Richard Letrero

Acquired Resistance to BRAF Inhibitors Mediated by a RAF Kinase Switch in Melanoma Can Be Overcome by Cotargeting MEK and IGF-1R/PI3K

BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAF(V)⁶⁰⁰(E) melanoma cells with the BRAF inhibitor SB-590885; these cells are cross-resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors.

HIF-α Effects on c-Myc Distinguish Two Subtypes of Sporadic VHL-Deficient Clear Cell Renal Carcinoma

von Hippel-Lindau (VHL) tumor suppressor loss results in hypoxia-inducible factor alpha (HIF-alpha) stabilization and occurs in 70% of sporadic clear cell renal carcinomas (ccRCCs). To determine whether opposing influences of HIF-1alpha and HIF-2alpha on c-Myc activity regulate human ccRCC progression, we analyzed VHL genotype and HIF-alpha expression in 160 primary tumors, which segregated into three groups with distinct molecular characteristics. Interestingly, ccRCCs with intact VHL, as well as pVHL-deficient HIF-1alpha/HIF-2alpha-expressing ccRCCs, exhibited enhanced Akt/mTOR and ERK/MAPK signaling. In contrast, pVHL-deficient ccRCCs expressing only HIF-2alpha displayed elevated c-Myc activity, resulting in enhanced proliferation and resistance to replication stress. These reproducible distinctions in ccRCC behavior delineate HIF-alpha effects on c-Myc in vivo and suggest molecular criteria for selecting targeted therapies.

Common variation in KITLG and at 5q31.3 predisposes to testicular germ cell cancer

Testicular germ cell tumors (TGCT) have been expected to have a strong underlying genetic component. We conducted a genome-wide scan among 277 TGCT cases and 919 controls and found that seven markers at 12p22 within KITLG (c-KIT ligand) reached genome-wide significance (P < 5.0 × 10−8 in discovery). In independent replication, TGCT risk was increased threefold per copy of the major allele at rs3782179 and rs4474514 (OR = 3.08, 95% CI = 2.29–4.13; OR = 3.07, 95% CI = 2.29–4.13, respectively). We found associations with rs4324715 and rs6897876 at 5q31.3 near SPRY4 (sprouty 4; P < 5.0 × 10−6 in discovery). In independent replication, risk of TGCT was increased nearly 40% per copy of the major allele (OR = 1.37, 95% CI = 1.14–1.64; OR = 1.39, 95% CI = 1.16–1.66, respectively). All of the genotypes were associated with both seminoma and nonseminoma TGCT subtypes. These results demonstrate that common genetic variants affect TGCT risk and implicate KITLG and SPRY4 as genes involved in TGCT susceptibility.

CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations

Here, we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E- and D594G-mutated melanomas were found to exhibit constitutive levels of phospho-extracellular signal-regulated kinase (pERK) and low levels of phospho-mitogen-activated protein kinase/ERK kinase (pMEK) and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis and associated with mitochondrial depolarization and relocalization of apoptosis-inducing factor, whereas the BRAF-V600E-mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal through CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2 and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a time-dependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E-mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity.

A second independent locus within DMRT1 is associated with testicular germ cell tumor susceptibility

Susceptibility to testicular germ cell tumors (TGCT) has a significant heritable component, and genome-wide association studies (GWASs) have identified association with variants in several genes, including KITLG, SPRY4, BAK1, TERT, DMRT1 and ATF7IP. In our GWAS, we genotyped 349 TGCT cases and 919 controls and replicated top hits in an independent set of 439 cases and 960 controls in an attempt to find novel TGCT susceptibility loci. We identified a second marker (rs7040024) in the doublesex and mab-3-related transcription factor 1 (DMRT1) gene that is independent of the previously described risk allele (rs755383) at this locus. In combined analysis that mutually conditions on both DMRT1 single nucleotide polymorphism markers, TGCT cases had elevated odds of carriage of the rs7040024 major A allele [per-allele odds ratio (OR) = 1.48, 95% confidence interval (CI) 1.23, 1.78; P = 2.52 × 10(-5)] compared with controls, while the association with rs755383 persisted (per allele OR = 1.26, 95% CI 1.08, 1.47, P = 0.0036). In similar analyses, the association of rs7040024 among men with seminomatous tumors did not differ from that among men with non-seminomatous tumors. In combination with KITLG, the strongest TGCT susceptibility locus found to date, men with TGCT had greatly elevated odds (OR = 14.1, 95% CI 5.12, 38.6; P = 2.98 × 10(-7)) of being double homozygotes for the risk (major) alleles at DMRT (rs7040024) and KITLG (rs4474514) when compared with men without TGCT. Our findings continue to corroborate that genes influencing male germ cell development and differentiation have emerged as the major players in inherited TGCT susceptibility.

