Biology subjects

Gonzalez, E. A.

Publications and source records attributed to Gonzalez, E. A..

4 recordsLinked to original sources

bioRxiv2026

Pluripotency and Transcriptional Pausing Disrupt Circadian Rhythms to Facilitate the Enrichment of Cancer Stem Cells

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype, enriched for cancer stem cells (CSCs), which are the cause of tumor recurrence. CSCs are responsible for tumor initiation and propagation; however, the molecular mechanisms underlying their formation remain largely unclear. We show that carboplatin treated TNBC cells lose their circadian rhythms and promote the enrichment of CSCs. Notably, genetic ablation of the circadian clock by itself, without carboplatin treatment, facilitated the formation of CSCs along with their ability to generate 3D tumorspheres and mouse tumors enriched with CSCs. Mechanistically, we identified an antagonistic interplay between the circadian clock and pluripotency, whereby the core pluripotent factor OCT4 disrupts the expression of circadian timekeeping genes to disable the circadian clock. Furthermore, we uncovered a transcriptional pausing program, controlling the circadian clock, to be perturbed in carboplatin treated TNBC cells. Concordantly, based on gene expression analysis from The Cancer Genome Atlas (TCGA), we found that the uncoupling of transcriptional pausing and the circadian clock correlated with low survivability across diverse cancer types. Moreover, cancer patients with poor prognosis exhibit low expression of the core timekeeping genes Clock, Npas2, Bmal1 and Rorc. Lastly, we restored circadian rhythms in Oct4 deficient TNBC cells and impaired their ability to generate tumorspheres and decreased the number of CSCs in mouse tumors. Overall, our findings demonstrate an unprecedented mechanism for the formation of CSCs that is dependent on the loss of circadian rhythms and thereby has eminent implications for developing new cancer therapies.

cancer biology↗

bioRxiv2024

CAR-T cells targeting CCR9 and CD1a for the treatment of T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by high rates of induction failure and relapse, and effective targeted immunotherapies are lacking. Despite promising clinical progress with genome-edited CD7-directed CAR-T cells, which present significant logistical and regulatory issues, CAR-T cell therapy in T-ALL remains challenging due to the shared antigen expression between malignant and healthy T cells. This can result in CAR-T cell fratricide, T cell aplasia, and the potential for blast contamination during CAR-T cell manufacturing. Recently, CAR-T cells have been described that target non-pan-T antigens, absent on healthy T cells but expressed on specific T-ALL subsets. These antigens include CD1a (NCT05679895), which is expressed in cortical T-ALL, and CCR9. We show that CCR9 is expressed on >70% of T-ALL patients (132/180) and is maintained at relapse, with a safe expression profile in healthy hematopoietic and non-hematopoietic tissues. Further analyses showed that dual targeting of CCR9 and CD1a could benefit [~]86% of patients with T-ALL, with a greater blast coverage than single CAR-T cell treatments. We therefore developed, characterized, and preclinically validated a novel humanized CCR9-specific CAR with robust and specific antileukemic activity as a monotherapy in vitro and in vivo against cell lines, primary T-ALL samples, and patient-derived xenografts. Importantly, CCR9/CD1a dual-targeting CAR-T cells showed higher efficacy than single-targeting CAR-T cells, particularly in T-ALL cases with phenotypically heterogeneous leukemic populations. Dual CCR9/CD1a CAR-T therapy may prevent T cell aplasia and obviate the need for allogeneic transplantation and regulatory-challenging genome engineering approaches in T-ALL.

immunology↗

bioRxiv2024

Integrative single-cell multi-omics of CD19CARpos and CARneg T cells suggest drivers of immunotherapy response in B-cell neoplasias

How phenotypic, clonal, and functional heterogeneity of CAR-T-cells impact clinical outcomes remain understudied. Here, we integrated clonal kinetics with transcriptomic heterogeneity resolved by single-cell omics to explore cellular dynamics response of both non-transduced (CARneg) and transduced (CARpos)T-cells. CARneg and CARposT-cells were longitudinally interrogated in the manufactured infusion product (IP) and in-vivo at CAR-T cell expansion peak in five B-ALL patients treated with CD19CAR-T-cells (varni-cel). Significant differences were found in the cellular dynamics between CARpos and CARnegT-cells in response to therapy. CARposT-cells in the IP exhibited a significant higher CD4:CD8 ratio than CARnegT-cells, and the CD4:CD8 CARposT-cell composition impacted therapy outcome as confirmed in a larger cohort of 24 varni-cel-treated B-ALL patients. Conversely, an inverted trend in the CD4:CD8 CARposT-cell ratio was consistently observed at the expansion peak, with clonally expanding CD8+ effector memory and cytotoxic T-cells being the most abundant populations. Expanded cytotoxic CARpos{gamma}{delta}T cells emerged at the expansion peak, and the extent of their in-vivo expansion positively correlated with treatment efficacy, which was validated in a large cohort of B-ALL patients (n=18) treated with varni-cell and B-cell lymphoma patients (n=58) treated with either lisa-cel or axi-cel. Our data provide insights into the complexity and diversity of T-cell responses following CAR-T cell therapy and suggest drivers of immunotherapy response.

immunology↗

bioRxiv2017

Control of Mast Cell Regulated Exocytosis by Munc13 Proteins

Mast cells (MCs) are involved in pathogen defense and inflammatory reactions. Upon stimulation, they release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 proteins play essential roles in regulated exocytosis. We found that MCs express Munc13-2 and -4, and we studied their roles using global and conditional knockout (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and histological evidence of defective MC degranulation, and not with changes in MC development, distribution, numbers or morphology. In vitro assays revealed that the defective MC response in the absence of Munc13-4 was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants with different expression levels of Munc13-4 in their MCs showed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and the total amount of exocytosis follows. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and electron microscopy studies showed that the number of multigranular compound events (granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that while Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full IgE-mediated anaphylactic response.

cell biology↗