Radiotherapy-activated NBTXR3 nanoparticles promote ferroptosis through induction of lysosomal membrane permeabilization.

PURPOSE: Radiotherapy-activated NBTXR3 (NBTXR3 + RT) has demonstrated superior efficacy in cancer cell destruction and tumor growth control, compared to radiotherapy (RT), in preclinical and clinical settings. Previous studies highlighted the immunomodulatory properties of NBTXR3 + RT, such as modification of tumor cell immunogenicity/adjuvanticity, producing an effective local tumor control and abscopal effect, related to an enhanced antitumor immune response. Furthermore, NBTXR3 + RT has shown potential in restoring anti-PD1 efficacy in a refractory tumor model. However, the early events leading to these results, such as NBTXR3 endocytosis, intracellular trafficking and primary biological responses induced by NBTXR3 + RT remain poorly understood. METHODS: We analyzed by transmission electron microscopy endocytosis and intracellular localization of NBTXR3 nanoparticles after endocytosis in various cell lines, in vitro and in vivo. A kinetic of NBTXR3 endocytosis and its impact on lysosomes was conducted using LysoTracker staining, and a RNAseq analysis was performed. We investigated the ability of NBTXR3 + RT to induce lysosomal membrane permeabilization (LMP) and ferroptosis by analyzing lipid peroxidation. Additionally, we evaluated the recapture by cancer cells of NBTXR3 released from dead cells. RESULTS: NBTXR3 nanoparticles were rapidly internalized by cells mainly through macropinocytosis and in a less extend by clathrin-dependent endocytosis. NBTXR3-containing endosomes were then fused with lysosomes. The day following NBTXR3 addition, we measured a significant increase in LysoTracker lysosome labeling intensity, in vitro as in vivo. Following RT, a significant lysosomal membrane permeabilization (LMP) was measured exclusively in cells treated with NBTXR3 + RT, while RT had no effect. The day post-irradiation, a significant increase in lipid peroxidation, a biomarker of ferroptosis, was measured with NBTXR3 + RT compared to RT. Moreover, we demonstrated that NBTXR3 nanoparticles released from dead cells can be recaptured by cancer cells. CONCLUSIONS: Our findings provide novel insights into the early and specific biological effects induced by NBTXR3 + RT, especially LMP, not induced by RT in our models. The subsequent significant increase in lipid peroxidation partially explains the enhanced cancer cell killing capacity of NBTXR3 + RT compared to RT, potentially by promoting ferroptosis. This study improves our understanding of the cellular mechanisms underlying NBTXR3 + RT and highlights its potential as an agnostic therapeutic strategy for solid cancers treatment.

Industrializing AI-powered drug discovery: lessons learned from the Patrimony computing platform.

INTRODUCTION: As a mid-size international pharmaceutical company, we initiated 4 years ago the launch of a dedicated high-throughput computing platform supporting drug discovery. The platform named 'Patrimony' was built up on the initial predicate to capitalize on our proprietary data while leveraging public data sources in order to foster a Computational Precision Medicine approach with the power of artificial intelligence. AREAS COVERED: Specifically, Patrimony is designed to identify novel therapeutic target candidates. With several successful use cases in immuno-inflammatory diseases, and current ongoing extension to applications to oncology and neurology, we document how this industrial computational platform has had a transformational impact on our R&D, making it more competitive, as well time and cost effective through a model-based educated selection of therapeutic targets and drug candidates. EXPERT OPINION: We report our achievements, but also our challenges in implementing data access and governance processes, building up hardware and user interfaces, and acculturing scientists to use predictive models to inform decisions.

Genes involved in the WNT and vesicular trafficking pathways are associated with melanoma predisposition.

Multifactorial predisposition to melanoma includes genes involved in pigmentation, immunity and DNA repair. Nonetheless, missing heritability in melanoma is still important. We studied the role of 335 candidate SNPs in melanoma susceptibility by using a dedicated chip and investigating 110 genes involved in different pathways. A discovery set was comprised of 1069 melanoma patients and 925 controls from France. Data were replicated using validation phases II (1085 cases and 801 controls from Spain) and III (1808 cases and 1894 controls from Germany and a second set of Spanish samples). In addition, an exome sequencing study was performed in three high-risk French melanoma families. Nineteen SNPs in 17 genes were initially associated with melanoma in the French population. Six SNPs were replicated in phase II, including two new SNPs in the WNT3 (rs199524) and VPS41 (rs11773094) genes. The role of VPS41 and WNT3 was confirmed in a meta-analysis (3940 melanoma cases and 3620 controls) with two-side p values of 0.002, (OR = 0.86) and 4.07 × 10(-10) (OR = 0.80), respectively. Exome sequencing revealed a non-synonymous VPS41 variant in one family that was shown to be strongly associated with familial melanoma (OR = 4.46, p = 0.001) in an independent sample of 178 melanoma families. WNT3 belongs to WNT pathway known to play a crucial role in melanoma, whereas VPS41 regulates vesicular trafficking and is thought to play a role in pigmentation. Our work identified two new pathways involved in melanoma predisposition. These results may be useful in the future for identifying individuals highly predisposed to melanoma.

Polytherapy with a combination of three repurposed drugs (PXT3003) down-regulates Pmp22 over-expression and improves myelination, axonal and functional parameters in models of CMT1A neuropathy.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited sensory and motor peripheral neuropathy. It is caused by PMP22 overexpression which leads to defects of peripheral myelination, loss of long axons, and progressive impairment then disability. There is no treatment available despite observations that monotherapeutic interventions slow progression in rodent models. We thus hypothesized that a polytherapeutic approach using several drugs, previously approved for other diseases, could be beneficial by simultaneously targeting PMP22 and pathways important for myelination and axonal integrity. A combination of drugs for CMT1A polytherapy was chosen from a group of authorised drugs for unrelated diseases using a systems biology approach, followed by pharmacological safety considerations. Testing and proof of synergism of these drugs were performed in a co-culture model of DRG neurons and Schwann cells derived from a Pmp22 transgenic rat model of CMT1A. Their ability to lower Pmp22 mRNA in Schwann cells relative to house-keeping genes or to a second myelin transcript (Mpz) was assessed in a clonal cell line expressing these genes. Finally in vivo efficacy of the combination was tested in two models: CMT1A transgenic rats, and mice that recover from a nerve crush injury, a model to assess neuroprotection and regeneration. Combination of (RS)-baclofen, naltrexone hydrochloride and D-sorbitol, termed PXT3003, improved myelination in the Pmp22 transgenic co-culture cellular model, and moderately down-regulated Pmp22 mRNA expression in Schwannoma cells. In both in vitro systems, the combination of drugs was revealed to possess synergistic effects, which provided the rationale for in vivo clinical testing of rodent models. In Pmp22 transgenic CMT1A rats, PXT3003 down-regulated the Pmp22 to Mpz mRNA ratio, improved myelination of small fibres, increased nerve conduction and ameliorated the clinical phenotype. PXT3003 also improved axonal regeneration and remyelination in the murine nerve crush model. Based on these observations in preclinical models, a clinical trial of PTX3003 in CMT1A, a neglected orphan disease, is warranted. If the efficacy of PTX3003 is confirmed, rational polytherapy based on novel combinations of existing non-toxic drugs with pleiotropic effects may represent a promising approach for rapid drug development.

The FOXE1 locus is a major genetic determinant for familial nonmedullary thyroid carcinoma.

Thyroid cancer is the most common endocrine malignancy and nonmedullary thyroid carcinoma (NMTC) represents 90% of all cases. NMTC risk in first-degree relatives of affected cases is elevated fivefold to ninefold. Familial NMTC (FNMTC) accounts for about 3-7% of all thyroid tumors and is a more aggressive clinical entity than its sporadic counterparts. Linkage analysis on high-risk families performed a decade ago mapped several susceptibility loci, but did not lead to the identification of high-penetrance causal germline mutations. More recently, a genome-wide association study (GWAS) identified common single nucleotide polymorphisms (SNPs) affecting the risk of sporadic NMTC. We sought to verify if the newly identified genetic risk factors for NMTC are relevant for FNMTC as well. We genotyped 23 SNPs at 11 candidate loci in 672 subjects belonging to 133 pedigrees with at least two NMTC cases. Statistical analysis was performed using family-based association tests, modified quasi-likelihood score and logistic-normal models. SNPs at 9q22.33 near FOXE1 showed convincing evidence of association with NMTC risk in these high-risk families. The other tested loci resulted negative. These findings confirm the importance of the SNPs identified by recent GWAS on sporadic NMTC on FNMTC as well. However, the proposed FOXE1 causal variants do not show the strongest association signal. Moreover, mutation screening of the FOXE1 coding sequence in the FNMTC cases did not identify rarer causal variants, suggesting that other yet unidentified variants at this locus are involved in FNMTC etiology.

