Resistance to BRAF inhibitors induces glutamine dependency in melanoma cells

BRAF inhibitors can extend progression-free and overall survival in melanoma patients whose tumors harbor mutations in BRAF. However, the majority of patients eventually develop resistance to these drugs. Here we show that BRAF mutant melanoma cells that have developed acquired resistance to BRAF inhibitors display increased oxidative metabolism and increased dependency on mitochondria for survival. Intriguingly, the increased oxidative metabolism is associated with a switch from glucose to glutamine metabolism and an increased dependence on glutamine over glucose for proliferation. We show that the resistant cells are more sensitive to mitochondrial poisons and to inhibitors of glutaminolysis, suggesting that targeting specific metabolic pathways may offer exciting therapeutic opportunities to treat resistant tumors, or to delay emergence of resistance in the first-line setting

Plasma total cell-free DNA (cfDNA) is a surrogate biomarker for tumour burden and a prognostic biomarker for survival in metastatic melanoma patients

Introduction Tumour burden is a prognostic biomarker in metastatic melanoma. However, tumour burden is difficult to measure and there are currently no reliable surrogate biomarkers to easily and reliably determine it. The aim of this study was to assess the potential of plasma total cell free DNA as biomarker of tumour burden and prognosis in metastatic melanoma patients. Materials and methods A prospective biomarker cohort study for total plasma circulating cell-free DNA (cfDNA) concentration was performed in 43 metastatic melanoma patients. For 38 patients, paired blood collections and scan assessments were available before treatment and at first response evaluation. Tumour burden was calculated as the sum of volumes from three-dimensional radiological measurements of all metastatic lesions in individual patients. Results Baseline cfDNA concentration correlated with pre-treatment tumour burden (ρ = 0.52, P < 0.001). Baseline cfDNA levels correlated significantly with hazard of death and overall survival, and a cut off value of 89 pg/μl identified two distinct prognostic groups (HR = 2.22 for high cfDNA, P = 0.004). Patients with cfDNA ≥89 pg/μl had shorter OS (10.0 versus 22.7 months, P = 0.009; HR = 2.22 for high cfDNA, P = 0.004) and the significance was maintained when compared with lactic dehydrogenase (LDH) in a multivariate analysis. We also found a correlation between the changes of cfDNA and treatment-related changes in tumour burden (ρ = 0.49, P = 0.002). In addition, the ratio between baseline cfDNA and tumour burden was prognostic (HR = 2.7 for cfDNA/tumour volume ≥8 pg/(μl*cm3), P = 0.024). Conclusions We have demonstrated that cfDNA is a surrogate marker of tumour burden in metastatic melanoma patients, and that it is prognostic for overall survival.Fil: Valpione, S.. University of Manchester; Reino Unido. Christie NHS Foundation Trust; Reino UnidoFil: Gremel, G.. University of Manchester; Reino UnidoFil: Mundra, P.. University of Manchester; Reino UnidoFil: Middlehurst, P.. University of Manchester; Reino UnidoFil: Galvani, E.. Christie NHS Foundation Trust; Reino Unido. University of Manchester; Reino UnidoFil: Girotti, Maria Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. University of Manchester; Reino UnidoFil: Lee, R.J.. University of Manchester; Reino UnidoFil: Garner, G.. University of Manchester; Reino UnidoFil: Dhomen, N.. University of Manchester; Reino UnidoFil: Lorigan, P.C.. Christie NHS Foundation Trust; Reino UnidoFil: Marais, R.. University of Manchester; Reino Unid

Targeting TMEM176B Enhances Antitumor Immunity and Augments the Efficacy of Immune Checkpoint Blockers by Unleashing Inflammasome Activation.

Although immune checkpoint blockers have yielded significant clinical benefits in patients with different malignancies, the efficacy of these therapies is still limited. Here, we show that disruption of transmembrane protein 176B (TMEM176B) contributes to CD8+ T cell-mediated tumor growth inhibition by unleashing inflammasome activation. Lack of Tmem176b enhances the antitumor activity of anti-CTLA-4 antibodies through mechanisms involving caspase-1/IL-1β activation. Accordingly, patients responding to checkpoint blockade therapies display an activated inflammasome signature. Finally, we identify BayK8644 as a potent TMEM176B inhibitor that promotes CD8+ T cell-mediated tumor control and reinforces the antitumor activity of both anti-CTLA-4 and anti-PD-1 antibodies. Thus, pharmacologic de-repression of the inflammasome by targeting TMEM176B may enhance the therapeutic efficacy of immune checkpoint blockers.Uruguay INNOVA 2, Fondo Maria Viñas and Clemente Estable from ANII, as well as grants from CABBIO, PEDECIBA, ECOS-SUD and FOCEM (MERCOSUR Structural Convergence Fund), COF 03/11 to MH, The Harry J Lloyd Foundation to MRG and the Instituto Nacional del Cancer to YDM, Agencia de Promoción Científica y Tecnológica to GAR and MRG, Fundación Bunge & Born and Fundación Sales to GA

Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naive cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGF beta/SMAD (transforming growth factor-beta/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naive cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.Fapesp-grant number 2012/04194-1, 2013/05172-4, 2014/24400-0 and 2015/10821-7, CNPq-grant number 150447/2013-2 and 471512/2013-3 and PRODOC-grant no 3193-32/2010. Work in the lab of KS Smalley was supported by the National Institutes of Health grants R01 CA161107, R21 CA198550, and Skin SPORE grant P50 CA168536info:eu-repo/semantics/publishedVersio

Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma

We generated cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)-SRC family kinase (SFK)-STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo, and monotherapy with the broad specifi city tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo. We analyzed tumors from patients with intrinsic or acquired resistance to vemurafenib and observed increased EGFR and SFK activity. Furthermore, dasatinib blocked the growth and metastasis of one of the resistant tumors in immunocompromised mice. Our data show that BRAF inhibitor-mediated activation of EGFR-SFK-STAT3 signaling can mediate resistance in patients with BRAF-mutant melanoma. We describe 2 treatments that seem to overcome this resistance and could deliver therapeutic effi cacy in patients with drug-resistant BRAF-mutant melanoma. SIGNIFICANCE: Therapies that target the driver oncogenes in cancer can achieve remarkable responses if patients are stratifi ed for treatment. However, as with conventional therapies, patients often develop acquired resistance to targeted therapies, and a proportion of patients are intrinsically resistant and fail to respond despite the presence of an appropriate driver oncogene mutation. We found that the EGFR/SFK pathway mediated resistance to vemurafenib in BRAF -mutant melanoma and that BRAF and EGFR or SFK inhibition blocked proliferation and invasion of these resistant tumors, providing potentially effective therapeutic options for these patients. \ua9 2012 American Association for Cancer Research

Photo-Oxidation of Unilamellar Vesicles by a Lipophilic Pterin: Deciphering Biomembrane Photodamage

Pterins are natural products that can photosensitize the oxidation of DNA, proteins, and phospholipids. Recently, a new series of decyl-chain (i.e., lipophilic) pterins were synthesized and their photophysical properties were investigated. These decyl-pterins led to efficient intercalation in large unilamellar vesicles and produced, under UVA irradiation, singlet molecular oxygen, a highly oxidative species that react with polyunsaturated fatty acids (PUFAs) to form hydroperoxides. Here, we demonstrate that the association of 4-(decyloxy)pteridin-2-amine (O-decyl-Ptr) to lipid membranes is key to its ability to trigger phospholipid oxidation in unilamellar vesicles of phosphatidylcholine rich in PUFAs used as model biomembranes. Our results show that O-decyl-Ptr is at least 1 order of magnitude more efficient photosensitizer of lipids than pterin (Ptr), the unsubstituted derivative of the pterin family, which is more hydrophilic and freely passes across lipid membranes. Lipid peroxidation photosensitized by O-decyl-Ptr was detected by the formation of conjugated dienes and oxidized lipids, such as hydroxy and hydroperoxide derivatives. These primary products undergo a rapid conversion into short-chain secondary products by cleavage of the fatty-acid chains, some of which are due to subsequent photosensitized reactions. As a consequence, a fast increase in membrane permeability is observed. Therefore, lipid oxidation induced by O-decyl-Ptr could promote cell photodamage due to the biomembrane integrity loss, which in turn may trigger cell death.Fil: Vignoni, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Urrutia, María Noel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Junqueira, Helena C.. Universidade de Sao Paulo; BrasilFil: Greer, Alexander. City University of New York; Estados UnidosFil: Reis, Ana. University of Porto; PortugalFil: Baptista, Mauricio S.. Universidad de Porto; PortugalFil: Itri, Rosangela. Universidade de Sao Paulo; BrasilFil: Thomas, Andrés Héctor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentin

Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors

RAF inhibitor therapy yields significant reductions in tumour burden in the majority of V600E-positive melanoma patients; however, resistance occurs within 2-18 months. Here we demonstrate that the mixed lineage kinases (MLK1-4) are MEK kinases that reactivate the MEK/ERK pathway in the presence of RAF inhibitors. Expression of MLK1-4 mediates resistance to RAF inhibitors and promotes survival in V600E-positive melanoma cell lines. Furthermore, we observe upregulation of the MLKs in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In summary, our data demonstrate a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors