Grazia Saturno

Paradox-Breaking RAF Inhibitors that Also Target SRC Are Effective in Drug-Resistant BRAF Mutant Melanoma

Summary BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.

Application of Sequencing, Liquid Biopsies, and Patient-Derived Xenografts for Personalized Medicine in Melanoma

UNLABELLED Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell-derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma. SIGNIFICANCE Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments.

BRAF Inhibitors Induce Metastasis in RAS Mutant or Inhibitor-Resistant Melanoma Cells By Reactivating MEK and ERK Signaling

When therapy leads to cancer metastasis, knowing where else to target in the pathway may be the key to successful treatment. Blocking Melanoma Metastasis Although inhibitors of the mutant BRAF kinase are effective in some melanoma patients, intrinsic or acquired resistance to the drug is common. Furthermore, the growth of melanoma tumors with concomitant mutations in guanosine triphosphatase RAS, which activated kinases in the RAF family, is paradoxically accelerated by BRAF inhibition. RAF is the first kinase in a three-kinase cascade [the RAF–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal–regulated kinase) pathway] that is involved in cell proliferation. Using proteomics, patient material, and mouse models, Sanchez-Laorden et al. found that BRAF inhibition paradoxically stimulated MEK and ERK signaling to induce metastasis of melanoma cells with mutant BRAF, resistance to a BRAF inhibitor, or mutant RAS. Combined treatment with a MEK inhibitor prevented BRAF inhibitor–induced metastasis in mice. Thus, combination therapies may be best to prevent both primary tumor growth and drug-induced metastasis. Melanoma is a highly metastatic and lethal form of skin cancer. The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF–ERK (extracellular signal–regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)–ERK pathway, increased expression and secretion of interleukin 8, and induction of protease-dependent invasion. These events were accompanied by a cell morphology switch from predominantly rounded to predominantly elongated cells. We also observed similar responses in BRAF inhibitor–resistant melanoma cells. These data show that BRAF inhibitors can induce melanoma cell invasion and metastasis in tumors that develop resistance to these drugs.

Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer‐related mortality. Despite significant advances made in the treatment of other cancers, current chemotherapies offer little survival benefit in this disease. Pancreaticoduodenectomy offers patients the possibility of a cure, but most will die of recurrent or metastatic disease. Hence, preventing metastatic disease in these patients would be of significant benefit. Using principal component analysis (PCA), we identified a LOX/hypoxia signature associated with poor patient survival in resectable patients. We found that LOX expression is upregulated in metastatic tumors from Pdx1‐Cre KrasG12D/+ Trp53R172H/+ (KPC) mice and that inhibition of LOX in these mice suppressed metastasis. Mechanistically, LOX inhibition suppressed both migration and invasion of KPC cells. LOX inhibition also synergized with gemcitabine to kill tumors and significantly prolonged tumor‐free survival in KPC mice with early‐stage tumors. This was associated with stromal alterations, including increased vasculature and decreased fibrillar collagen, and increased infiltration of macrophages and neutrophils into tumors. Therefore, LOX inhibition is able to reverse many of the features that make PDAC inherently refractory to conventional therapies and targeting LOX could improve outcome in surgically resectable disease.

No longer an untreatable disease: How targeted and immunotherapies have changed the management of melanoma patients

The discovery that BRAF is a driver oncogene in cancer, and complementary improvements in our understanding of the immune system have resulted in new targeted and immune‐therapies for metastatic melanoma. Targeted therapies achieve impressive clinical results in carefully selected patients but the development of resistance seems inevitable in most cases. Conversely, immune‐checkpoints inhibitors can achieve long‐term remission and cures, but in a smaller proportion of patients, and biomarkers to predict which patients will respond are not available. Nevertheless, melanoma has led the evolution of cancer treatment from relatively nonspecific cytotoxic agents to highly selective therapies and here we review the lessons from this paradigm shift in treatment and the opportunities for further improvements in outcomes for melanoma patients.

Whole genome sequencing reveals complex mechanisms of intrinsic resistance to BRAF inhibition

We used a combination of whole-genome sequencing and in vitro validation to show that mutations that activated at least two pro-growth/survival pathways mediated intrinsic resistance to BRAF inhibition in a melanoma patient. These data demonstrate how in-depth analysis can reveal intrinsic resistance to standard of care, providing an opportunity for alternative therapeutic strategies for patients who are likely to fail first-line treat-575 ment.

Lysyl oxidase drives tumour progression by trapping EGF receptors at the cell surface.

Lysyl oxidase (LOX) remodels the tumour microenvironment by cross-linking the extracellular matrix. LOX overexpression is associated with poor cancer outcomes. Here, we find that LOX regulates the epidermal growth factor receptor (EGFR) to drive tumour progression. We show that LOX regulates EGFR by suppressing TGFβ1 signalling through the secreted protease HTRA1. This increases the expression of Matrilin2 (MATN2), an EGF-like domain-containing protein that traps EGFR at the cell surface to facilitate its activation by EGF. We describe a pharmacological inhibitor of LOX, CCT365623, which disrupts EGFR cell surface retention and delays the growth of primary and metastatic tumour cells in vivo. Thus, we show that LOX regulates EGFR cell surface retention to drive tumour progression, and we validate the therapeutic potential of inhibiting this pathway with the small molecule inhibitor CCT365623.