An array CGH based genomic instability index (G2I) is predictive of clinical outcome in breast cancer and reveals a subset of tumors without lymph node involvement but with poor prognosis.

BACKGROUND: Despite entering complete remission after primary treatment, a substantial proportion of patients with early stage breast cancer will develop metastases. Prediction of such an outcome remains challenging despite the clinical use of several prognostic parameters. Several reports indicate that genomic instability, as reflected in specific chromosomal aneuploidies and variations in DNA content, influences clinical outcome but no precise definition of this parameter has yet been clearly established. METHODS: To explore the prognostic value of genomic alterations present in primary tumors, we performed a comparative genomic hybridization study on BAC arrays with a panel of breast carcinomas from 45 patients with metastatic relapse and 95 others, matched for age and axillary node involvement, without any recurrence after at least 11 years of follow-up. Array-CGH data was used to establish a two-parameter index representative of the global level of aneusomy by chromosomal arm, and of the number of breakpoints throughout the genome. RESULTS: Application of appropriate thresholds allowed us to distinguish three classes of tumors highly associated with metastatic relapse. This index used with the same thresholds on a published set of tumors confirms its prognostic significance with a hazard ratio of 3.24 [95CI: 1.76-5.96] p = 6.7 x 10(-5) for the bad prognostic group with respect to the intermediate group. The high prognostic value of this genomic index is related to its ability to individualize a specific group of breast cancers, mainly luminal type and axillary node negative, showing very high genetic instability and poor outcome. Indirect transcriptomic validation was obtained on independent data sets. CONCLUSION: Accurate evaluation of genetic instability in breast cancers by a genomic instability index (G2I) helps individualizing specific tumors with previously unexpected very poor prognosis.

MC1R, SLC45A2 and TYR genetic variants involved in melanoma susceptibility in southern European populations: results from a meta-analysis.

BACKGROUND AND METHODS: Seven genetic biomarkers previously associated with melanoma were analysed in a meta-analysis conducted in three South European populations: five red hair colour (RHC) MC1R alleles, one SLC45A2 variant (p.Phe374Leu) and one thermosensitive TYR variant (p.Arg402Gln). The study included 1639 melanoma patients and 1342 control subjects. RESULTS: The estimated odds ratio (OR) associated with carrying at least one MC1R RHC variant was 2.18 (95% confidence interval (CI): 1.86-2.55; p-value=1.02×10(-21)), with an additive effect for carrying two RHC variants (OR: 5.02, 95% CI: 2.88-8.94, p-value=3.91×10(-8)). The SLC45A2 variant, p.Phe374Leu, was significantly and strongly protective for melanoma in the three South European populations studied, with an overall OR value of 0.41 (95% CI: 0.33-0.50; p-value=3.50×10(-17)). The association with melanoma of the TYR variant p.Arg402Gln was also statistically significant (OR: 1.50; 95% CI: 1.11-2.04; p-value=0.0089). Adjustment for all clinical potential confounders showed that melanoma risks attributable to MC1R and SLC45A2 variants strongly persisted (OR: 2.01 95% CI: 1.49-2.72 and OR: 0.50, 95% CI: 0.31-0.80, respectively), while the association of TYR p.Arg402Gln was no longer significant. In addition, stratification of clinical melanoma risk factors showed that the risk of melanoma was strong in those individuals who did not have clinical risk factors. CONCLUSION: In conclusion, our results show without ambiguity that in South European populations, MC1R RHC and SCL45A2 p.Phe374Leu variants are strong melanoma risk predictors, notably in those individuals who would not be identified as high risk based on their phenotypes or exposures alone. The use of these biomarkers in clinical practice could be promising and warrants further discussion.

