Gottumukkala, N. V. | Search bioRxiv

bioRxiv2026

RASAL3 regulates RAC/CDC42 GTPases, SAPK/JNK signaling, IL-2 gene activity, and directed motility in human T cells

RASAL3 acts as a negative regulator of small cellular GTPases in hematopoietic cells. In immune cells, it primarily modulates the RAS/MAPK signaling pathway and affects cellular events including proliferation, differentiation, survival, and migration. Due to its inhibitory role in T cells, RASAL3 may represent a potential modulatory target for improving therapeutic strategies such as cell-based immunotherapy. However, most existing knowledge about RASAL3 function is derived from murine models, and its role in human T-cell signaling remains insufficiently characterized. To address this gap, we systematically investigated the function of RASAL3 in human primary T cells and T-cell line. For this purpose, we employed RASAL3 overexpression, CRISPR/Cas9-mediated deletion, and siRNA-mediated knockdown to thoroughly analyze the effects of RASAL3 on T-cell signaling, proliferation, and migration. Our data demonstrate that RASAL3 modulates primarily CDC42 and RAC1/RAC2 GTPases activity, SAPK/JNK phosphorylation, c-Fos and c-Jun expression, and IL-2 gene promoter activation. In addition, RASAL3 regulates actin polymerization and T-cell migration. Notably, loss of RASAL3 increases Jurkat T cells motility in vivo and potentiates their homing to the spleen. Collectively, these findings identify RASAL3 as an important regulator of human T-cell activation and motility and highlight its application potential for improving CAR-T cell therapy.

cell biology↗

bioRxiv2026

Genome-wide CRISPR Screening Reveals Cullin-1 as a Therapeutic Target Enhancing Efficacy of CD19-directed Immunotherapy

Cancer immunotherapy targeting B-cell specific CD19 antigen meant a major breakthrough in the treatment of B-cell malignancies. Yet, vast proportion of treated patients experience relapse and failure of the therapy. Although multiple mechanisms of CD19-immunotherapy failure have been described, CD19-negative relapses represent the major hurdle in achieving higher and durable response rates. Our established in vitro co-culture models revealed that suboptimal CAR-T cell performance, inefficient to mediate target cell killing, results in robust downregulation of CD19 target antigen. Using genome-wide CRISPR screening, we addressed the mechanisms responsible for such CD19 downregulation and identified Cullin-1 and CD81 playing instrumental role in negative and positive regulation of CD19 expression, respectively. Inhibiting Cullin-1 activity with pevonedistat prevents the loss of CD19 under immunotherapeutic pressure, results in higher CD19 surface levels and consequently enhances the efficacy of target cell killing by CD19-CAR-T cells, CD19-CAR-NK cells and CD19-targeting antibody treatment. Mechanistically, we show that pevonedistat blocks the degradation of CD19 upon its internalization and allows its recycling back to the plasma membrane. CD81 chaperone protein is critically involved in this process as the absence of CD81 abrogates the effect of pevonedistat. In summary, we identify Cullin-1 as a novel and druggable regulator of CD19 protein stability. Cullin-1 inhibition augments CD19 surface expression, thereby improving the efficiency of CD19-targeting immunotherapies, thus arguing for potential incorporation of pevonedistat into novel combination therapies. Key PointsO_LICullin-1 inhibition stabilizes CD19 surface expression, preventing its loss under immunotherapeutic pressure C_LIO_LIPevonedistat treatment enhances the efficacy of CD19-CAR-T cells, CAR-NK cells and CD19-targeting antibodies C_LI

cancer biology↗