Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation.

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

Divergent local and systemic antitumor response in primary uveal melanomas.

Uveal melanoma (UM) is the most common cancer of the eye. The loss of chromosome 3 (M3) is associated with a high risk of metastases. M3 tumors are more infiltrated by T-lymphocytes than low-risk disomic-3 (D3) tumors, contrasting with other tumor types in which T cell infiltration correlates with better prognosis. Whether these T cells represent an antitumor response and how these T cells would be primed in the eye are both unknown. Herein, we characterized the T cells infiltrating primary UMs. CD8+ and Treg cells were more abundant in M3 than in D3 tumors. CD39+PD-1+CD8+ T cells were enriched in M3 tumors, suggesting specific responses to tumor antigen (Ag) as confirmed using HLA-A2:Melan-A tetramers. scRNAseq-VDJ analysis of T cells evidenced high numbers of proliferating CD39+PD1+CD8+ clonal expansions, suggesting in situ antitumor Ag responses. TCRseq and tumor-Ag tetramer staining characterized the recirculation pattern of the antitumor responses in M3 and D3 tumors. Thus, tumor-Ag responses occur in localized UMs, raising the question of the priming mechanisms in the absence of known lymphatic drainage.

Familial uveal melanoma and other tumors in 25 families with monoallelic germline MBD4 variants.

BACKGROUND: Monoallelic germline MBD4 pathogenic variants were recently reported to cause a predisposition to uveal melanoma, associated with a specific tumor mutational signature and good response to immunotherapy. Monoallelic tumor pathogenic variants have also been described in brain tumors, breast cancers, and myxofibrosarcomas, whereas biallelic germline MBD4 pathogenic variants have been involved in a recessive hereditary adenomatous polyposis and a specific type of acute myeloid leukemia. METHODS: We analyzed MBD4 for all patients with a diagnosis of uveal melanoma at Institut Curie since July 2021 and in the 3240 consecutive female probands explored at the Institut Curie for suspicion of predisposition to breast cancer between July 2021 and February 2023. RESULTS: We describe 25 families whose probands carry a monoallelic germline pathogenic variant in MBD4. Eighteen of these families presented with uveal melanoma (including a case patient with multiple uveal melanoma), and 7 families presented with breast cancer. Family histories showed the first familial case of uveal melanoma in monoallelic MBD4 pathogenic variant carriers and other various types of cancers in relatives, especially breast, renal, and colorectal tumors. CONCLUSIONS: Monoallelic MBD4 pathogenic variant may explain some cases of familial and multiple uveal melanoma as well as various cancer types, expanding the tumor spectrum of this predisposition. Further genetic testing in relatives combined with molecular tumor analyses will help define the tumor spectrum and estimate each tumor's risk.

Mosaic BRCA1 promoter methylation contribution in hereditary breast/ovarian cancer pedigrees.

PURPOSE: Mosaic BRCA1 promoter methylation (BRCA1meth) increases the risk of early-onset breast cancer, triple-negative breast cancer and ovarian cancer. As mosaic BRCA1meth are believed to occur de novo, their role in family breast/ovarian cancer has not been assessed. PATIENTS: Blood-derived DNA from 20 unrelated affected cases from families with aggregation of breast/ovarian cancer, but with no germline pathogenic variants in BRCA1/2, PALB2 or RAD51C/D, were screened by methylation-sensitive high-resolution melting. CpG analysis was performed by pyrosequencing on blood and buccal swab. Two probands carried a pathogenic variant in a moderate-penetrance gene (ATM and BARD1), and 8 of 18 others (44%) carried BRCA1meth (vs none of the 20 age-matched controls). Involvement of BRCA1 in tumourigenesis in methylated probands was demonstrated in most tested cases by detection of a loss of heterozygosity and a homologous recombination deficiency signature. Among the eight methylated probands, two had relatives with breast cancer with detectable BRCA1meth in blood, including one with high methylation levels in two non-tumour tissues. CONCLUSIONS: The high prevalence of mosaic BRCA1meth in patients with breast/ovarian cancer with affected relatives, as well as this first description of a family aggregation of mosaic BRCA1meth, shows how this de novo event can contribute to hereditary breast/ovarian cancer pedigrees.

