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Nicholas Hayward and colleagues sequenced eight metastatic melanoma exomes and identified frequent somatic mutations in two MAP kinase family genes, MAP3K5 and MAP3K9 . Mutation in MAP3K9 may confer resistance to temozolomide, a common chemotherapeutic drug. We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9 . Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

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. B-Raf is mutated in many melanomas but treatment of the disease with small molecules targeting the mutant protein often results in tumour resistance. Here, the authors show that mixed lineage kinases (MLK1-4) can reactivate the B-Raf signalling pathway in the presence of inhibitors, resulting in drug resistance.

Endometrial cancer is the most commonly diagnosed gynaecological malignancy. Unfortunately, 15–20% of women demonstrate persistent or recurrent tumours that are refractory to current chemotherapies. We previously identified activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid ECs and found that these mutations are associated with shorter progression‐free and cancer‐specific survival. Although FGFR inhibitors are undergoing clinical trials for treatment of several cancer types, little is known about the mechanism by which they induce cell death. We show that treatment with BGJ398, AZD4547 and PD173074 causes mitochondrial depolarization, cytochrome c release and impaired mitochondrial respiration in two FGFR2‐mutant EC cell lines (AN3CA and JHUEM2). Despite this mitochondrial dysfunction, we were unable to detect caspase activation following FGFR inhibition; in addition, the pan‐caspase inhibitor Z‐VAD‐FMK was unable to prevent cell death, suggesting that the cell death is caspase‐independent. Furthermore, while FGFR inhibition led to an increase in LC3 puncta, treatment with bafilomycin did not further increase lipidated LC3, suggesting that FGFR inhibition led to a block in autophagosome degradation. We confirmed that cell death is mitochondrial‐dependent as it can be blocked by overexpression of Bcl‐2 and/or Bcl‐XL. Importantly, we show that combining FGFR inhibitors with the BH3 mimetics ABT737/ABT263 markedly increased cell death in vitro and is more effective than BGJ398 alone in vivo, where it leads to marked tumour regression. This work may have implications for the design of clinical trials to treat a wide range of patients with FGFR‐dependent malignancies. Bcl‐2 family members bound to Bax and Bak in FGFR‐mutant cancer cells maintain mitochondrial function and promote survival. FGFR inhibitor BGJ398 induces Bim expression which sequesters Bcl‐2 family members from Bax and Bak, leading to MOMP and cytochrome c release. Combining BGJ398 with the Bcl‐2/XL inhibitor ABT263 lowers the apoptotic threshold and enhances mitochondrial‐dependent cell death.

David Adams, Julia Newton-Bishop, Timothy Bishop, Nicholas Hayward and colleagues identify loss-of-function variants in POT1 in several families with early onset multiple primary melanoma. They further show that these variants disrupt telomere binding by POT1 and are associated with increased telomere length. Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases 1 , and rare variants in CDK4 , BRCA2 , BAP1 and the promoter of TERT have also been linked to the disease 2 , 3 , 4 , 5 . Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene ( POT1 ), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.

We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma. Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases. The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating. In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants. Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

Mutations in multiple oncogenes including KRAS, CTNNB1, PIK3CA and FGFR2 have been identified in endometrial cancer. The aim of this study was to provide insight into the clinicopathological features associated with patterns of mutation in these genes, a necessary step in planning targeted therapies for endometrial cancer. 466 endometrioid endometrial tumors were tested for mutations in FGFR2, KRAS, CTNNB1, and PIK3CA. The relationships between mutation status, tumor microsatellite instability (MSI) and clinicopathological features including overall survival (OS) and disease-free survival (DFS) were evaluated using Kaplan-Meier survival analysis and Cox proportional hazard models. Mutations were identified in FGFR2 (48/466); KRAS (87/464); CTNNB1 (88/454) and PIK3CA (104/464). KRAS and FGFR2 mutations were significantly more common, and CTNNB1 mutations less common, in MSI positive tumors. KRAS and FGFR2 occurred in a near mutually exclusive pattern (p = 0.05) and, surprisingly, mutations in KRAS and CTNNB1 also occurred in a near mutually exclusive pattern (p = 0.0002). Multivariate analysis revealed that mutation in KRAS and FGFR2 showed a trend (p = 0.06) towards longer and shorter DFS, respectively. In the 386 patients with early stage disease (stage I and II), FGFR2 mutation was significantly associated with shorter DFS (HR = 3.24; 95% confidence interval, CI, 1.35-7.77; p = 0.008) and OS (HR = 2.00; 95% CI 1.09-3.65; p = 0.025) and KRAS was associated with longer DFS (HR = 0.23; 95% CI 0.05-0.97; p = 0.045). In conclusion, although KRAS and FGFR2 mutations share similar activation of the MAPK pathway, our data suggest very different roles in tumor biology. This has implications for the implementation of anti-FGFR or anti-MEK biologic therapies.