Phase II Trial of Temozolomide and Sorafenib in Advanced Melanoma Patients with or without Brain Metastases

Purpose: The combination of the oral alkylating agent temozolomide and the oral multikinase inhibitor sorafenib was evaluated in advanced melanoma patients. Experimental Design: Patients with metastatic melanoma (n = 167) were treated on four arms. All patients received sorafenib at 400 mg p.o. twice daily without interruption. Patients without brain metastases or prior temozolomide were randomized between arm A: extended dosing of temozolomide (75 mg/m2 temozolomide daily for 6 of every 8 weeks) and arm B: standard dosing (150 mg/m2 temozolomide daily for 5 of every 28 days). Patients previously treated with temozolomide were enrolled on arm C: extended dosing of temozolomide. Patients with brain metastases and no prior temozolomide were assigned to arm D: standard dosing. The primary end point was 6-month progression-free survival (PFS) rate. Secondary end points included response rate, toxicity rates, and the rates of BRAF or NRAS mutations. Results: The 6-month PFS rate for arms A, B, C, and D were 50%, 40%, 11%, and 23%. The median PFS for patients on arm A, B, C, and D was 5.9, 4.2, 2.2, and 3.5 months, respectively. No significant differences were observed between arms A and B in 6-month PFS rate, median PFS, or response rates. Treatment was well tolerated in all arms. No significant differences in toxicity were observed between arms A and B except for more grade 3 to 4 lymphopenia in arm A. Conclusion: Temozolomide plus sorafenib was well tolerated and showed activity in melanoma patients without prior history of temozolomide. The activity of this combination regimen warrants further investigation. (Clin Cancer Res 2009;15(24):7711–8)

Tumor genetic analyses of patients with metastatic melanoma treated with the BRAF inhibitor dabrafenib (GSK2118436)

Purpose: Dabrafenib is a selective inhibitor of V600-mutant BRAF kinase, which recently showed improved progression-free survival (PFS) as compared with dacarbazine, in metastatic melanoma patients. This study examined potential genetic markers associated with response and PFS in the phase I study of dabrafenib. Experimental Design: Baseline (pretreatment or archival) melanoma samples were evaluated in 41 patients using a custom genotyping melanoma-specific assay, sequencing of PTEN, and copy number analysis using multiplex ligation amplification and array-based comparative genomic hybridization. Nine patients had on-treatment and/or progression samples available. Results: All baseline patient samples had BRAFV600E/K confirmed. Baseline PTEN loss/mutation was not associated with best overall response to dabrafenib, but it showed a trend for shorter median PFS [18.3 (95% confidence interval, CI, 9.1–24.3) vs. 32.1 weeks (95% CI, 24.1–33), P = 0.059]. Higher copy number of CCND1 (P = 0.009) and lower copy number of CDKN2A (P = 0.012) at baseline were significantly associated with decreased PFS. Although no melanomas had high-level amplification of BRAF, the two patients with progressive disease as their best response had BRAF copy gain in their tumors. Conclusions: Copy number changes in CDKN2A, CCND1, and mutation/copy number changes in PTEN correlated with the duration of PFS in patients treated with dabrafenib. The results suggest that these markers should be considered in the design and interpretation of future trials with selective BRAF inhibitors in advanced melanoma patients. Clin Cancer Res; 19(17); 4868–78. ©2013 AACR.

Correlation of somatic mutations and clinical outcome in melanoma patients treated with carboplatin, paclitaxel, and sorafenib