Abstract 4167: Ultraviolet radiation cooperates with individual oncogenes to drive melanomagenesis through distinct molecular mechanisms

The contribution ultraviolet radiation (UVR) makes to genetically distinct melanoma subtypes is unclear. Melanoma driven by oncogenic BRAF or oncogenic NRAS differ in their epidemiological, clinicopathological and genetic features. NRAS melanoma in humans occurs in older individuals and presents at sites most exposed to UVR damage such as the head and neck. In this study we show excessive sun exposure and NRAS-driven melanoma can co-occur by juxtaposing the epidemiological, clinical and genetic characteristics of a G12DNRAS-driven human melanoma case subject to extreme UVR exposure. To model the interaction of UVR with specific oncogenes and mimic human disease, we used mouse melanoma models driven by V600EBRAF or G12DNRAS. We previously reported that UVR cooperates with V600EBRAF by targeting the tumour suppressor TP53. In this study we confirm that UVR cooperates with G12DNRAS to drive melanoma and, as observed in humans, murine UVR-G12DNRAS melanoma occurs in older animals that accumulate higher lifetime exposure to UVR. Furthermore, we find murine UVR-G12DNRAS melanomas are histologically and genetically heterogeneous, and distinct to UVR-V600EBRAF murine and human melanomas. UVR-G12DNRAS melanomas present more UVR-induced mutations, a longer latency to tumour development and secondary driver mutations distinct from UVR-V600EBRAF melanoma. We investigated whether UVR cooperates equally with other RAS isoforms, and exposed G12DKRAS animals to UVR. These animals presented a different latency to melanoma development and specific cooperating secondary targets distinct from UVR-V600EBRAF and UVR- G12DNRAS. This implies that different RAS isoforms activate oncogenic pathways differently. Critically, UVR-V600EBRAF, UVR-G12DNRAS, UVR-G12DKRAS tumors present similar mutation rates when UVR exposure is held constant, which suggests that the differences in tumour latency might be explained if melanomas driven by different oncogenes require varying numbers of subsequent co-operators. Finally, we present preclinical evidence showing how targeting the UVR-induced mutations found in the “long tail” of UVR-related mutations in NRAS melanoma can provide therapeutic options for NRAS mutant melanoma patients. The insight gained begins to provide a molecular explanation for distinct associations between UVR and individual oncogenes in melanomagenesis, and we show how UVR-induced damage can be exploited to stratify patients for personalised therapy approaches. Citation Format: Amaya Viros, Malin Pedersen, Simon J. Furney, Maria Romina Girotti, Grazia Saturno, Elena Galvani, Berta Sanchez-Laorden, Kate Hogan, Charlotte Ng, Jorge S. Reis-Filho, Paul Lorigan, Martin Cook, Richard Marais. Ultraviolet radiation cooperates with individual oncogenes to drive melanomagenesis through distinct molecular mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4167.

Abstract LB-212: Therapeutic efficacy of the paradox-breaking panRAF and SRC drug CCT3833/BAL3833 in KRAS-driven cancer models

KRAS is mutated in ∼80% of pancreatic ductal adenocarcinoma (PDAC), ∼35% of colorectal cancer (CRC) and ∼20% of non-small-cell lung cancer (NSCLC). KRAS remains an intractable drug target and targeting the downstream pathway component is ineffective because feedback mechanisms or parallel pathways provide alternative routes to cell proliferation and/or survival. Here we show that a new panRAF/SRC inhibitor, CCT3833, is active in KRAS-mutant PDAC, CRC and NSCLC. We demonstrate that CCT3833 inhibits tumor growth in several KRAS-driven cancers via inhibition of RAF and SRC, eliciting therapeutic efficacy at well-tolerated doses in mouse models of human cancer. CCT3833 has already entered clinical trials (NCT02437227) for BRAF mutant and BRAF inhibitor-resistant melanomas and these new data show that it is also effective in KRAS mutant cancers, potentially providing a new therapeutic option for these patients. Citation Format: Grazia Saturno, Filipa Lopes, Maria Romina Girotti, Ion Niculescu-Duvaz, Dan Niculescu-Duvaz, Alfonso Zambon, Lawrence Davies, Louise Johnson, Natasha Preece, Amaya Viros, Malin Pedersen, Robert McLeary, Ruth Knight, Rebecca Lee, Denys Holovanchuk, Paul Lorigan, Nathalie Dhomen, Richard Marais, Caroline Springer. Therapeutic efficacy of the paradox-breaking panRAF and SRC drug CCT3833/BAL3833 in KRAS-driven cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-212.

Abstract 470: Application of sequencing, liquid biopsies and patient derived xenografts for personalized medicine in melanoma

BRAF/MEK inhibitors and immunotherapies have revolutionized care for patients with advanced melanoma, improving expected median survival from 9 months to 25-30 months, but the majority of patients still die of their disease. Personalized medicine strives to individualize and improve patient care. To individualize treatment decisions in advanced melanoma we analyzed 364 samples from 214 patients. We performed whole exome sequencing (WES) and circulating tumor DNA (ctDNA) analysis, and we developed patient derived xenografts (PDX). WES and targeted sequencing of ctDNA allowed us to predict responses to therapy and to identify and monitor mechanisms of resistance. WES of tumors revealed new therapeutic strategies in BRAF V600 wild-type and BRAF inhibitor-resistant melanoma and we validated these in patient derived xenografts (PDX). Thus, we describe a powerful combination of techniques for personalized medicine to improve the management of melanoma patients. Citation Format: Maria R. Girotti, Gabriela Gremel, Rebecca Lee, Elena Galvani, Dominic Rothwell, Amaya Viros, Amit Kumar Mandal, Kok Haw Jonathan Lim, Grazia Saturno, Simon J Furney, Franziska Baenke, Malin Pedersen, Jane Rogan, Jacqueline Swan, Matthew Smith, Alberto Fusi, Deemesh Oudit, Nathalie Dhomen, Ged Brady, Caroline Dive, Richard Marais. Application of sequencing, liquid biopsies and patient derived xenografts for personalized medicine in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 470.