The key role of the p53 protein in tumor suppression is highlighted by its frequent mutation in human cancers and by the completely penetrant cancer predisposition of p53 null mice. Beyond providing definitive evidence for the critical function of p53 in tumor suppression, genetically engineered mouse models have offered numerous additional insights into p53 function. p53 knock-in mice expressing tumor-derived p53 mutants have revealed that these mutants display gain-of-function activities that actively promote carcinogenesis. The generation of p53 knock-in mutants with alterations in different domains of p53 has helped further elucidate the cellular and biochemical activities of p53 that are most fundamental for tumor suppression. In addition, modulation of p53 post-translational modification (PTM) status by generating p53 knock-in mouse strains with mutations in p53 PTM sites has revealed a subtlety and complexity to p53 regulation. Analyses of mouse models perturbing upstream regulators of p53 have solidified the notion that the p53 pathway can be compromised by means other than direct p53 mutation. Finally, switchable p53 models that allow p53 reactivation in tumors have helped evaluate the potential of p53 restoration therapy for cancer treatment. Collectively, mouse models have greatly enhanced our understanding of physiological p53 function and will continue to provide new biological and clinical insights in future investigations.
Regulated expression of miRNAs influences development in a wide variety of contexts. We report here that miR290-5p (100049710) and miR292-5p (100049711) are induced at the pre-B stage of murine B cell development and that they influence assembly of the Igκ light chain gene (243469) by contributing to the activation of germline Igκ transcription (κGT). We found that upon forced over-expression of miR290-5p/292-5p in Abelson Murine Leukemia Virus (AMuLV) transformed pro-B cells, two known activators of κGT, E2A (21423) and NF-κB (19697), show increased chromosomal binding to the kappa intronic enhancer. Conversely, knockdown of miR290-5p/292-5p in AMuLV pro-B cells blunts drug-induced activation of κGT. Furthermore, miR290-5p/292-5p knockdown also diminishes κGT activation, but not Rag1/2 (19373, 19374) expression, in an IL-7 dependent primary pro-B cell culture system. In addition, we identified a deficiency in κGT induction in miR290 cluster knockout mice. We hypothesize that increased expression of miR290-5p and miR292-5p contributes to the induction of κGT at the pre-B stage of B cell development through increased binding of NF-κB and E2A to kappa locus regulatory sequences.
To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation.
e14551 Background: An allogeneic approach for CAR T-Cell therapy can significantly streamline manufacturing, provide more accessible options to patients as well as enhance safety by reducing the possibility of graft-versus-host-disease. The T-Cell Receptor (TCR) is comprised of multiple subunits and functions to activate T-cells by a signal transduction cascade that is initiated upon antigen binding. Eliminating expression of the endogenous TCR on modified CAR-T-Cells may eliminate the ability to recognize major and minor histocompatibility antigens in the recipient. This study assessed if simultaneous expression of multiple short hairpin RNAs that knockdown levels of individual TCR subunits could result in the loss of TCR expression and TCR-mediated T-Cell activation. Methods: Recombinant DNA producing short hairpin RNAs against the TCR complex was transfected into T-cells. Cell surface TCR was analyzed by FACS. Following CD3 activation or co-culture with B-cells, T-Cell activation was quantified by measuring the levels of IL-2 by ELISA and QPCR. Results: When expressed individually, each shRNA inhibited it’s cognate TCR subunit by up to 93% of the endogenous levels. However, upon simultaneous expression of the shRNAs against the different subunits from the same vector, we observed a near complete depletion of the TCR complex from the cell surface ( > 99%) as measured by FACS analyses. Furthermore, TCR functionality was inhibited when treated cells were stimulated with either CD3 or in B cell co-cultures with Staphylococcal enterotoxins. IL-2 secretion was inhibited to undetectable levels by ELISA by the multi-shRNA treatment and > 98% by qPCR. Conclusions: Though knockdown of any single component of TCR never exceeded 93%, simultaneous knockdown of several TCR subunits abrogated surface TCR and downstream activation suggesting that disruption of stoichiometric expression the subunits prevented TCR formation. The small size of the shRNA expression cassette ( < 2 Kb) permits co-expression from the same lentiviral vector as the CAR. Altogether, these data point to a viable strategy towards generating a single vector approach for the production of allogeneic T-Cells for immunotherapies against certain cancers.
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