Male breast cancer: No evidence for mosaic BRCA1 promoter methylation involvement.

Breast cancers (BC) are rare in men and are often caused by constitutional predisposing factors. In women, mosaic BRCA1 promoter methylations (MBPM) are frequent events, detected in 4-8% of healthy subjects. This constitutional epimutation increases risk of early-onset and triple-negative BC. However, the role of MBPM in male BC predisposition has never been assessed. We screened 40 blood samples from men affected by BC, and performed extensive tumour analysis on MBPM-positive patients. We detected two patients carrying MBPM. Surprisingly, tumour analysis revealed that neither of these two male BCs were caused by the constitutional BRCA1 epimutations carried by the patients.

Shallow whole genome sequencing approach to detect Homologous Recombination Deficiency in the PAOLA-1/ENGOT-OV25 phase-III trial.

The bevacizumab (bev)/olaparib (ola) maintenance regimen was approved for BRCA1/2-mutated (BRCAmut) and Homologous Recombination Deficient (HRD) high-grade Advanced Ovarian Cancer (AOC) first line setting, based on a significantly improved progression-free survival (PFS) compared to bev alone in the PAOLA-1/ENGOT-ov25 trial (NCT02477644), where HRD was detected by MyChoice CDx PLUS test. The academic shallowHRDv2 test was developed based on shallow whole-genome sequencing as an alternative to MyChoice. Analytical and clinical validities of shallowHRDv2 as compared to MyChoice on 449 PAOLA-1 tumor samples are presented. The overall agreement between shallowHRDv2 and MyChoice was 94% (369/394). Less non-contributive tests were observed with shallowHRDv2 (15/449; 3%) than with MyChoice (51/449; 11%). Patients with HRD tumors according to shallowHRDv2 (including BRCAmut) showed a significantly prolonged PFS with bev+ola versus bev (median PFS: 65.7 versus 20.3 months, hazard ratio (HR): 0.36 [95% CI: 0.24-0.53]). This benefit was significant also for BRCA1/2 wild-type tumors (40.8 versus 19.5 months, HR: 0.45 [95% CI: 0.26-0.76]). ShallowHRDv2 is a performant, clinically validated, and cost-effective test for HRD detection.

Multi-omics comparison of malignant and normal uveal melanocytes reveals molecular features of uveal melanoma.

Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

Immunohistochemical characterisation of the immune landscape in primary uveal melanoma and liver metastases.

BACKGROUND: The immune landscape of uveal melanoma liver metastases (UMLM) has not been sufficiently studied. METHODS: Immune cell infiltrates (ICIs), PD-1 and PD-L1 were characterised in 62 UMLM and 28 primary uveal melanomas (PUM). ICI, PD-1 and PD-L1 were scored as: (1) % tumoral area occupied by tumour-infiltrating lymphocytes or macrophages (TILs, TIMs) and (2) % perTumoral (perT) area. ICIs and other variables including histopathologic growth patterns (HGPs), replacement and desmoplastic, of UMLM were analysed for their prognostic value. RESULTS: ICIs recognised by haematoxylin-eosin-saffron (HES) and IHC (e.g., T cells (CD3), B cells (CD20). Macrophages (CD68), (CD163), were primarily localised to the perT region in PUM and UMLM and were more conspicuous in UMLM. HES, CD3, CD4, FoxP3, CD8, CD20, PD-1 TILs were scant (<5%). TIMs were more frequent, particularly in UMLM than in PUM. Both CD68+ TIMs and HGPs remained significant on multivariate analysis, influencing overall (OS) and metastasis-specific overall survival (MSOS). CD68 + , CD163+ and CD20+ perT infiltrates in UMLM predicted increased OS and MSOS on univariate analysis. CONCLUSIONS: TILs and PD-L1 have no predictive value in PUM or UMLM. CD68+ and CD163+TIMs, CD20+ perT lymphocytes, and HGPs are important prognostic factors in UMLMs.

