutations, might influence the evolution of benign lesions to more proliferative and malignant tumors. Clin Cancer Res; 18(1); 263–72. ©2011 AACR.

Association of Vitiligo With Tumor Response in Patients With Metastatic Melanoma Treated With Pembrolizumab

IMPORTANCE Vitiligo is an autoimmune skin disorder that reacts against melanocytes. The association of vitiligo with tumor response in patients with melanoma who undergo immunotherapy has been reported but is still controversial. OBJECTIVE To prospectively evaluate the appearance of vitiligo in patients receiving pembrolizumab, a monoclonal antibody directed against the programmed death cell receptor. DESIGN, SETTING, AND PARTICIPANTS This prospective observational study was conducted from January 1, 2012, through September 24, 2013, in a single tertiary care hospital with a unit dedicated to patients with melanoma. Sixty-seven patients with metastatic melanoma who received pembrolizumab treatment in the context of a phase 1 study were included and screened for the emergence of vitiligo. Data were collected from January 1, 2012, to February 28, 2014, and analyzed from February through December 2014. MAIN OUTCOMES AND MEASURES Objective tumor response with regard to the occurrence of vitiligo in patients receiving pembrolizumab therapy. Correlation between vitiligo occurrence and overall survival was also estimated using the Kaplan-Meier product-limit method and compared with a log-rank test. To prevent guarantee- or lead-time bias, a landmark analysis approach after 12, 16, and 20 weeks of treatment was retained. RESULTS Of the 67 patients included in the study, 17 (25%) developed vitiligo during pembrolizumab treatment and 50 (75%) did not. An objective (complete or partial) response to treatment was associated with a higher occurrence of vitiligo (12 of 17 [71%] vs 14 of 50 [28%]; P = .002). The time to onset of vitiligo ranged from 52 to 453 (median, 126) days from the start of treatment. Of the 17 patients with vitiligo, 3 (18%) had a complete response, 9 (53%) had a partial response, 3 (18%) had stable disease, and 2 (12%) had progressive disease at the final follow-up. All the patients treated with pembrolizumab who developed vitiligo were alive at the time of analysis, with a median follow-up of 441 days. CONCLUSIONS AND RELEVANCE Vitiligo, a clinically visible immune-related adverse event could be associated with clinical benefit in the context of pembrolizumab treatment.

Vemurafenib and Radiosensitization

IMPORTANCE The BRAF inhibitor, vemurafenib, was recently approved for the treatment of patients with BRAFV600 metastatic melanoma. Wider use of this drug and longer follow-up periods of treatment are resulting in the emergence of a growing number of reports detailing new adverse effects. Cutaneous adverse effects are preeminent with UV-A-dependent phototoxicity, hyperkeratotic folliculitis, hand-foot skin reaction, hair changes, verrucous papillomas, keratoacanthomas, and squamous cell carcinomas. OBSERVATIONS We report 2 cases of dermatitis occurring on a previously irradiated skin area in patients treated with vemurafenib for a BRAFV600-mutated metastatic melanoma. The first case occurred 10 days after a low dose of radiation was delivered that usually does not induce any radiodermatitis, suggesting radiosensitization by vemurafenib. The second case occurred 30 days after radiotherapy and was diagnosed as radiation recall dermatitis. CONCLUSIONS AND RELEVANCE Vemurafenib should be considered a potential cutaneous radiosensitizer and an inducer of radiation recall dermatitis. However, these adverse effects are easily managed with topical corticosteroids. Dose reduction or interruption of vemurafenib is not required. Further studies and reports will enlighten us as to whether this pharmacodynamic interaction between x-rays and vemurafenib is also seen with other BRAF or MEK inhibitors on the same mitogen-activated protein kinase pathway currently under development.

Vemurafenib Cooperates with HPV to Promote Initiation of Cutaneous Tumors

Treatment with RAF inhibitors such as vemurafenib causes the development of cutaneous squamous cell carcinomas (cSCC) or keratoacanthomas as a side effect in 18% to 30% of patients. It is known that RAF inhibitors activate the mitogen-activated protein kinase (MAPK) pathway and stimulate growth of RAS-mutated cells, possibly accounting for up to 60% of cSCC or keratoacanthoma lesions with RAS mutations, but other contributing events are obscure. To identify such events, we evaluated tumors from patients treated with vemurafenib for the presence of human papilloma virus (HPV) DNA and identified 13% to be positive. Using a transgenic murine model of HPV-driven cSCC (K14-HPV16 mice), we conducted a functional test to determine whether administration of RAF inhibitors could promote cSCC in HPV-infected tissues. Vemurafenib treatment elevated MAPK markers and increased cSCC incidence from 22% to 70% in this model. Furthermore, 55% of the cSCCs arising in vemurafenib-treated mice exhibited a wild-type Ras genotype, consistent with the frequency observed in human patients. Our results argue that HPV cooperates with vemurafenib to promote tumorigenesis, in either the presence or absence of RAS mutations.

Dramatic response to radiotherapy combined with vemurafenib. Is vemurafenib a radiosensitizer

Activating BRAFV600E mutations occur in about 43% of primary melanomas [1]. An early clinical experience with a novel class I RAF-selective inhibitor, vemurafenib, demonstrated an unprecedented 48% antitumor response rate among patients with BRAFV600E-positive melanomas [2].Although vemurafenib sensitizes melanoma cells to irradiation in vitro [3], it has never been tested combined with radiotherapy in a clinical trial. We report dramatic responses to radiotherapy in two patients who exhibited a [...]