Independent replication and meta analysis of association studies establish TNFSF4 as a susceptibility gene preferentially associated with the subset of anticentromere-positive patients with systemic sclerosis.

OBJECTIVE: Independent replication with large cohorts and metaanalysis of genetic associations are necessary to validate genetic susceptibility factors. The known tumor necrosis factor (ligand) superfamily, member 4 gene (TNFSF4) systemic lupus erythematosus (SLE) risk locus has been found to be associated with systemic sclerosis (SSc) in 2 studies, but with discrepancies between them for genotype-phenotype correlation. Our objective was to validate TNFSF4 association with SSc and determine the subset with the higher risk. METHODS: Known SLE and SSc TNFSF4 susceptibility variants (rs2205960, rs1234317, rs12039904, rs10912580, and rs844648) were genotyped in 1031 patients with SSc and 1014 controls of French white ancestry. Genotype-phenotype association analysis and meta analysis of available data were performed, providing a population study of 4989 patients with SSc and 4661 controls, all of European white ancestry. RESULTS: Allelic and genotypic associations were observed for the 5 single-nucleotide polymorphisms (SNP) with the subset of patients with SSc who are positive for anticentromere antibodies (ACA) and only a trend for association with SSc and limited cutaneous SSc. Rs2205960 exhibited the strongest allelic association in ACA+ patients with SSc [p = 0.0015; OR 1.37 (1.12-1.66)], with significant intra-cohort association when compared to patients with SSc positive for ACA. Metaanalysis confirmed overall association with SSc but also raised preferential association with the ACA+ subset and strongest effect with rs2205960 [T allele p = 0.00013; OR 1.33 (1.15-1.54) and TT genotype p = 0.00046; OR 2.02 (1.36-2.98)]. CONCLUSION: We confirm TNFSF4 as an SSc susceptibility gene and rs2205960 as a putative causal variant with preferential association in the ACA+ SSc subphenotype.

A reliable method for the selection of exploitable melanoma archival paraffin embedded tissues for transcript biomarker profiling.

The source tissue for biomarkers mRNA expression profiling of tumors has traditionally been fresh-frozen tissue. The adaptation of formalin-fixed, paraffin-embedded (FFPE) tissues for routine mRNA profiling would however be invaluable in view of their abundance and the clinical information related to them. However, their use in the clinic remains a challenge due to the poor quality of RNA extracted from such tissues. Here, we developed a method for the selection of melanoma archival paraffin-embedded tissues that can be reliably used for transcript biomarker profiling. For that, we used qRT-PCR to conduct a comparative study in matched pairs of frozen and FFPE melanoma tissues of the expression of 25 genes involved in angiogenesis/tumor invasion and 15 housekeeping genes. A classification method was developed that can select the samples with a good frozen/FFPE correlation and identify those that should be discarded on the basis of paraffin data for four reference genes only. We propose therefore a simple and inexpensive assay which improves reliability of mRNA profiling in FFPE samples by allowing the identification and analysis of "good" samples only. This assay which can be extended to other genes would however need validation at the clinical level and on independent tumor series.

Assessment of tyrosinase variants and skin cancer risk in a large cohort of French subjects.

BACKGROUND: Tyrosinase (TYR) is a key pigmentation gene that is highly polymorphic and responsible for the most common form of autosomal recessive albinism, OCA1. OBJECTIVE: To assess the role of frequent and rare TYR variants in predisposition to skin cancer (SK) in the French population. METHODS: We genotyped a frequent TYR variant (p.R402Q) in 1273 patients {1047 cutaneous melanoma (CM) and 226 basal cell carcinoma (BCC)} and 925 controls, and the full coding region of TYR was sequenced in 287 patients suspected of genetic predisposition to SK (familial and/or multiple SK and/or onset before 40 years) and 187 controls. RESULTS: The homozygous p.R402Q variant was significantly associated with SK risk (P value=0.008; OR=1.57), and was mostly associated with multiple CM risk (P value=0.021; OR=2.50) and familial CM risk (P value=0.022; OR=2.16). In addition, 19 rare TYR variants, mainly albinism mutations, were identified in 15 patients and 8 controls. Among these, 3 clearly deleterious mutations (1 non-sense and 2 affecting mRNA splicing) were identified in 3 patients, one of which was homozygous. CONCLUSION: Our data confirmed the association of TYR p.R402Q with SK risk in the French population, and support that rare deleterious TYR variants may also play a role in multi-factorial genetic predisposition to SK. These results should be confirmed by replications studies.

