The goal of this project is to determine the role that mutations in the PPM1D gene has in the development of myelodysplastic syndrome (MDS), and how the mutations affect the PPM1D protein. PPM1D is a gene that is commonly mutated in MDS, particularly in individuals who have received prior chemotherapy, so called therapy related MDS (t-MDS). t-MDS is particularly difficult to treat, and new biological insights and therapies are urgently required. To achieve these goals, I proposed using two novel mouse models of PPM1D, one in which the gene is removed, and one in which the gene is mutated and therefore activated, to study the role of PPM1D in blood cell and MDS development. I also proposed to define the mechanism by which mutations in PPM1D lead to activation of the protein.

In the past year, I have continued to make significant progress in year three in both aims of this grant (see section III). This includes a co-first author publication in Nature Communications describing the structure and function of PPM1D.  For the mouse models (Aim 1), I have now successfully assessed the role of PPM1D in hematopoiesis at baseline, in the presence of chemotherapy, in the presence of radiation, and in the presence of inflammatory stress. I have been able to do so with multiple different mouse models including both the Ppm1d activating mutation and the Ppm1d knockout, with both constitutive Cre-recombinases (Vav-Cre) and inducible ones (Mx-Cre). I have also compared these phenotypes with P53 models (the mouse Trp53- R172H) in direct competition and by creating compound genetic backgrounds. I have performed pilot experiments to introduce NRAS into the mouse models to try to model neoplasia and am currently setting up genetic crosses with other mouse models of myeloid neoplasia to determine how PPM1D influences these processes.

For the role of the ubiquitin proteasome system in regulating PPM1D (Aim 2), we have been unable to confirm that UBE3C regulates PPM1D levels specifically through its N-terminus (as opposed to a more general biology, perhaps related to the unfolded protein response). We are continuing to determine if this is indeed the case. In the meantime we have repeated multiple CRISPR screens using our PPM1D reporter systems and are now pursuing additional hits to determine if they are E3 ligases the ubiquitinate PPM1D. In parallel we have completed our generation of a series of PPM1D truncation reporter vectors to determine which amino acids in the C-terminus are regulating PPM1D degradation and will be performing the full experiment in the next 2-3 months. Finally, we have generated additional reporter and tagged vectors to look for proteins that interact with PPM1D.