In Vitro Testing of the Virus-Like Drug Conjugate Belzupacap Sarotalocan (AU-011) on Uveal Melanoma Suggests BAP1-Related Immunostimulatory Capacity.

Purpose: The virus-like drug conjugate belzupacap sarotalocan (AU-011), currently under clinical investigation for first-line treatment of primary uveal melanoma (UM), shows enhanced tumor specificity by targeting heparan sulfate proteoglycans (HSPG). Such a treatment may potentially lead to systemic immune responses. We studied the potential of AU-011 treatment to induce immunogenic cell death as the first step to induce systemic immunity. Methods: We determined binding and uptake of AU-011 in ten primary and metastatic UM cell lines. The subcellular location of AU-011 was assessed by fluorescence microscopy. Following light activation (wavelength 690 nm) of AU-011, the half-maximal effective concentration (EC50) of AU-011 treatment and exposure of damage-associated molecular patterns (DAMPs) were assessed using flow cytometry. DAMPs were measured by RNAseq. Results: Fluorescence microscopy revealed most of the AU-011 was present in the cytoplasm. AU-011 binding and uptake by UM cells increased over time, with a lower uptake in BAP1-negative than in BAP1-positive cell lines. AU-011 activation induced cell death across all UM cell lines with EC50 values at picomolar concentrations. The AU-011 concentration and total light dose (J/cm2) were the most important parameters for the observed cytotoxicity. Finally, light-activated AU-011 induced exposure of DAMPs calreticulin (CRT) and HSP90. CRT exposure by light-activated AU-011 as well as CRT RNA exposure were lower in BAP1-negative compared to BAP1-positive UM cell lines. Conclusions: AU-011 treatment at low picomolar range induces immunogenic cell death in all 10 UM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs (HSP90 and CRT), suggesting AU-011 may contribute to the development of systemic immunity and be a suitable candidate for combination with immunotherapy in vivo. AU-011 treatment was more effective against BAP1-positive cell lines, with a lower EC50 and higher CRT exposure.

FAK Inhibitor-Based Combinations with MEK or PKC Inhibitors Trigger Synergistic Antitumor Effects in Uveal Melanoma.

Uveal Melanoma (UM) is a rare and malignant intraocular tumor with dismal prognosis. Even if radiation or surgery permit an efficient control of the primary tumor, up to 50% of patients subsequently develop metastases, mainly in the liver. The treatment of UM metastases is challenging and the patient survival is very poor. The most recurrent event in UM is the activation of Gαq signaling induced by mutations in GNAQ/11. These mutations activate downstream effectors including protein kinase C (PKC) and mitogen-activated protein kinases (MAPK). Clinical trials with inhibitors of these targets have not demonstrated a survival benefit for patients with UM metastasis. Recently, it has been shown that GNAQ promotes YAP activation through the focal adhesion kinase (FAK). Pharmacological inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in UM both in vitro and in vivo. In this study, we have evaluated the synergy of the FAK inhibitor with a series of inhibitors targeting recognized UM deregulated pathways in a panel of cell lines. The combined inhibition of FAK and MEK or PKC had highly synergistic effects by reducing cell viability and inducing apoptosis. Furthermore, we demonstrated that these combinations exert a remarkable in vivo activity in UM patient-derived xenografts. Our study confirms the previously described synergy of the dual inhibition of FAK and MEK and identifies a novel combination of drugs (FAK and PKC inhibitors) as a promising strategy for therapeutic intervention in metastatic UM.

Homologous recombination deficiency derived from whole-genome sequencing predicts platinum response in triple-negative breast cancers.

