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Tumor infiltration by T cells is paramount for effective anti-cancer immune responses. We hypothesized that the T cell receptor (TCR) repertoire of tumor infiltrating T lymphocytes could therefore be indicative of the functional state of these cells and determine disease course at different stages in cancer progression. Here we show that the diversity of the TCR of tumor infiltrating T cell at baseline is prognostic in various cancers, whereas the TCR clonality of T cell infiltrating metastatic melanoma pre-treatment is predictive for activity and efficacy of PD1 blockade immunotherapy. Precision medicine needs prognostic markers to select the patients that will benefit more from targeted therapy. Authors show here that high level of baseline T cell receptor diversity is an indicator of favourable prognosis in multiple cancer types, and monoclonal expansion of T-cells correlates with good response to immune checkpoint blockade therapy in metastatic melanoma patients.

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. Lysyl oxidase is able to remodel the extracellular matrix and its expression correlates with poor prognosis. Here the authors show that this protein modulates trapping of the epidermal growth factor receptor at the cell surface, causing persistent signalling activation and tumour progression.

The Microphthalmia-associated transcription factor (MITF) is an important regulator of cell-type specific functions in melanocytic cells. MITF is essential for the survival of pigmented cells, but whereas high levels of MITF drive melanocyte differentiation, lower levels are required to permit proliferation and survival of melanoma cells. MITF is phosphorylated by ERK, and this stimulates its activation, but also targets it for degradation through the ubiquitin-proteosome pathway, coupling MITF degradation to its activation. We have previously shown that because ERK is hyper-activated in melanoma cells in which BRAF is mutated, the MITF protein is constitutively down-regulated. Here we describe another intriguing aspect of MITF regulation by oncogenic BRAF in melanoma cells. We show oncogenic BRAF up-regulates MITF transcription through ERK and the transcription factor BRN2 (N-Oct3). In contrast, we show that in melanocytes this pathway does not exist because BRN2 is not expressed, demonstrating that MITF regulation is a newly acquired function of oncogenic BRAF that is not performed by the wild-type protein. Critically, in melanoma cells MITF is required downstream of oncogenic BRAF because it regulates expression of key cell cycle regulatory proteins such as CDK2 and CDK4. Wild-type BRAF does not regulate this pathway in melanocytes. Thus, we show that oncogenic BRAF exerts exquisite control over MITF on two levels. It downregulates the protein by stimulating its degradation, but then counteracts this by increasing transcription through BRN2. Our data suggest that oncogenic BRAF plays a critical role in regulating MITF expression to ensure that its protein levels are compatible with proliferation and survival of melanoma cells. We propose that its ability to appropriate the regulation of this critical factor explains in part why BRAF is such a potent oncogene in melanoma.

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. •We examine the metabolic requirements in melanoma cell lines resistant to BRAF inhibitor PLX4720.•Cells that are resistant to BRAF inhibitors are more dependent on glutamine than glucose as their carbon source.•Combined glutaminolysis and BRAF inhibition was effective to promote a delay in the onset of resistance.

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 Kras G12D/+ Trp53 R172H/+ (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. Synopsis Lysyl oxidase (LOX) is identified as a therapeutic target in pancreatic ductal adenocarcinoma (PDAC). Inhibition of LOX resulted in increased drug efficacy and stromal changes and reduction in metastasis. A signature of hazardous and protective genes in PDAC was defined. High expression of hypoxia‐associated genes, including LOX, was associated with poor patient prognosis. Using transgenic mouse models of PDAC, LOX was found to be overexpressed in metastatic disease and its expression was required for PDAC cell invasion. Inhibition of LOX in transgenic mice inhibited metastasis, while combination therapy with LOX inhibition and gemcitabine induced stromal alterations, immune cell infiltration and tumor necrosis and improved survival. Graphical Abstract Lysyl oxidase (LOX) is identified as a therapeutic target in pancreatic ductal adenocarcinoma (PDAC). Inhibition of LOX resulted in increased drug efficacy and stromal changes and reduction in metastasis.

Although identified as the key environmental driver of common cutaneous melanoma, the role of ultraviolet radiation (UVR)-induced DNA damage in mucosal melanoma is poorly defined. We analyze 10 mucosal melanomas of conjunctival origin by whole genome sequencing and our data shows a predominance of UVR-associated single base substitution signature 7 (SBS7) in the majority of the samples. Our data shows mucosal melanomas with SBS7 dominance have similar genomic patterns to cutaneous melanomas and therefore this subset should not be excluded from treatments currently used for common cutaneous melanoma. Mucosal melanomas are not thought to be UV radiation (UVR)-driven, yet some present UVR-related mutations. Here the authors show that some mucosal melanomas, especially conjunctival, present mutations characteristic of UVR exposure, yet share structural variants typical of other mucosal melanomas.

