👤 Philippe Chevalier

Pan-fibroblast growth factor receptor (FGFR) inhibitors, targeting FGFR1-3 or FGFR1-4, are Food and Drug Administration-approved for FGFR2-driven cholangiocarcinoma. However, acquired resistance and dose-limiting toxicities from systemic FGFR inhibition constrain efficacy. Lirafugratinib (RLY-4008), a first-in-class FGFR2-selective inhibitor with activity against resistance-associated FGFR2 kinase domain mutations, shows promise in patients with FGFR2-altered solid tumors (ReFocus trial, NCT04526106). Defining acquired resistance mechanisms to selective FGFR2 targeting is essential for therapeutic development. Circulating tumor DNA (ctDNA) samples from 28 patients treated with lirafugratinib (16 FGFR inhibitor-naive, 12 FGFR inhibitor-refractory) were analyzed using targeted next-generation sequencing. Genomic alterations observed were compared with those reported in prior studies of pan-FGFR inhibitor resistance and validated in preclinical models. Polyclonal FGFR2 kinase domain mutations and receptor tyrosine kinase-mitogen activated protein kinase (RTK-MAPK) bypass alterations emerged as common lirafugratinib resistance mechanisms in the FGFR inhibitor-naive context (8/16 and 9/16 patients, respectively). Resistance profiles differed markedly from pan-FGFR inhibitors, with decreased FGFR2 V565F/L and N550H/K mutations, increased M538I and L618F mutations, and more frequent RTK-MAPK bypass alterations. The variant allele fraction was typically higher for FGFR2 kinase domain mutations, consistent with these alterations serving as primary resistance drivers. Preclinical studies confirmed differential sensitivity of these FGFR2 mutations to lirafugratinib. Importantly, lirafugratinib demonstrated clinical efficacy in the FGFR inhibitor-refractory setting, with ctDNA dynamics showing resolution of multiple FGFR2 mutations and persistence or emergence of others. Lirafugratinib retains activity against multiple mutations that confer clinical resistance to pan-FGFR inhibitors. However, diverse resistance mechanisms, including various kinase domain mutations and RTK-MAPK bypass alterations, remain challenges in the treatment of FGFR2-altered tumors, even with selective FGFR2 kinase inhibition. Show less

Radiation-induced bystander effects are induced changes in cells that were not themselves directly irradiated but were in the vicinity of a radiation path. Such effects, which occur in the microenvironment of an irradiated tumor, remain poorly understood and depend on the cell type and irradiation quality. This study aimed to evaluate bystander effects in non-irradiated chondrocytes that received conditioned medium from irradiated chondrosarcoma cells. SW1353 chondrosarcoma cells were irradiated with X-rays and carbon ions, each at 0.1 Gy and 2 Gy, and the conditioned media of the irradiated cells were transferred to T/C-28A2 chondrocytes and Human Umbilical Venous Endothelial Cells (HUVECs). The whole proteome of bystander chondrocytes was analyzed by label-free mass spectrometry, and a comparative study was performed by dose and irradiation quality. HUVECs were evaluated for inflammatory cytokine secretion. The bystander response of chondrocytes to X-ray irradiation primarily affected the protein translation pathway (DHX36, EIF3B, EIF3D, EIF3M, EIF5, RPL6, RPLP0, RPS24, SYNCRIP), IL-12 (AIP, BOLA2, MIF, GAS6, MIF, PDGFRB) and the oxidative stress pathway (MGST3, PRDX2, PXDN, SOD2, TXN, TXNL1). Following carbon-ion irradiation, the G1/S pathway (PCBP4, PSMD12, PSME, XIAP) and mitotic G2 DNA damage checkpoint pathway (MRE11, TAOK1, UIMC1) were engaged. Changes in the regulation of chromosome separation (BCL7C, BUB3, CENPF, DYNC1LI1, SMARCA4, SMC4) were associated with only low-dose X-ray and carbon-ion irradiation. Modification of the protein translation pathway represented at least 30% of bystander effects and could play a role, possibly along with stress granules, in reduction in cellular metabolism to protect proteins. Stress granules were significantly enriched according to an interaction map. All these accessions corresponded to a window of the proteins modulated in response to the bystander effect. Our chondrosarcoma model clarified the nature of the bystander response of chondrocytes and may suggest several interesting new mechanisms that are specific to particular irradiation doses and qualities. Show less

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiomyopathy, historically believed to affect 1 of 500 people. MYBPC3 pathogenic variations are the most frequent cause of familial HCM and more than 90% of them introduce a premature termination codon. The current study aims to determine the prevalence of deep intronic MYBPC3 pathogenic variations that could lead to splice mutations. To improve molecular diagnosis, a next-generation sequencing (NGS) workflow based on whole MYBPC3 sequencing of a cohort of 93 HCM patients, for whom no putatively causative point mutations were identified after NGS sequencing of a panel of 48 cardiomyopathy-causing genes, was performed. Our approach led us to reconsider the molecular diagnosis of six patients of the cohort (6.5%). These HCM probands were carriers of either a new large MYBPC3 rearrangement or splice intronic variations (five cases). Four pathogenic intronic variations, including three novel ones, were detected. Among them, the prevalence of one of them (NM₀₀₀₂₅₆.3:c.1927+ 600 C>T) was estimated at about 0.35% by the screening of 1,040 unrelated HCM individuals. This study suggests that deep MYBPC3 splice mutations account for a significant proportion of HCM cases (6.5% of this cohort). Consequently, NGS sequencing of MYBPC3 intronic sequences have to be performed systematically. Show less

