Biology subjects

Cancellieri, S.

Publications and source records attributed to Cancellieri, S..

3 recordsLinked to original sources

bioRxiv2026

NFATC2 in pancreatic cancer-associated fibroblasts predicts treatment response and facilitates ERBB-targeted therapies

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy, with therapeutic resistance influenced by a dense desmoplastic stroma dominated by cancer-associated fibroblasts (CAF). Using single-cell RNA-sequencing and gene regulatory network modeling of 42 PDAC tumors, we identified a CAF subpopulation characterized by elevated NFATC2 expression that is enriched in patients with improved therapeutic response and survival. NFATC2+ CAFs exhibited tumor-suppressive features, including enhanced apoptotic signaling and suppression of ERBB pathway activity. Co-culture experiments demonstrated that NFATC2+ CAFs restrain pancreatic cancer cell growth and enhance chemotherapy-induced apoptosis, increasing sensitivity to standard-of-care chemotherapy regimens and synergizing with ERBB-targeted therapies. The favorable effect of NFATC2+ CAFs on chemotherapy response was validated in two other PDAC cohorts and in rectal cancer. Together, these findings identify NFATC2+ CAFs as a therapy-conditioned stromal state linked to improved treatment response and uncover a context-dependent vulnerability within the tumor microenvironment that may be exploited to rationally optimize combination therapies.

cancer biology↗

bioRxiv2026

Classical enhancers couple cis-regulatory logic with transcriptional condensates and 3D genome architecture

Deciphering the regulatory logic of enhancers remains a central question in understanding cell- and tissue-specific gene expression in multicellular organisms. This is particularly pertinent at multipartite enhancer clusters such as super-enhancers, where multiple enhancers contribute to the expression of a single gene. Gene expression has been studied largely through sequence-dependent recruitment of transcription factors (TF) and co-activators, whereas 3D chromatin structure has been attributed to architectural proteins such as cohesion and CTCF1-3. However, the contribution of DNA sequence encoded in enhancers to shaping higher-order genome organization remains poorly understood. Here we show that classical enhancers, embedded within multipartite super-enhancer structures, act as determinant regulatory elements that initiate the gene regulatory cascade by linking DNA sequence recognition to 3D chromatin architecture. Classical enhancers are more evolutionarily conserved and display stronger regulatory activity than facilitator elements, which lack intrinsic enhancer activity but potentiate classical enhancer function. We show that classical enhancers are selectively bound by specific TFs with strong intrinsically disordered regions, such as NFE2L2 in liver cancer cells, capable of driving transcriptional condensate formation through phase separation. NFE2L2 depletion reduced enhancer activity and induced widespread chromatin reorganization, characterized by increased cohesin and CTCF occupancy at super-enhancer boundaries and beyond. This "cohesin clogging" impaired DNA loop extrusion, led to formation of smaller topologically associated domains, and weakened enhancer-promoter contacts. These findings highlight that sequence-specific TFs have multifaceted roles beyond transcriptional control, establishing a direct mechanistic link between enhancer sequence, TF binding, condensate formation, and 3D genome organization, with the regulatory logic being encoded in the DNA sequence itself.

genomics↗

bioRxiv2021

Human genetic diversity modifies therapeutic gene editing off-target potential

CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to nominate and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and {beta}-thalassemia1. We find that the top candidate off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF)=4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels ([~]9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. The CRISPRme tool highlights alternative allele-specific off-target editing as a prevalent risk of gRNAs considered for therapeutic gene editing. Our report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We suggest that variant-aware off-target assessment should be considered in therapeutic genome editing efforts going forward and provide a powerful approach for comprehensive off-target nomination.

genetics↗