Functional characterization of 5p15.33 risk locus in uveal melanoma reveals rs452384 as a functional variant and NKX2.4 as an allele-specific interactor.

The TERT/CLPTM1L risk locus on chromosome 5p15.33 is a pleiotropic cancer risk locus in which multiple independent risk alleles have been identified, across well over ten cancer types. We previously conducted a genome-wide association study in uveal melanoma (UM), which uncovered a role for the TERT/CLPTM1L risk locus in this intraocular tumor and identified multiple highly correlated risk alleles. Aiming to unravel the biological mechanisms in UM of this locus, which contains a domain enriched in active chromatin marks and enhancer elements, we demonstrated the allele-specific enhancer activity of this risk region using reporter assays. In UM, we identified the functional variant rs452384, of which the C risk allele is associated with higher gene expression, increased CLPTM1L expression in UM tumors, and a longer telomere length in peripheral blood mononuclear cells. Electrophoretic mobility shift assays and quantitative mass spectrometry identified NKX2.4 as an rs452384-T-specific binding protein, whereas GATA4 preferentially interacted with rs452384-C. Knockdown of NKX2.4 but not GATA4 resulted in increased TERT and CLPTM1L expression. In summary, the UM risk conferred by the 5p locus is at least partly due to rs452384, for which NKX2.4 presents strong differential binding activity and regulates CLPTM1L and TERT expression. Altogether, our work unraveled some of the complex regulatory mechanisms at the 5p15.33 susceptibility region in UM, and this might also shed light on shared mechanisms with other tumor types affected by this susceptibility region.

MBD4 deficiency is predictive of response to immune checkpoint inhibitors in metastatic uveal melanoma patients.

BACKGROUND: MBD4 mutations have been reported in uveal melanomas, acute myeloid leukemias, colorectal adenocarcinomas, gliomas, and spiradenocarcinomas and cause a hypermutated phenotype. Although metastatic uveal melanomas (mUM) are usually resistant to immune checkpoint inhibitors (ICI), the first reported MBD4-mutated (MBD4m) patient responded to ICI, suggesting that MBD4 mutation may predict response to ICI. METHODS: Retrospective cohort of mUM patients treated with ICI. MBD4 was sequenced in a subset of these patients. RESULTS: Three hundred mUM patients were included. Median follow-up was 17.3 months. Ten patients with an objective response and 20 cases with stable disease for >12 months were observed, corresponding to an objective response rate of 3.3% and a clinical benefit (i.e., responder patients and stable disease) rate of 10%. Of the 131 tumors sequenced for MBD4, five (3.8%) were mutated. MBD4 mutation was associated with a better objective response rate as three out of five MBD4m versus 4% of MBD4 wild-type patients responded (p < 0.001). Of these five responders, three presented progressive disease at 2.8, 13.9, and 22.3 months. Median PFS was 4.0 months in MBD4 wild-type and 22.3 months in MBD4m patients (HR = 0.22; p = 0.01). Median OS in MBD4def patients was unreached as compared to 16.6 months in MBD4pro (HR = 0.11; 95% CI: 0.02-0.86; log-rank p-test = 0.04; Fig. 2e). CONCLUSIONS: In mUM patients, MBD4 mutation is highly predictive for the response, PFS, and overall survival benefit to ICI. MBD4 could be a tissue-agnostic biomarker and should be sequenced in mUM, and other tumor types where MBD4 mutations are reported.

Different Pigmentation Risk Loci for High-Risk Monosomy 3 and Low-Risk Disomy 3 Uveal Melanomas.