The high frequency of homologous recombination deficiency (HRD) is the main rationale of testing platinum-based chemotherapy in triple-negative breast cancer (TNBC), however, the existing methods to identify HRD are controversial and there is a medical need for predictive biomarkers. We assess the in vivo response to platinum agents in 55 patient-derived xenografts (PDX) of TNBC to identify determinants of response. The HRD status, determined from whole genome sequencing, is highly predictive of platinum response. BRCA1 promoter methylation is not associated with response, in part due to residual BRCA1 gene expression and homologous recombination proficiency in different tumours showing mono-allelic methylation. Finally, in 2 cisplatin sensitive tumours we identify mutations in XRCC3 and ORC1 genes that are functionally validated in vitro. In conclusion, our results demonstrate that the genomic HRD is predictive of platinum response in a large cohort of TNBC PDX and identify alterations in XRCC3 and ORC1 genes driving cisplatin response.

Circulating Tumor DNA as a Prognostic Factor in Patients With Resectable Hepatic Metastases of Uveal Melanoma.

OBJECTIVE: We report here the results of a prospective study of circulating tumor DNA (ctDNA) detection in patients undergoing uveal melanoma (UM) liver metastases resection (NCT02849145). BACKGROUND: In UM patients, the liver is the most common and often only site of metastases. Local treatments of liver metastases, such as surgical resection, have a likely benefit in selected patients. METHODS: Upon enrollment, metastatic UM patients eligible for curative liver surgery had plasma samples collected before and after surgery. GNAQ / GNA11 mutations were identified in archived tumor tissue and used to quantify ctDNA by droplet digital polymerase chain reaction which was then associated with the patient's surgical outcomes. RESULTS: Forty-seven patients were included. Liver surgery was associated with a major increase of cell-free circulating DNA levels, with a peak 2 days after surgery (∼20-fold). Among 40 evaluable patients, 14 (35%) had detectable ctDNA before surgery, with a median allelic frequency of 1.1%. These patients experienced statistically shorter relapse-free survival (RFS) versus patients with no detectable ctDNA before surgery (median RFS: 5.5 vs 12.2 months; hazard ratio=2.23, 95% CI: 1.06-4.69, P =0.04), and had a numerically shorter overall survival (OS) (median OS: 27.0 vs 42.3 months). ctDNA positivity at postsurgery time points was also associated with RFS and OS. CONCLUSIONS: This study is the first to report ctDNA detection rate and prognostic impact in UM patients eligible for surgical resection of their liver metastases. If confirmed by further studies in this setting, this noninvasive biomarker could inform treatment decisions in UM patients with liver metastases.

Noncanonical splicing junctions between exons and transposable elements represent a source of immunogenic recurrent neo-antigens in patients with lung cancer.

Although most characterized tumor antigens are encoded by canonical transcripts (such as differentiation or tumor-testis antigens) or mutations (both driver and passenger mutations), recent results have shown that noncanonical transcripts including long noncoding RNAs and transposable elements (TEs) can also encode tumor-specific neo-antigens. Here, we investigate the presentation and immunogenicity of tumor antigens derived from noncanonical mRNA splicing events between coding exons and TEs. Comparing human non-small cell lung cancer (NSCLC) and diverse healthy tissues, we identified a subset of splicing junctions that is both tumor specific and shared across patients. We used HLA-I peptidomics to identify peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides were immunogenic in vitro, and CD8+ T cells specific for junction-encoded epitopes were present in tumors and tumor-draining lymph nodes from patients with NSCLC. We conclude that noncanonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in patients with NSCLC.

Epigenetically controlled tumor antigens derived from splice junctions between exons and transposable elements.

Oncogenesis often implicates epigenetic alterations, including derepression of transposable elements (TEs) and defects in alternative splicing. Here, we explore the possibility that noncanonical splice junctions between exons and TEs represent a source of tumor-specific antigens. We show that mouse normal tissues and tumor cell lines express wide but distinct ranges of mRNA junctions between exons and TEs, some of which are tumor specific. Immunopeptidome analyses in tumor cell lines identified peptides derived from exon-TE splicing junctions associated to MHC-I molecules. Exon-TE junction-derived peptides were immunogenic in tumor-bearing mice. Both prophylactic and therapeutic vaccinations with junction-derived peptides delayed tumor growth in vivo. Inactivation of the TE-silencing histone 3-lysine 9 methyltransferase Setdb1 caused overexpression of new immunogenic junctions in tumor cells. Our results identify exon-TE splicing junctions as epigenetically controlled, immunogenic, and protective tumor antigens in mice, opening possibilities for tumor targeting and vaccination in patients with cancer.

