PROJECT SUMMARY/ABSTRACT Acute lymphoblastic leukemia (ALL) is an aggressive blood cancer with high unmet needs, as high cure rates achieved with intensive chemoradiation come at the expense of high toxicity rates and risk of secondary malignancies. Still, up to 25% pediatric ALL patients fail or relapse post-frontline chemoradiation, while response rates for relapsed disease are dismal (~20%). Whole-genome sequencing capabilities have shed new light on oncogenic processes, but identifying “clean” targets remain elusive (easily druggable proteins, dysregulated in cancer but not in healthy cells). We hypothesize that oncogenic transformation is driven by aberrant activity of oncogene-associated chromatin (epigenetic) modifying partners. These changes create a chromatin environment unique to the malignant state. Our preliminary data, including the ones published in Clinical Cancer Research, provides strong evidence that two chromatin oncogenic partners in ALL, JMJD3, an epigenetic player coopted by oncogenes such as NOTCH1, and the deubiquitinase USP7, represent novel therapeutic targets in T-ALL. USP7 appears to control tumor growth and JMJD3 and NOTCH1 stabilization is at least one potential mechanism. Inhibition of each of JMJD3 and USP7 using small molecules appears to significantly inhibit growth of leukemia cells in vitro. We propose to further characterize the pro-oncogenic roles of JMJD3 and USP7 in relevant human and murine models, model USP7 mutations in cancer and conduct preclinical analyses of small molecule inhibitors against these two targets (used as single agents or in combinations). Other mechanistic studies will include a proteomic screen for USP7 interactors and global histone changes. We will evaluate study genomic and chemical inhibition of JMJD3 and USP7 (individual and combined), in human T-ALL lines, primary patient samples, and mouse:human xenograft disease models with respect to: 1) transcriptional and epigenetic changes, 2) cellular viability, and 3) animal survival. Absent any targeted therapies currently in use for T cell ALL, identification of two novel targets for inhibitor cocktails could provide a useful concept for future drug development. Our biopharma collaborators have already generated chemical inhibitors for both JMJD3 and USP7. We anticipate our mechanistic analyses could serve as solid foundation for future toxicology analyses, along with further chemical optimization.