The continuous renewal of the skin relies on stem and progenitor cells, yet their differential susceptibility to oncogenic mutations in cutaneous squamous cell carcinoma (cSCC) remains unclear. Rapid cSCC develops in melanoma patients on BRAF inhibitors due to paradoxical MAPK activation. To model this in mice, we use two complementary approaches: HRAS G12V with a BRAF inhibitor to mimic paradoxical MAPK activation, and BRAF V600E , which drives MAPK hyperactivation without further treatment. We target these mutations to the interfollicular stem and differentiation-committed progenitors of the basal epidermis. While stem cells rapidly form tumours, progenitors exhibit long-latency resistance despite retaining mutations and repopulating the basal layer. Ultimately, both populations produce similar tumours, showing a shared transformation process. However, SOX2 is uniquely upregulated in progenitor-derived tumours and is expressed in 20% of human cSCC, indicating it might mark tumours arising from committed progenitors. Here, we show that SOX2 overexpression, along with MAPK activation, in progenitors induces a stem-like state and renders this otherwise resistant population permissive to rapid transformation. The oncogenic potential of interfollicular stem and progenitor cells in cutaneous squamous cell carcinoma (cSCC) remains poorly understood. Here, the authors modelled rapidly growing cSCC driven by the hyperactivation of the MAPK signalling pathway in mice and showed that SOX2 overexpression renders progenitor cells prone to transformation.

Congenital disorders of glycosylation (CDG) are rare genetic disorders with a spectrum of clinical manifestations caused by abnormal N -glycosylation of secreted and cell surface proteins. Over 130 genes are implicated and next generation sequencing further identifies potential disease drivers in affected individuals. However, functional testing of these variants is challenging, making it difficult to distinguish pathogenic from non-pathogenic events. Using proximity labelling, we identified OST48 as a protein that transiently interacts with lysyl oxidase (LOX), a secreted enzyme that cross-links the fibrous extracellular matrix. OST48 is a non-catalytic component of the oligosaccharyltransferase (OST) complex, which transfers glycans to substrate proteins. OST48 is encoded by DDOST , and 43 variants of DDOST are described in CDG patients, of which 34 are classified as variants of uncertain clinical significance (VUS). We developed an assay based on LOX N -glycosylation that confirmed two previously characterised DDOST variants as pathogenic. Notably, 39 of the 41 remaining variants did not have impaired activity, but we demonstrated that p.S243F and p.E286del were functionally impaired, consistent with a role in driving CDG in those patients. Thus, we describe a rapid assay for functional testing of clinically relevant CDG variants to complement genome sequencing and support clinical diagnosis of affected individuals.

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through Braf V600E or Kras G12D knockin) and TGFβ signalling ablation (through Tgfbr1 deletion) in LGR5 +ve stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5 +ve cells also results in cSCC development. These findings indicate that LGR5 +ve stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis. Cutaneous squamous cell carcinomas is a growing problem but the driver genes causing this remain poorly defined. Here, the authors demonstrate that inactivating driver mutations in TGFBR1 and TGFBR2 occur in vemurafenib-induced and sporadic cutaneous squamous cell carcinomas.

Although immune checkpoint inhibitors (ICIs) have achieved unprecedented results in melanoma, the biological features of the durable responses initiated by these drugs remain unknown. Here we show the genetic and phenotypic changes induced by treatment with programmed cell death-1 (PD-1) blockade in a genetically engineered mouse model of melanoma driven by oncogenic BRAF. In this controlled system anti-PD-1 treatment yields responses in ~35% of the tumors, and prolongs survival in ~27% of the animals. We identify increased stroma remodeling and reduced expression of proliferation markers as features associated with prolonged response. These traits are corroborated in two independent early on-treatment anti-PD-1 melanoma patient cohorts. These insights into the biological responses of tumors to ICI provide a strategy for identification of durable response early during the course of treatment and could improve patient stratification for checkpoint inhibitory drugs. Identification of clinical relevant biomarkers to predict response to immune checkpoint blockade (ICB) in melanoma remains challenging. Here, the authors show that stroma remodelling and reduced cell division are associated with durable response to anti-PD1 in a mouse model of melanoma and in ICB-treated patients.