Loss of epithelial polarity and gain in invasiveness by carcinoma cells are critical events in the aggressive progression of cancers and depend on phenotypic transition programs such as the epithelial-to-mesenchymal transition (EMT). Many studies have reported the aberrant expression of voltage-gated sodium channels (Na Show less

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease with an estimated prevalence of 1/500. More than 40 genes have been reported to cause HCM. Among them, CSRP3 is usually included on HCM gene panels used for molecular diagnosis by next-generation sequencing (NGS). To provide new insights into the pathophysiology of hypertrophic cardiomyopathy, a NGS workflow based on a panel of 48 cardiomyopathies-causing genes was analyzed on a cohort of 542 HCM patients. As expected, this molecular approach led to identify most pathogenic or likely pathogenic variants into prevalent HCM-causing genes: MYBPC3 (123/542; 22.7%), MYH7 (48/542; 8.9%), TNNT2 (12/542; 2.2%), and TNNI3 (10/542; 1.8%). Among MYBPC3 variants, 96 led to a premature stop codon (78%). More surprisingly, our molecular study led also to detect, for the first time, homozygous CSRP3 truncating variants in two unrelated HCM probands. Meta-analysis of rare previously reported CSRP3 variants on HCM probands using ACMG guidelines indicate that only one variation (p.Cys58Gly) could be considered as likely pathogen. By combining meta-analysis results and identification of two unrelated HCM patients with homozygous CSRP3 truncating variants, we suggest that the association of CSRP3 as a validated HCM-causing gene require additional studies and those CSRP3 variants could result in HCM with an autosomal recessive inheritance rather than with an autosomal dominant transmission as usually reported on HCM (OMIM 612124). Show less

Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches. Show less

Hypertrophic cardiomyopathy (HCM), a common and clinically heterogeneous disease characterized by unexplained ventricular myocardial hypertrophy and a high risk of sudden cardiac death, is mostly caused by mutations in MYH7 and MYBPC3 genes. As 70% of MYBPC3 mutations introduce a premature termination codon, the purpose of the current study was to report the prevalence of large MYBPC3 rearrangements. A large French cohort of 100 HCM patients, for whom no putatively causative point mutations were identified previously in the most prevalent HCM-causing genes, was investigated using an MLPA methodology. One HCM patient was identified to carry a large MYBPC3 rearrangement (<1%). This patient presents a 3505-bp deletion, which begins in the intron 27 and ends 485 bp after the MYBPC3 stop codon (g.47309385₄₇₃₁₂₈₈₉del). It was originated by recombination of a 296 bp AluSz sequence located in intron 27 and a 300 bp AluSx sequence located immediately downstream of exon 35. This study allowed the characterization of the first large MYBPC3 deletion reported in the literature. However, it appears that MLPA strategy, that moderates the identification of large MYBPC3 rearrangements, might confirm a clinical diagnosis only in a small number of patients (<1%). Show less

Hypertrophic Cardiomyopathy (HCM), a common and clinically heterogeneous disease characterized by unexplained ventricular myocardial hypertrophy and a high risk of sudden cardiac death, is mostly caused by mutations in sarcomeric genes but modifiers genes may also modulate the phenotypic expression of HCM mutations. The aim of the current study was to report the frequency of single and multiple gene mutations in a large French cohort of HCM patients and to evaluate the influence of polymorphisms previously suggested to be potential disease modifiers in this myocardial pathology. We report the molecular screening of 192 unrelated HCM patients using denaturing high-performance liquid chromatography/sequencing analysis of the MYBPC3, MYH7, TNNT2 and TNNI3 genes. Genotyping of 6 gene polymorphisms previously reported as putative HCM modifiers (5 RAAS polymorphisms and TNF-α -308 G/A) was also performed. Seventy-five mutations were identified in 92 index patients (48%); 32 were novel. MYBPC3 mutations (25%) represent the most prevalent cause of inherited HCM whereas MYH7 mutations (12%) rank second in the pathogenesis. The onset age was older in patients carrying MYBPC3 mutations than in those with MYH7 mutations. The MYBPC3 IVS20-2A>G splice mutation was identified in 7% of our HCM population. Multiple gene mutations were identified in 9 probands (5%), highlighting the importance of screening other HCM-causing genes even after a first mutation has been identified, particularly in young patients with a severe phenotype. No single or cumulative genetic modifier effect could be evidenced in this HCM cohort. Show less