PROJECT 3 ABSTRACT PI: Ronald Hoffman, MD First generation JAK2 inhibitor therapy is currently widely employed to treat myelofibrosis (MF) patients. Its use, however, has not substantially altered the risk for disease progression or interrupted the inevitable evolution to MPN-blast phase (MPN-BP), likely due to an inability to selectively deplete MF stem/progenitor cells (HSPC). MF HSPCs are characterized by upregulation of HDM2 which promotes the proteosomal degradation of wild- type (WT) p53. We have focused on implementing strategies that upregulate WT p53 activity in order to deplete MF HSPCs. We subsequently have shown that treatment with the HDM2 antagonist, nutlin-3 antagonizes the interaction between WT p53 and HDM2 resulting in the upregulation of p53 and the depletion of MF but not normal HSCs. Furthermore, p53 dependent signaling is known to play a role in tumor suppression by acting in a non-cell autonomous manner, such that down regulation of p53 leads to upregulation of NFκB resulting in the increased elaboration of abundant amounts of pro-inflammatory cytokines. Subsequent trials of nutlin therapy in MPN patients have resulted in an improvement not only in clinical parameters and symptom scores but also rapid reductions in the variant allele frequencies of MPN driver mutations and other associated myeloid gene mutations, as well as the reversal of marrow fibrosis. We hypothesize that HDM2 antagonist therapy actually depletes MF HSPCs and downregulates NFκB in MF cells and components of the tumor microenvironment. The long-term administration of HDM2 antagonists has, however, proven problematic due to gastrointestinal toxicity. MPN p53 activity can also be downregulated by activating mutations or overexpression of PPM1D, a phosphatase which dephosphorylates p53. To further optimize the therapeutic upregulation of WT p53 and possibly lessen the gastrointestinal toxicity associated with HDM2 antagonist therapy, we hypothesize that adding drugs that either reduce PPMID activity or antagonize the action of BET proteins (epigenetic regulators which downregulate events downstream of p53) will result in further upregulation of p53 activity, greater depletion of MF HSPCs and additional correction of the MF tumor microenvironment. Since HDM2 can also be recruited to chromatin in a p53 independent manner to increase NFκB, HIF1α, and polycomb repressor complex 2 activity, as well as orchestrate the global metabolic reprogramming of cancer cells, we hypothesize that agents that effectively eliminate both the p53 dependent and independent consequences of increased HDM2 copy number, might result in even greater depletion of MF HSPCs. To test these hypotheses, we will pursue the following Specific Aims (1) Determine the degree of malignant MF HSCs depletion achieved by up-regulating WT p53 activity with single agent HDM2 antagonist treatment; (2) Determine if dual targeting of HDM2 and PPM1D or HDM2 and BRD4 leads to further upregulation of p53 ac...