Posttranslational modifications (PTMs) of transcription regulatory proteins allow the integration of various signaling and environmental cues into highly dynamic and controlled responses, thereby achieving coordinated gene expression programs essential for cell proliferation or differentiation.The transcription factor BCL11B/CTIP2 was independently isolated as an interacting partner of chicken ovalbumin upstream promoter transcription factor (COUP-TF) in neurons and as a tumor suppressor gene in mouse models of gamma ray-induced thymic lymphomas (1-3). Besides its expression in the central nervous system (CNS), BCL11B was shown to be widely expressed in all T-cell subsets, starting from the double-negative stage 2 (DN2 stage) and to be involved in various aspects of development, function, and survival of T cells (4). Indeed, BCL11B is a focal point essential for several checkpoints involved in T-cell commitment in early progenitors, selection at the DN2 stage, and differentiation of peripheral T cells (5-9). Furthermore, monoallelic BCL11B deletions or missense mutations have been identified in the major molecular subtypes of T-cell acute lymphoblastic leukemia (10). Therefore, these observations together with the occurrence of deletions and mutations in gamma ray-induced thymomas in mice identify BCL11B as a haploinsufficient tumor suppressor gene (11).BCL11B is essential for T-cell development and is considered a "guardian of T cell fate" (12). Its closely related paralog BCL11A is essential for normal lymphopoiesis and hemoglobin switching during erythroid differentiation (13-15). Thus, these two transcription factors appear to be key regulators of fundamental differentiation programs during normal hematopoiesis.BCL11B represses transcription of its target genes through interaction with several chromatin remodelling complexes and notably recruits NuRD complexes (nuclear remodeling and deacetylation complexes) via interaction with MTA1 and MTA2 (4,11,[16][17][18]. Although originally characterized as a sequence-specific transcriptional repressor, BCL11B also behaves as a context-dependent transcriptional activator of the IL-2 and Cot kinase genes in CD4 ϩ T-cell activation (19, 20). This dual behavior of BCL11B as a transcriptional repressor and activator is not fully understood but clearly relies on a dynamic cross talk between BCL11B PTMs. Indeed, mass spectrometry analyses of thymocytes isolated from 4-to 8-week-old mice and stimulated with a mixture of phorbol ester and calcium ionophore used as an in vitro model mimicking T-cell receptor (TCR) activation identified several mitogen-activated protein kinase (MAPK) phosphorylation sites of BCL11B and confirmed its

The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor mediating the p53-dependent apoptotic response to irreparable DNA double-strand breaks (DSBs) through direct transcriptional repression of SIRT1. HIC1 is also essential for DSB repair as silencing of endogenous HIC1 in BJ-hTERT fibroblasts significantly delays DNA repair in functional Comet assays. HIC1 SUMOylation favours its interaction with MTA1, a component of NuRD complexes. In contrast with irreparable DSBs induced by 16-hours of etoposide treatment, we show that repairable DSBs induced by 1 h etoposide treatment do not increase HIC1 SUMOylation or its interaction with MTA1. Furthermore, HIC1 SUMOylation is dispensable for DNA repair since the non-SUMOylatable E316A mutant is as efficient as wt HIC1 in Comet assays. Upon induction of irreparable DSBs, the ATM-mediated increase of HIC1 SUMOylation is independent of its effector kinase Chk2. Moreover, irreparable DSBs strongly increase both the interaction of HIC1 with MTA1 and MTA3 and their binding to the SIRT1 promoter. To characterize the molecular mechanisms sustained by this increased repression potential, we established global expression profiles of BJ-hTERT fibroblasts transfected with HIC1-siRNA or control siRNA and treated or not with etoposide. We identified 475 genes potentially repressed by HIC1 with cell death and cell cycle as the main cellular functions identified by pathway analysis. Among them, CXCL12, EPHA4, TGFβR3 and TRIB2, also known as MTA1 target-genes, were validated by qRT-PCR analyses. Thus, our data demonstrate that HIC1 SUMOylation is important for the transcriptional response to non-repairable DSBs but dispensable for DNA repair.