Background Oesophageal adenocarcinoma (EAC) incidence is increasing and has a poor survival rate. Barrett’s oesophagus (BE) is a precursor condition that is associated with EAC and often occurs in conjunction with chronic gastro-oesophageal reflux, however many individuals diagnosed with BE never progress to cancer. An understanding of the genomic features of BE and EAC may help with the early identification of at-risk individuals. Methods In this study, we assessed the genomic features of 16 BE samples using whole-genome sequencing. These included non-dysplastic samples collected at two time-points from two BE patients who had not progressed to EAC over several years. Seven other non-dysplastic samples and five dysplastic BE samples with high-grade dysplasia were also examined. We compared the genome profiles of these 16 BE samples with 22 EAC samples. Results We observed that samples from the two non-progressor individuals had low numbers of somatic single nucleotide variants, indels and structural variation events compared to dysplastic and the remaining non-dysplastic BE. EAC had the highest level of somatic genomic variations. Mutational signature 17, which is common in EAC, was also present in non-dysplastic and dysplastic BE, but was not present in the non-progressors. Many dysplastic samples had mutations in genes previously reported in EAC, whereas only mutations in CDKN2A or in the fragile site genes appeared common in non-dysplastic samples. Rearrangement signatures were used to identify a signature associated with localised complex events such as chromothripsis and breakage fusion-bridge that are characteristic of EACs. Two dysplastic BE samples had a high contribution of this signature and contained evidence of localised rearrangements. Two other dysplastic samples also had regions of localised structural rearrangements. There was no evidence for complex events in non-dysplastic samples. Conclusions The presence of complex localised rearrangements in dysplastic samples indicates a need for further investigations into the role such events play in the progression from BE to EAC.

Background Little is known about the association between signet ring cell (SRC) differentiation and response to neoadjuvant chemotherapy (nCT) or neoadjuvant chemoradiotherapy (nCRT) in patients with esophageal and junctional adenocarcinoma (EAC). We aimed to assess if SRC differentiation is associated with survival and response to nCT or nCRT in patients with EAC. Methods Patients who underwent nCT and nCRT followed by surgery for EAC from 2000 until 2016 were identified from two institutional prospectively maintained databases. The pretreatment biopsy report or surgical resection specimen was used to differentiate patients into an SRC or non-SRC group. Results Overall, 129 (19%) of 689 patients included had SRCs (nCT: n  = 64; nCRT: n  = 65). The SRC group had a more advanced ypT stage ( p  = 0.003), a higher number of positive lymph nodes in the resection specimen {median (interquartile range [IQR]) 2 [0–5] vs. 1 [0–3]; p  = 0.002} and a higher rate of R1/R2 resections (19.4% vs. 12%; p  = 0.026). SRC differentiation was not an independent prognostic factor for overall survival (OS) or disease-free survival (DFS). Following nCT, the SRC group had significantly shorter DFS (median [IQR] 12 [5–50] vs. 23 [8–164]; p  = 0.013), but not OS, compared with the non-SRC group. In contrast, no differences according to SRC status for OS or DFS were found in patients who underwent nCRT. Conclusions SRC differentiation was not independently associated with worse OS in patients with EAC who underwent neoadjuvant therapy and surgery. However, nCRT was associated with greater tumor downstaging and better DFS.

Mutations in Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) and Cyclin-Dependent Kinase 4 (CDK4) contribute to susceptibility in approximately 40% of high-density cutaneous melanoma (CMM) families and about 2% of unselected CMM cases. BRCA-1 associated protein-1 (BAP1) has been more recently shown to predispose to CMM and uveal melanoma (UMM) in some families; however, its contribution to CMM development in the general population is unreported. We sought to determine the contribution of these genes to CMM susceptibility in a population-based sample of cases from Australia. We genotyped 1,109 probands from Queensland families and found that approximately 1.31% harbored mutations in CDKN2A, including some with novel missense mutations (p.R22W, p.G35R and p.I49F). BAP1 missense variants occurred in 0.63% of cases but no CDK4 variants were observed in the sample. This is the first estimate of the contribution of BAP1 and CDK4 to a population-based sample of CMM and supports the previously reported estimate of CDKN2A germline mutation prevalence.

Germline mutations in the exonuclease domain of POLE have been shown to predispose to colorectal cancers and adenomas. POLE is an enzyme involved in DNA repair and chromosomal DNA replication. In order to assess whether such mutations might also predispose to cutaneous melanoma, we interrogated whole-genome and exome data from probands of 34 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A , CDK4 , BAP1 , TERT , POT1 , ACD and TERF2IP . We found a novel germline mutation, POLE p.(Trp347Cys), in a 7-case cutaneous melanoma family. Functional assays in S . pombe showed that this mutation led to an increased DNA mutation rate comparable to that seen with a Pol ε mutant with no exonuclease activity. We then performed targeted sequencing of POLE in 1243 cutaneous melanoma cases and found that a further ten probands had novel or rare variants in the exonuclease domain of POLE. Although this frequency is not significantly higher than that in unselected Caucasian controls, we observed multiple cancer types in the melanoma families, suggesting that some germline POLE mutations may predispose to a broad spectrum of cancers, including melanoma. In addition, we found the first mutation outside the exonuclease domain, p.(Gln520Arg), in a family with an extensive history of colorectal cancer.