PTPN22 R620W genotype-phenotype correlation analysis and gene-environment interaction study in early rheumatoid arthritis: results from the ESPOIR cohort.

OBJECTIVES: To investigate genotype-phenotype correlation and gene-environment interaction between PTPN22 R620W environmental factors such as tobacco/hormonal treatments in an inception cohort of RA patients. METHODS: An intra-cohort study including 532 Caucasian RA patients genotyped for the PTPN22 rs2476601 polymorphism was performed. Anti-CCP and RF status at baseline, presence of bone erosions at 1 year, HLADR1 and/or DR4 status, demography, comorbidities, exposure to tobacco with the cumulative dose in pack-years, hormonal treatments and treatments received for RA were collected. Logistic regression was performed to estimate the ORs and multiplicative interaction with adjustment for confounding factors. Gene-environment interaction was estimated by the relative excess risk due to interaction (RERI), attributable proportion (AP) and synergy index (SI). RESULTS: PTPN22 620W risk allele was associated with ACPA production [odds ratio (OR) = 2.21, 95% CI 1.4, 3.4, P < 0.0001]. Hormonal treatment exposition and smoking were found to act with a protective effect against ACPA production (OR = 0.44, 95% CI 0.3, 0.7, P = 0.001) and early bone erosion (OR = 0.56, 95% CI 0.4-0.8, P = 0.003), respectively, and independently of HLADR and PTPN22 status. No evidence for a gene-environment interaction was detected. CONCLUSION: These data provide new insights into the pathogenesis of RA, underlying the pivotal key role of environmental factors in the typical heterogeneity of RA.

Molecular apocrine differentiation is a common feature of breast cancer in patients with germline PTEN mutations.

INTRODUCTION: Breast carcinoma is the main malignant tumor occurring in patients with Cowden disease, a cancer-prone syndrome caused by germline mutation of the tumor suppressor gene PTEN characterized by the occurrence throughout life of hyperplastic, hamartomatous and malignant growths affecting various organs. The absence of known histological features for breast cancer arising in a PTEN-mutant background prompted us to explore them for potential new markers. METHODS: We first performed a microarray study of three tumors from patients with Cowden disease in the context of a transcriptomic study of 74 familial breast cancers. A subsequent histological and immunohistochemical study including 12 additional cases of Cowden disease breast carcinomas was performed to confirm the microarray data. RESULTS: Unsupervised clustering of the 74 familial tumors followed the intrinsic gene classification of breast cancer except for a group of five tumors that included the three Cowden tumors. The gene expression profile of the Cowden tumors shows considerable overlap with that of a breast cancer subgroup known as molecular apocrine breast carcinoma, which is suspected to have increased androgenic signaling and shows frequent ERBB2 amplification in sporadic tumors. The histological and immunohistochemical study showed that several cases had apocrine histological features and expressed GGT1, which is a potential new marker for apocrine breast carcinoma. CONCLUSIONS: These data suggest that activation of the ERBB2-PI3K-AKT pathway by loss of PTEN at early stages of tumorigenesis promotes the formation of breast tumors with apocrine features.

Clinically relevant characterization of lung adenocarcinoma subtypes based on cellular pathways: an international validation study.