BACKGROUND: Uveal melanoma (UM), a rare malignant tumor of the eye, is predominantly observed in populations of European ancestry. UMs carrying a monosomy 3 (M3) frequently relapse mainly in the liver, whereas UMs with disomy 3 (D3) are associated with more favorable outcome. Here, we explored the UM genetic predisposition factors in a large genome-wide association study (GWAS) of 1142 European UM patients and 882 healthy controls . METHODS: We combined 2 independent datasets (Global Screening Array) with the dataset described in a previously published GWAS in UM (Omni5 array), which were imputed separately and subsequently merged. Patients were stratified according to their chromosome 3 status, and identified UM risk loci were tested for differential association with M3 or D3 subgroups. All statistical tests were 2-sided. RESULTS: We recapitulated the previously identified risk locus on chromosome 5 on CLPTM1L (rs421284: odds ratio [OR] =1.58, 95% confidence interval [CI] = 1.35 to 1.86; P = 1.98 × 10-8) and identified 2 additional risk loci involved in eye pigmentation: IRF4 locus on chromosome 6 (rs12203592: OR = 1.76, 95% CI = 1.44 to 2.16; P = 3.55 × 10-8) and HERC2 locus on chromosome 15 (rs12913832: OR= 0.57, 95% CI = 0.48 to 0.67; P = 1.88 × 10-11). The IRF4 rs12203592 single-nucleotide polymorphism was found to be exclusively associated with risk for the D3 UM subtype (ORD3 = 2.73, 95% CI = 1.87 to 3.97; P = 1.78 × 10-7), and the HERC2 rs12913832 single-nucleotide polymorphism was exclusively associated with risk for the M3 UM subtype (ORM3 = 2.43, 95% CI = 1.79 to 3.29; P = 1.13 × 10-8). However, the CLPTM1L risk locus was equally statistically significant in both subgroups. CONCLUSIONS: This work identified 2 additional UM risk loci known for their role in pigmentation. Importantly, we demonstrate that UM tumor biology and metastatic potential are influenced by patients' genetic backgrounds.

Germline MBD4 Mutations and Predisposition to Uveal Melanoma.

BACKGROUND: Uveal melanoma (UM) arises from malignant transformation of melanocytes in the uveal tract of the eye. This rare tumor has a poor outcome with frequent chemo-resistant liver metastases. BAP1 is the only known predisposing gene for UM. UMs are generally characterized by low tumor mutation burden, but some UMs display a high level of CpG>TpG mutations associated with MBD4 inactivation. Here, we explored the incidence of germline MBD4 variants in a consecutive series of 1093 primary UM case patients and a series of 192 UM tumors with monosomy 3 (M3). METHODS: We performed MBD4 targeted sequencing on pooled germline (n = 1093) and tumor (n = 192) DNA samples of UM patients. MBD4 variants (n = 28) were validated by Sanger sequencing. We performed whole-exome sequencing on available tumor samples harboring MBD4 variants (n = 9). Variants of unknown pathogenicity were further functionally assessed. RESULTS: We identified 8 deleterious MBD4 mutations in the consecutive UM series, a 9.15-fold (95% confidence interval = 4.24-fold to 19.73-fold) increased incidence compared with the general population (Fisher exact test, P = 2.00 × 10-5, 2-sided), and 4 additional deleterious MBD4 mutations in the M3 cohort, including 3 germline and 1 somatic mutations. Tumors carrying deleterious MBD4 mutations were all associated with high tumor mutation burden and a CpG>TpG hypermutator phenotype. CONCLUSIONS: We demonstrate that MBD4 is a new predisposing gene for UM associated with hypermutated M3 tumors. The tumor spectrum of this predisposing condition will likely expand with the addition of MBD4 to diagnostic panels. Tumors arising in such a context should be recognized because they may respond to immunotherapy.

DNA repair functional analyses of NBN hypomorphic variants associated with NBN-related infertility.

Nijmegen breakage syndrome caused by biallelic pathogenic variants of the DNA-damage response gene NBN, is characterized by severe microcephaly, cancer proneness, infertility, and karyotype abnormalities. We previously reported NBN variants in siblings suffering from fertility defects. Here, we identify a new founder NBN variant (c.442A>G, p.(Thr148Ala)) in Lebanese patients associated with isolated infertility. Functional analyses explored preserved or altered functions correlated with their remarkably mild phenotype. Transcript and protein analyses supported the use of an alternative transcript with in-frame skipping of exons 4-5, leading to p84-NBN protein with a preserved forkhead-associated (FHA) domain. The level of NBN was dramatically reduced and the MRN complex delocalized to the cytoplasm. Interestingly, ataxia-elangiectasia mutated (ATM) also shifted from the nucleus to the cytoplasm, suggesting some interaction between ATM and the MRN complex at a steady state. The ATM pathway activation, attenuated in typical patients with NBS, appeared normal under camptothecin treatment in these new NBN-related infertile patients. Cell cycle checkpoint defect was present in these atypical patients, although to a lesser extent than in typical patients with NBS. In conclusion, we report three new NBN-related infertile patients and we suggest that preserved FHA domain could be responsible for the mild phenotype and intermediate DNA-damage response defects.