Characterization of the Role of Nemo-like Kinase in Normal and Diamond Blackfan Anemia Models of Erythropoiesis.

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Summary / Abstract Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome associated with physical malformations and defects in early erythroid progenitors. Over 80% of patients carry mutations in one of over twenty ribosomal genes, leading to haploinsufficiency and defective global ribosome biogenesis, but the mechanism by which this leads to erythropoiesis defects is poorly understood. I observed that Nemo-like Kinase (NLK) is activated in erythroid progenitors with ribosome-insufficiency, irrespective of the driving ribosomal mutation. Suppression of NLK improves erythroid expansion of hematopoietic stem and progenitor cells (HSPCs) from DBA patients and mouse models in vitro. The overarching goal of this proposal is to define the role of NLK in the pathogenesis of DBA and identify novel upstream regulators and downstream substrates of NLK. Through the acquisition of new skills in state-of-the-art technologies pioneered by my mentoring team, I foresee the successful resolution of the proposed research aims and the development of the skillset and preliminary data necessary to establish my own independent research program. In Aim 1, I will use CRISPR/Cas9 to knock out NLK in ribosome- insufficient donor HSPCs and examine engraftment after transplantation into recipient mice. As NLK is activated in DBA models irrespective of the driving mutation, this represents a gene therapy approach for autologous stem cell transplantation with the potential to cure the hematological impacts of the disease. In Aim 2, I will identify and characterize downstream substrates of NLK in DBA. In Aim 3, I will identify and characterize deregulated proteins upstream of NLK activation in DBA. The last two aims include characterization of preliminary candidates, complimented by genome wide screens to identify novel factors. Collectively, these studies have the potential to identify new therapeutic targets and improve outcomes for DBA patients. This proposed work will also provide me with the necessary tools and expertise to successfully transition to an independent career. Bench skills I will acquire include mouse stem cell transplantation, CRISPR/Cs9-mediated gene therapy of stem cells, ribosome and mitochondrial biogenesis, translational analysis and genome-wide kinome analysis. Coursework covering bench skills (e.g. RNA biology, applied computational tools, and bioinformatics) and career development (e.g. mentorship, personnel management and faculty transitioning) will compliment guidance from my mentoring team. Dr. Sakamoto has an exemplary track record of producing leaders and is committed to continued guidance as I take on more autonomy. Dr. Sakamoto and Stanford have demonstrated exceptional commitment to my professional development throughout my fellowship and instructor training and I have no doubt their continued support towards our shared goal of developing a world class independent research program dedicated to understanding the pathogenesis of nonmalignant hematological disorders such as DBA.