Protecting Normal Intestinal Cells From Chemotherapy: PUMA

How to spare normal intestinal cells in chemotherapy. Interfering with the p53-PUMA axis by repressing PUMA can protect the normal cells while leaving p53-mediated killing of cancer cells intact.  This presumably is for p53 wild-type cells.  Many cancers lose p53 expression and for those this would likely be irrelevant.  Abstract:

The gastrointestinal (GI) epithelium is the fastest renewing adult tissue and is maintained by tissue-specific stem cells. Treatment-induced GI side effects are a major dose-limiting factor for chemotherapy and abdominal radiotherapy and can decrease the quality of life in cancer patients and survivors. p53 is a key regulator of the DNA damage response, and its activation results in stimulus- and cell type-specific outcomes via distinct effectors. We demonstrate that p53-dependent PUMA induction mediates chemotherapy-induced intestinal injury in mice. Genetic ablation of Puma, but not of p53, protects against chemotherapy-induced lethal GI injury. Blocking chemotherapy-induced loss of LGR5+ stem cells by Puma KO or a small-molecule PUMA inhibitor (PUMAi) prevents perturbation of the stem cell niche, rapid activation of WNT and NOTCH signaling, and stem cell exhaustion during repeated exposures. PUMAi also protects human and mouse colonic organoids against chemotherapy-induced apoptosis and damage but does not protect cancer cells in vitro or in vivo. Therefore, targeting PUMA is a promising strategy for normal intestinal chemoprotection because it selectively blocks p53-dependent stem cell loss but leaves p53-dependent protective effects intact.

p53 Inhibits CRISPR-Cas9 Engineering In Human Pluripotent Stem Cells

Human pluripotent stem cells are difficult to engineer with the CRSPR system compared to other cell types, which is unfortunate given the great potential in being able to modify human stem cells.  The p53 gene protects against cancer and it seems that p53 function interferes with CRISPR modification of these human stem cell types by making the genetic alterations induced by CRISPR relatively toxic to the cells.  Human stem cells can acquire p53 mutations – which can lead to cancer – and it would seem that human stem cells that have been successfully engineered with CRISPR may be at higher risk for having p53 mutations since the presence of such mutations, inactivating toxicity to CRISPR, may have been the reason the cells were successfully engineered in the first place.  This is an important finding suggesting caution in using CRISPR in such cells and also suggests the need to find novel approaches that can increase CRISPR efficiency in these cells while still maintaining p53 and its anti-cancer functions.  Abstract:

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells1-3. Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells3-13. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction 3 . The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations 14 , cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.