Lung adenocarcinoma (AD) represents a predominant type of lung cancer demonstrating significant morphologic and molecular heterogeneity. We sought to understand this heterogeneity by utilizing gene expression analyses of 432 AD samples and examining associations between 27 known cancer-related pathways and the AD subtype, clinical characteristics and patient survival. Unsupervised clustering of AD and gene expression enrichment analysis reveals that cell proliferation is the most important pathway separating tumors into subgroups. Further, AD with increased cell proliferation demonstrate significantly poorer outcome and an increased solid AD subtype component. Additionally, we find that tumors with any solid component have decreased survival as compared to tumors without a solid component. These results lead to the potential to use a relatively simple pathological examination of a tumor in order to determine its aggressiveness and the patient's prognosis. Additional results suggest the ability to use a similar approach to determine a patient's sensitivity to targeted treatment. We then demonstrated the consistency of these findings using two independent AD cohorts from Asia (N = 87) and Europe (N = 89) using the identical analytic procedures.

Variants of the xeroderma pigmentosum variant gene (POLH) are associated with melanoma risk.

PURPOSE: Xeroderma pigmentosum variant (XPV) is a rare recessive autosomal genodermatosis predisposing to multiple early onset skin cancers, including melanoma. XPV results from mutations of the POLH gene that encodes a DNA translesion polymerase. In this work, we tested the hypothesis that POLH variants could be associated with melanoma risk. EXPERIMENTAL DESIGN: A common non-synonymous POLH variant, c.1783A>G p.M595V, was genotyped in 1075 melanoma patients and in 1091 ethnic-matched controls from France. In addition, we searched for rare POLH variants by sequencing the entire coding sequence in 201 patients having a familial history of melanoma (n=123), sporadic multiple melanomas (n=65) and a melanoma associated with a skin carcinoma (n=13). RESULTS: Overall, the c.1783G, p.595V allele was statistically associated with melanoma (respective allelic frequencies, 0.040 versus 0.022, P-value=1.17 x 10(-3), odds ratio (OR)=1.86 [1.27-2.71]), which was further confirmed by a meta-analysis including 274 patients and 174 matched controls from Italy (P-value=7.7 x 10(-4), OR=1.84 [1.29-2.63]). Interestingly, three non-synonymous POLH variants were identified in three patients (c.295G>A p.V99M, c.815T>C p.I272T and c.1745C>T p.S582L) which were absent in 352 chromosome controls from healthy subjects. CONCLUSIONS: Besides severe deficiencies in translesion synthesis which are major risks factors for skin carcinomas and melanomas, less deleterious POLH variants could act as low penetrance melanoma predisposing alleles. The ongoing identification of genetic markers implied in skin cancer predisposition could help to identify high-risk subjects as targets for clinical follow-up. Replication studies in other populations are awaited to assess these data.

Variants of the MATP/SLC45A2 gene are protective for melanoma in the French population.

In this study, we investigated whether variants in three key pigmentation genes-MC1R, MATP/SLC45A2, and OCA2--were involved in melanoma predisposition. A cohort comprising 1,019 melanoma patients (MelanCohort) and 1,466 Caucasian controls without skin cancers were studied. A total of 10 polymorphisms, including five functional MC1R alleles (p.Asp84Glu, p.Arg142His, p.Arg151Cys, p.Arg160Trp, and p.Asp294His), two nonsynonymous SLC45A2 variants (p.Phe374Leu and p.Glu272Lys), and three intronic OCA2 variants previously shown to be strongly associated with eye color (rs7495174 T>C, rs4778241 G>T, and rs4778138 T>C) were genotyped. As expected, MC1R variants were closely associated with melanoma risk (P value <2.20.10(-16); odds ratio [OR]=2.29 [95% confidence interval, CI=1.85-2.82 and OR=3.3 [95% CI=2.00-5.45], for the presence of one or two variants, respectively). Interestingly, the SLC45A2 variant p.Phe374Leu was significantly and strongly protective for melanoma (P-value=2.12.10(-15); OR=0.35 [95% CI=0.26-0.46] and OR=0.32 [95% CI=0.24-0.43], considering the genotypes Phe/Leu and Leu/Leu, respectively). MC1R and SLC45A2 variants had additive effects on melanoma risk, and after adjusting for pigmentation characteristics, the risk was persistent, even though both genes had a strong impact on pigmentation. Future studies may show whether genetic information could provide a useful complement to physical examination in predicting melanoma risk.