Purpose: Sorafenib is an inhibitor of VEGF receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and RAF kinases, amongst others. We assessed the association of somatic mutations with clinicopathologic features and clinical outcomes in patients with metastatic melanoma treated on E2603, comparing treatment with carboplatin, paclitaxel ± sorafenib (CP vs. CPS). Experimental Design: Pretreatment tumor samples from 179 unique individuals enrolled on E2603 were analyzed. Genotyping was performed using a custom iPlex panel interrogating 74 mutations in 13 genes. Statistical analysis was performed using Fisher exact test, logistic regression, and Cox proportional hazards models. Progression-free survival (PFS) and overall survival were estimated using Kaplan–Meier methods. Results: BRAF and NRAS mutations were found at frequencies consistent with other metastatic melanoma cohorts. BRAF-mutant melanoma was associated with worse performance status, increased number of disease sites, and younger age at diagnosis. NRAS-mutant melanoma was associated with better performance status, fewer sites of disease, and female gender. BRAF and NRAS mutations were not significantly predictive of response or survival when treated with CPS versus CP. However, patients with NRAS-mutant melanoma trended toward a worse response and PFS on CP than those with BRAF-mutant or WT/WT melanoma, an association that was reversed for this group on the CPS arm. Conclusions: This study of somatic mutations in melanoma is the last prospectively collected phase III clinical trial population before the era of BRAF-targeted therapy. A trend toward improved clinical response in patients with NRAS-mutant melanoma treated with CPS was observed, possibly due to the effect of sorafenib on CRAF. Clin Cancer Res; 20(12); 3328–37. ©2014 AACR.

The FBXO4 tumor suppressor functions as a barrier to BRAFV600E-dependent metastatic melanoma.

ABSTRACT Cyclin D1–cyclin-dependent kinase 4/6 (CDK4/6) dysregulation is a major contributor to melanomagenesis. Clinical evidence has revealed that p16INK4A, an allosteric inhibitor of CDK4/6, is inactivated in over half of human melanomas, and numerous animal models have demonstrated that p16INK4A deletion promotes melanoma. FBXO4, a specificity factor for the E3 ligase that directs timely cyclin D1 proteolysis, has not been studied in melanoma. We demonstrate that Fbxo4 deficiency induces Braf-driven melanoma and that this phenotype depends on cyclin D1 accumulation in mice, underscoring the importance of this ubiquitin ligase in tumor suppression. Furthermore, we have identified a substrate-binding mutation, FBXO4 I377M, that selectively disrupts cyclin D1 degradation while preserving proteolysis of the other known FBXO4 substrate, TRF1. The I377M mutation and Fbxo4 deficiency result in nuclear accumulation of cyclin D1, a key transforming neoplastic event. Collectively, these data provide evidence that FBXO4 dysfunction, as a mechanism for cyclin D1 overexpression, is a contributor to human malignancy.

Abstract 5557: Mutation analysis of melanoma tumor samples from ECOG 2603 clinical trial

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The BRAF kinase, a member of the MAP kinase pathway, has emerged as an attractive molecule to target in melanoma. In an initial attempt to target BRAF in melanoma, a randomized, phase III trial (ECOG 2603) of carboplatin, paclitaxel, and sorafenib versus carboplatin, paclitaxel, and placebo was performed. Patients enrolled on the trial had either unresectable, locally advanced Stage III or Stage IV melanoma. We analyzed patients’ tumor samples for 74 mutations in 13 genes using a custom iPlex (Sequenom). We have preliminarily analyzed 157 tumor samples from patients enrolled in this clinical trial. Analysis of the initial data set demonstrates that 62 samples (42%, 62/148, 95% CI 34%,50%) carried BRAF mutations - 60 samples with V600 and two samples having a BRAF V601 mutation. Thirty eight samples (26%, 38/148, 95% CI 19%,33%) were positive for NRAS mutations, with 33 with Q61, two G12 and three G13 mutations. With the exception of one tumor sample, the BRAF and NRAS mutations were negatively correlated with each other, consistent with prior observations. We also observed a number of rare mutations in our samples including one in AKT1, one in AKT3, four in CDK4, five in beta-catenin, two in GNAQ and three in KIT. The results of this study demonstrated no difference between the two treatment arms in OS and PFS and no association with treatment outcome and BRAF and NRAS mutations. Consequently, the treatment arms were collapsed, and we examined the relationship of BRAF and NRAS mutations with OS and PFS. Interestingly, our data demonstrated that the BRAF and NRAS mutations did not correlate with OS or PFS. This sample population is unique in that it provides a large data set of melanoma tumor samples, all of whom were entered on a clinical trial, unselected for pre-existing mutations. Using the Sequenom assay platform, we have identified a number of mutations in a subset of genes known to be involved in melanoma in patient tumor samples. In the future, these tumor samples will be informative as we continue to examine and identify additional genetic alterations in melanoma, providing information regarding the natural history and distribution of certain mutations. Future studies will use array comparative genomic hybridization to further investigate additional somatic mutations occurring in melanoma tumors and correlate these with clinical outcome, as well as provide insight into the pathogenesis of melanoma. These results may lead to further opportunities for clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5557. doi:1538-7445.AM2012-5557