Project Summary Multiple Myeloma (MM) is a plasma cell malignancy characterized by extensive structural and numerical chromosomal abnormalities. By comparing consecutive gene expression profiles (GEP) of samples at baseline, during intensive chemotherapy and at relapse, we have identified chromosomal instability (CIN) genes associated with poor prognosis in MM disease. One of the CIN genes, the serine-threonine kinase NEK2, is the most significantly upregulated gene in myeloma cells in patients in complete remission (CR) with minimal residual disease (MRD) and at relapse early during chemotherapy following tandem transplantations. We have shown that pharmacological or genetic inhibition of NEK2 delays tumor growth and induces cell apoptosis in myeloma and high expression of NEK2 has also been related to poor outcomes in many other cancers. The deubiquitinase (DUB) USP7 stabilizes NEK2 protein resulting in the activation of the NF- ?B/heparanase pathway, also involved in bone destruction. However, it is unknown whether NEK2 also activates USP7. In addition, we have found that NEK2 negatively regulates PD-L1 expression, most likely via epigenetic modification. High-risk myeloma cells show low levels of PD-L1 expression and are resistant to immune checkpoint blockade. Therefore, we hypothesize that NEK2 promotes myeloma cell survival, drug resistance, and disease progression by activating the deubiquitinase USP7 and the histone methyltransferase EZH2. In order to rigorously test this hypothesis, we will: (1) Identify the major deubiquitination targets regulated by the Nek2-Usp7 complex using Nek2-transgenic mice with varying levels of Nek2 expression and controls. We will focus on selecting probable proteins as a proof-of-principle to establish the role of Usp7 as the signaling hub in this complex. (2) Determine the mechanisms by which NEK2 inhibits responses to immune checkpoint blockade in myeloma cells using Nek2-knockout mice and biochemical approaches. And, (3) Develop novel immune therapies for drug-resistant and relapsed myelomas using primary myeloma cells, a genetic Nek2-knockout mouse model, and a myeloma mouse model. Supported by strong preliminary data that provide strong rationale for this application, the proposed research is poised to facilitate novel, targeted approaches to the prevention and treatment of myeloma progression and relapse. We predict that our results will extend beyond myeloma and also apply to other hematological and solid tumors.