Abstract 934: BRAF inhibitors induce skin and extra-cutaneous tumors via paradoxical activation of the MAPK pathway: Molecular study of 66 tumors and visualization of BRAF/CRAF protein dimers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA INTRODUCTION : BRAF inhibitors (BRAFI) are affective anti-melanoma agents in V600BRAF mutant melanoma but have one major adverse event, the paradoxical activation of MAPK pathway in WT BRAF cells, which favors the emergence of benign, borderline and malignant skin cancers: verrucous papillomas, keratoacanthomas (KA), squamous cell carcinomas (SCC) and even new melanomas. MATERIALS AND METHODS : Between January 2010 and October 2013 we collected 63 skin tumors, 1 vulvar tumor, 1 gingival tumor, and 1 urothelial tumor in 50 patients treated with BRAFI vemurafenib or dabrafenib. Tumors were characterized histologically, p16 and P-ERK expression were studied by immunohistochemistry. Mutation hotspots for HRAS, KRAS and NRAS were investigated by Sanger analysis; human papilloma virus (HPV) DNA was also investigated and genotyped by PCR. RESULTS: The 63 skin tumors consisted of 35 verrucous papillomas, 9 KA, 13 SCC, and 6 melanomas. The other tumors were 1 vulvar and 1 gingival SCC, and 1 urothelial carcinoma. All tumors showed a strong nuclear pERK staining. Using Proximity Ligation Assay (PLA), we visualized, for the first time in human tumors in situ, the dimerization between BRAF and CRAF proteins in BRAFI-induced secondary tumors. BRAF-CRAF dimers were significantly less abundant in control normal skin samples and skin tumors that were not induced by BRAFI. P16 staining, often associated with HPV infection in cervical SCCs, was positive in 37% of papillomas, 78% of KA and 100% of SCC, respectively. However, the prevalence of HPV was not correlated with p16 staining: 9, 11 and 13 %, respectively (vs 25% of a series of 10 KA controls occurring in the absence of any BRAF inhibitor treatment ). HRAS mutation was found in 3% of papillomas and 56% of KA and a KRAS mutation was found in 17% of papillomas. We found NRAS mutations in the secondary melanomas, an HRAS mutation in the vulvar SCC, and a KRAS mutation in the urothelial carcinoma. DISCUSSION BRAFI can induce both cutaneous and extra-cutaneous secondary tumors that might have been underestimated in previous studies. Paradoxical activation of the MAPK pathway facilitated by a RAS mutation can be visualized using PLA illustrating in situ the dimerization between BRAF and CRAF. Although most keratinocytic tumors harbor clinical and histological characteristics of viral warts), despite the intensity of the P16 staining, we found a relatively low prevalence of HPV. CONCLUSION BRAFI are oncogenic agents that can induce both skin and extra-cutaneous cancers. Differential levels of RAF protein multimers in BRAFI-induced tumors and in control similar “spontaneous” tumors as well as the relatively low incidence of HPV despite indirect virus presence stigmata demonstrate that oncogenesis induced by BRAFI involves mechanisms distinct from previously known cancer initiation pathways. Citation Format: Lise Boussemart, Isabelle Girault, Christine Mateus, Marina Thomas, Emilie Routier, Hugo Cazenave, Gorana Tomasic, Janine Wechlser, Nyam Kamsu-Kom, Severine Roy, Michel Favre, Ludovic Lacroix, Alexander Eggermont, Stephan Vagner, Caroline Robert. BRAF inhibitors induce skin and extra-cutaneous tumors via paradoxical activation of the MAPK pathway: Molecular study of 66 tumors and visualization of BRAF/CRAF protein dimers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 934. doi:10.1158/1538-7445.AM2014-934

Abstract A23: eIF4F is a key and targetable convergence nexus of multiple mechanisms of resistance to anti-BRAF and anti-MEK cancer therapies

The V600E mutation in BRAF is the most frequent oncogenic mutation in human cancers, and can be targeted by specific anti-BRAF agents. However, several mechanisms of resistance have been identified, leading to reactivation of the initially blocked MAP-kinase pathway (known as ERK-dependent mechanisms) or reactivation of alternative ones, like the PI3K-AKT-mTOR pathway, or inhibition of the apoptotic cascade (ERK-independent). These pathways converge to regulate the formation of the eIF4F translation initiation complex that binds to the 7-methylguanosine cap at the 5′ end of mRNAs, thereby modulating mRNA translation of specific mRNAs. We addressed the potential role of the eIF4F eukaryotic translation initiation complex in resistance or sensitivity to anti-BRAF and anti-MEK agents in several BRAF mutant cells, xenografts and tumors. Our data demonstrate that the formation of the eIF4F complex is associated with most resistance mechanisms to these targeted therapies, independently of their capacity to reactivate or not the MAP-kinase pathway. Formation of this complex can be explored in tumor samples using a new in situ technology. Inhibitors of the eIF4F complex synergize with anti-BRAF agents and can thus reverse resistance. Citation Format: Lise Boussemart, Helene Malka-Mahieu, Isabelle Girault, Delphine Allard, Oskar Hemmingson, Nyam Kamsu-Kom, Sandrine Agoussi, Alexander Eggermont, Laurent Desaubry, Caroline Robert, Stephan Vagner. eIF4F is a key and targetable convergence nexus of multiple mechanisms of resistance to anti-BRAF and anti-MEK cancer therapies. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A23.