Biology subjects

Bernasconi, M.

Publications and source records attributed to Bernasconi, M..

4 recordsLinked to original sources

bioRxiv2026

Transcription controls chromatin-nuclear lamina contacts through distinct Lamin A and LBR tethering mechanisms

Lamina-associated domains (LADs) are large genomic regions that interact with the nuclear lamina (NL). Much of the underlying "grammar" governing their positioning at the nuclear periphery remains unclear. LADs are composed of heterochromatin and typically harbor repressed genes, and their association with the NL is generally incompatible with strong transcriptional activity. The extent to which transcription globally shapes chromatin-NL interactions is not fully understood. Here, we combined acute transcription inhibition using Flavopiridol or Triptolide with genome-wide mapping of chromatin-NL contacts. We found that chromatin-NL interactions are rapidly rewired upon transcription inhibition. Changes in chromatin-NL contacts upon transcription shutdown are predictable based on transcriptional activity and the presence of H3K9me3-marked heterochromatin. This rewiring is reversible, as genome-NL interactions quickly return to baseline levels following drug wash-off. Notably, gain and loss of chromatin-NL interactions upon transcription shutdown reflect two distinct tethering mechanisms. Inter-LADs genomic regions (iLADs) enriched in highly active genes and located near stable LADs, which are tethered by Lamin A (LMNA/C), become re-attached to the NL following transcription inhibition. In parallel, H3K9-methylated regions tethered to the nuclear envelope by the Lamin B receptor (LBR) undergo extensive detachment from the NL. Strikingly, LMNA/C and LBR oppositely regulate transcription-sensitive LADs and are required for transcriptional control of chromatin-NL contacts. Together, our findings highlight the plasticity and dynamic nature of chromatin-NL interactions and provide the first evidence that LMNA/C- and LBR-mediated tethering mechanisms exhibit distinct sensitivities to transcription inhibition. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/738400v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@16781a2org.highwire.dtl.DTLVardef@6febd1org.highwire.dtl.DTLVardef@1e3220forg.highwire.dtl.DTLVardef@d137f7_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LITranscription inhibition alters chromatin-NL contacts rapidly and reversibly C_LIO_LIActive transcription prevents inter-LADs located near LMNA/C-tethered LADs from associating with the nuclear lamina. C_LIO_LILBR-tethered heterochromatin is repositioned away from the NL C_LIO_LITranscription-dependent modulation of chromatin-NL contacts is dependent on LMNA/C and partially on LBR C_LI

genomics↗

bioRxiv2026

L1CAM-CAR T cells with enhanced potency overcome low-density antigen expression in rhabdomyosarcoma

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, shows dismal survival in relapsed or metastatic alveolar disease. Chimeric antigen receptor (CAR) T cells are promising but limited by scarce tumor-selective antigens and suboptimal efficacy at low antigen density. We investigated L1 cell adhesion molecule (L1CAM) as a therapeutic target by profiling its expression by flow cytometry, immunoblotting, and immunohistochemistry in cell lines, patient-derived xenografts, and healthy tissues. Using the scFv derived from the CE7 antibody, we engineered L1CAM-CARs with distinct hinge and costimulatory domains and tested them in vitro and in orthotopic RMS mouse models against clinically tested CE7- and B7-H3-CARs. L1CAM was consistently expressed at moderate levels in RMS, especially alveolar subtypes, but very weakly expressed in healthy tissues. Flow cytometry revealed a moderate density typically limiting CAR activity. Among constructs, L1CAM.III (CE7-CAR with long hinge and CD28 domain) showed the strongest cytotoxicity and IFN-{gamma} release. In vivo, L1CAM.III-CAR T cells regressed tumors, prolonged survival, and persisted in orthotopic RMS models, showing greater efficacy in alveolar RMS and no off-tumor activity. These findings establish L1CAM as a rational RMS therapeutic target. Optimized L1CAM.III-CAR T cells overcome moderate antigen density, achieving potent and persistent antitumor activity comparable to B7-H3-CARs but with improved safety. This work supports CAR optimization for clinical translation to broaden pediatric sarcoma immunotherapy.

cancer biology↗

bioRxiv2023

CAR T cells recognizing CD276 and Dual-CAR T cells against CD276/FGFR4 promote rhabdomyosarcoma clearance in an orthotopic mouse model

BackgroundRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activity in vitro and in vivo. MethodsFour different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8 or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were tested in vitro. The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluated in vivo in three orthotopic RMS mouse models. ResultsCD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3z) showed the strongest killing of RMS cells, and the highest release of IFN-{gamma} and Granzyme B in vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3z) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumors in vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276low JR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed of Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice. ConclusionsCD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killing in vitro and eradication of CD276high RMS tumors in vivo. CD276low tumors escaped the therapy showing a correlation of antigen density and effectiveness. FGFR4-CAR Ts showed specific killing in vitro but could only delay RMS growth in vivo. Our results show that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.

cancer biology↗

bioRxiv2023

In vivo single-cell CRISPR uncovers distinct TNF-alphaprograms in clonal expansion and tumorigenesis

The tumor evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions occur frequently in human epithelia and can remodel almost entire tissues, the mechanisms behind why only a small number of clones transform into malignant tumors remain enigmatic. Here, we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing, guide capture and spatial transcriptomics to longitudinally monitor cell type-specific clonal expansions, document their underlying gene programs and contrast clonal expansions from tumor initiation. We uncover a TNF- signaling module that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF- signaling module is downregulated, and instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF- gene program. By analyzing clonally expanded perturbations and their frequency in tumors, we demonstrate that the autocrine TNF- gene program is associated with epithelial-mesenchymal transition (EMT) and is preexistent in a subpopulation of expanded epidermal stem cells, contributing to the predisposition for tumor initiation. Finally, we provide in vivo evidence that the epithelial TNF- gene program is sufficient to mediate invasive properties of epidermal stem cells and show that the TNF- signature correlates with shorter overall survival in human squamous cell carcinoma patients. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues and unveils distinct TNF- programs in tumor evolution. Understanding the biology of clonal expansions in phenotypically normal epithelia and the mechanisms governing their transformation will guide the development of novel strategies for early cancer detection and therapy.

cancer biology↗