Control of cell death/survival balance is an important feature to maintain tissue homeostasis. Dependence receptors are able to induce either survival or cell death in presence or absence of their ligand, respectively. However, their precise mechanism of action and their physiological importance are still elusive for most of them including the MET receptor. We evidence that pro-apoptotic fragment generated by caspase cleavage of MET localizes to the mitochondria-associated membrane region. This fragment triggers a calcium transfer from endoplasmic reticulum to mitochondria, which is instrumental for the apoptotic action of the receptor. Knock-in mice bearing a mutation of MET caspase cleavage site highlighted that p40MET production is important for FAS-driven hepatocyte apoptosis, and demonstrate that MET acts as a dependence receptor in vivo. Our data shed light on new signaling mechanisms for dependence receptors’ control of cell survival/death balance, which may offer new clues for the pathophysiology of epithelial structures.

Exon skipping mutations of the MET receptor tyrosine kinase (METex14), increasingly reported in cancers, occur in 3–4% of non–small‐cell lung cancer (NSCLC). Only 50% of patients have a beneficial response to treatment with MET‐tyrosine kinase inhibitors (TKIs), underlying the need to understand the mechanism of METex14 oncogenicity and sensitivity to TKIs. Whether METex14 is a driver mutation and whether it requires hepatocyte growth factor (HGF) for its oncogenicity in a range of in vitro functions and in vivo has not been fully elucidated from previous preclinical models. Using CRISPR/Cas9, we developed a METex14/WT isogenic model in nontransformed human lung cells and report that the METex14 single alteration was sufficient to drive MET‐dependent in vitro anchorage‐independent survival and motility and in vivo tumorigenesis, sensitising tumours to MET‐TKIs. However, we also show that human HGF (hHGF) is required, as demonstrated in vivo using a humanised HGF knock‐in strain of mice and further detected in tumour cells of METex14 NSCLC patient samples. Our results also suggest that METex14 oncogenicity is not a consequence of an escape from degradation in our cell model. Thus, we developed a valuable model for preclinical studies and present results that have potential clinical implication.

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Mutations affecting exon 14 splice sites of the gene encoding the MET receptor have been recently revealed in non-small cell lung cancer (NSCLC). These mutations induce MET exon 14 skipping (METex14), leading to receptor activation through deletion of a regulatory domain. Importantly, these mutations represent a promising therapeutic opportunity since MET tyrosine kinase inhibitors (TKI) are available. Nevertheless, these mutations raise several scientific and clinical questions. (i) Their functional consequences are still poorly understood,(ii) these mutations are highly heterogeneous which makes them difficult to detect, and (iii) efficacy of MET-TKI seems limited by largely unknown resistances. To address these issues, we first created by genome editing a panel of pulmonary cells expressing either METex14 or MET receptor mutated in each known active site of the regulatory domain. Comparison of signalling pathways, transcriptional landscapes and cellular responses revealed that METex14 activation is recapitulated by mutation of the CBL binding site involved in MET internalization, but provide also an unexpected resistance to apoptosis through abrogating its caspase cleavage. Second, in order to detect METex14 mutations in clinical routine practice, we developed an optimized targeted next generation sequencing panel covering the METex14 in addition to the usual targets. This panel revealed METex14 alterations in 2.2% NSCLC patients and presence of various concurrent alterations. Third, by further characterization of the concurrent alterations, we found high rate of PI3K pathway alterations in METex14 patients. In addition, MET-TKI treatment in 3 patients harboring these alterations had shown progressive disease, suggesting their involvement in resistance. Using a patient-derived cell line with primary resistance and cell lines in which MET or PI3K alterations were inserted, we confirmed involvement of PI3K activation in the resistance process, which was overcome with PI3K inhibitor. Overall, our integrated study reveals that METex14 mutations induce an original activation involving cooperation between regulatory mechanisms, but offering sensitivity to MET-TKI. Therefore, these mutations, now detectable in routine practice, are druggable by MET-TKI providing a novel therapeutic line for NSCLC, but have to face to innate resistance including PI3K alterations. Citation Format: Marie Fernandes, Philippe Jamme, Sonia Paget, Angela Morabito, Frédéric Leprêtre, Martin Figeac, Clotilde Descarpentries, Fabienne Escande, Simon Baldacci, Anne Chotteau-Lelièvre, Luca Grumolato, Marie-Christine Copin, Zoulika Kherrouche, Alexis B. Cortot, David Tulasne. MET exon 14 skipping mutations in lung cancer: Screening, functional and clinical impact [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3683.