Background: Apoptosis is a genetically programmed process of cell death, critical for development and homeostasis in the adult. The mitochondrial-mediated process of apoptosis is regulated by the BCL-2 (B-cell lymphoma 2) protein family, which includes pro-survival and pro-apoptotic members. The pro-apoptotic BH3 (Bcl-2 homolog region [BH] 3)-only proteins (BIM, PUMA, BID, BMF, BAD, BIK, NOXA, HRK) are the initial responders of apoptosis and play crucial roles in the killing of malignant cells by diverse anti-cancer agents. Several chemotherapeutics kill malignant cells by activation of the tumour suppressor p53. The BH3-only protein PUMA is a critical mediator of both p53-dependent and independent apoptosis and interacts with all the pro-survival BCL-2 family members. We aim to understand the regulation of PUMA by both p53-dependent and independent mechanisms with the aim to find novel ways to boost PUMA expression in malignant cells to enhance the efficacy of anti-cancer therapies.
Methods: We have engineered a novel reporter mouse that has the tdTomato gene replacing the PUMA encoding region but retains the endogenous mouse promoter and regulatory regions. We can generate mouse models of blood cancer using cells from the reporter mice and use them to monitor PUMA gene induction in malignant cells using FACS and microscopy. We will also identify novel regulators of PUMA expression by performing CRISPR/Cas9 whole genome knockout and activation screens in the reporter tumour cell lines.
Results: We have generated Acute Myeloid Leukemia (AML) tumours from PUMA/Bbc reporter hematopoietic stem cells and have derived cell lines from the tumours. Upregulated expression of PUMA in the AML cells expressing the reporter can be detected after treatment with diverse anti-cancer drugs. Furthermore, we have identified novel regulators of PUMA expression by carrying out a CRISPR gene knockout screen.
Conclusion: We have generated a PUMA dtTomato reporter mouse that allows us to monitor PUMA gene expression. By generating murine AML models carrying the reporter, we have been able to monitor PUMA induction in response to anti-cancer drugs. We also performed CRISPR whole genome knockout screen in the AML cell lines and have identified candidate genes that can induce or repress PUMA expression.