One of the most common causes of treatment failure in T-cell leukemia is dissemination of malignant cells. The overarching goal of this proposal is to test whether inhibition of CDK6 kinase holds promise as an effective therapeutic strategy for treatment of T-cell leukemia dissemination. Cyclin D3/CDK6 is the major type of cyclin D/CDK in T-cell acute lymphoblastic leukemia (T-ALL), and kinase activity of CDK6 in T-ALL is dramatically enhanced as its inhibitor proteins are mutated in over 50% of T-ALL cases. Targeting CDK6 for T-ALL therapy is promising as it prevents leukemia cell proliferation and induces T- ALL apoptosis. It is not clear whether or how CDK6 regulates T-ALL dissemination. In this study, we will elucidate the novel regulatory mechanism of cyclin D3/CDK6 in T-cell leukemia dissemination. We obtained substantial preliminary evidence to show that CDK6 plays an important role in T-ALL dissemination by regulating nuclear translocation and phosphorylation of PFKP. We also found that CDK6-dependent nuclear enrichment of PFKP may have a prognostic impact in T-cell lymphoma/leukemia. In the proposed work, we will extend these findings. In Aim 1, we will examine how cyclin D3/CDK6 regulates PFKP nuclear translocation to promote leukemia invasion. In Aim 2, we will determine how CDK6 mediated PFKP phosphorylation increases CXCR4 and PD-L1 expression to enhance leukemia cell dissemination. In Aim 3, we will perform a translational study to test the prognostic value of nuclear PFKP in T-cell lymphoma/leukemia using an expanded clinically well-annotated cohort of T cell lymphoma/leukemia patients, and a pre-clinical study of the therapeutic effect of a CDK6 specific degrader on T-ALL mouse models. The expected overall impact of this proposal is that it may elucidate the molecular function of CDK6 in T-ALL dissemination, and it may lead to novel targeted therapeutic strategies based on CDK6 inhibition.