Deep learning identifies morphological patterns of homologous recombination deficiency in luminal breast cancers from whole slide images.

Homologous recombination DNA-repair deficiency (HRD) is becoming a well-recognized marker of platinum salt and polyADP-ribose polymerase inhibitor chemotherapies in ovarian and breast cancers. While large-scale screening for HRD using genomic markers is logistically and economically challenging, stained tissue slides are routinely acquired in clinical practice. With the objectives of providing a robust deep-learning method for HRD prediction from tissue slides and identifying related morphological phenotypes, we first show that digital pathology workflows are sensitive to potential biases in the training set, then we propose a method to overcome the influence of these biases, and we develop an interpretation method capable of identifying complex phenotypes. Application to our carefully curated in-house dataset allows us to predict HRD with high accuracy (area under the receiver-operator characteristics curve 0.86) and to identify morphological phenotypes related to HRD. In particular, the presence of laminated fibrosis and clear tumor cells associated with HRD open new hypotheses regarding its phenotypic impact.

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.

Microsatellite instability detection in breast cancer using drop-off droplet digital PCR.

The use of conventional methods (immunohistochemistry, pentaplex PCR) for detecting microsatellite instability (MSI), a predictive biomarker of immunotherapy efficacy, is debated for cancers with low MSI prevalence, such as breast cancer (BC). We developed two multiplex drop-off droplet digital PCR (ddPCR) assays targeting four microsatellites, initially identified from public BC whole-genome sequencing dataset. Performances of the assays were investigated and 352 tumor DNA and 28 circulating cell-free DNA from BC patients, with unknown MSI status were blindly screened. Cross-validation of ddPCR MSI status with other MSI detection methods was performed. We then monitored circulating tumor DNA (ctDNA) dynamics before and during pembrolizumab immunotherapy in one patient with MSI-high (MSI-H) metastatic BC. The assays showed high analytical specificity and sensitivity (limit of detection = 0.16%). Among N = 380 samples, seven (1.8%) were found as MSI-H by ddPCR with six of them confirmed by next-generation sequencing (NGS). Specificity was 100% in N = 133 microsatellite stable BC submitted to NGS. In the patient with MSI-H metastatic BC, ctDNA monitoring revealed an early decrease of microsatellite mutant allelic frequencies during immunotherapy. These results demonstrated MSI detection by ddPCR, a non-invasive, fast and cost-effective approach, allowing for large pre-screening of BC patients who may benefit from immunotherapy.

Cytidine analogs are synthetic lethal with base excision repair default due to MBD4 deficiency.

Inactivating mutations of MBD4 have been reported in subsets of various tumors. A deficiency of this DNA glycosylase, recognizing specifically T:G mismatch resulting from the deamination of methyl-cytosine, results in a hypermutated phenotype due to the accumulation of CpG>TpG transitions. Here, we hypothesize that the difference in DNA metabolism consecutive to MBD4 deficiency may result in specific cytotoxicities in MBD4-deficient tumor cells in a synthetic lethality fashion. After a large-scale drug repurposing screen, we show in two isogenic MBD4 knock-out cell models that the inactivation of MBD4 sensitizes cancer cells to cytidine analogs. We further confirm the exquisite activity of gemcitabine in an MBD4-deficient co-clinical model as (i) it completely prevented the development of an MBD4-deficient uveal melanoma patient-derived xenograft and (ii) treatment in the corresponding patient resulted in an exceptional tumor response. These data suggest that patients harboring MBD4-deficient tumors may be treated efficiently by cytidine analogs.