PROJECT SUMMARY Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and a leading cause of cancer-related death during childhood. ALL can arise in both lymphoid lineages, with B-ALL and T-ALL accounting for 85% and 15% of this cancer. T-ALL is associated with more aggressive presenting features and historically inferior treatment outcomes compared to B-ALL. Current T-ALL therapy largely relies on cytotoxic chemotherapeutic agents. In recent years, further intensification of chemotherapy has led to incremental increases in the cure rate of T-ALL, but is likely to have reached a plateau due to excessive toxicities (especially in the relapse setting). Moreover, in relapsed T-ALL, leukemia is markedly resistant to cytotoxic drugs. Unlike high-risk B-ALL for which cellular therapy such as CAR-T is highly effective, there are no immunotherapies available for T-ALL and patients with relapsed disease have a dismal five-year survival rate below 25%. Therefore, novel and molecularly targeted therapeutics are needed to improve both survival and quality of life for children with T-ALL. We have previously observed that in 64 T-ALL cases (43 children and 21 adults) profiled thus far 38% showed a striking sensitivity to dasatinib in vitro. In particular, the proportion of T-ALL sensitive to dasatinib is markedly higher in children than in adults (49% vs 14%, respectively). Dasatinib LC50 (drug concentration that kills 50% of leukemia cells) in these T-ALL cases were on par with that observed in BCR-ABL1 B-ALL. However, none of the dasatinib-sensitive T-ALL cases had ABL fusion nor did they respond to a more ABL-specific inhibitor. We demonstrated that LCK activation and phosphorylation level of its downstream targets are responsible for this sensitivity, and, more importantly, can be used to predict the effectiveness of dasatinib treatment in T-ALL cases. During this project, we will develop a companion diagnostic panel that can be used to predict sensitivity to dasatinib and ponatinib in a personalized manner. The following aims will be completed in the proposal: Aim #1. Comprehensive characterization of phosphorylation and activation state of LCK by LC-MS in T-ALL cells. Aim #2. Development of the PRM-MS and immunoassay-based methods for rapid quantitative analysis of p-LCK, p-CD247, and p-ZAP70. By the completion of this project, a companion diagnostic panel will be developed and validated with cell culture samples. This feasibility portion will enable a much more extensive validation of this personalized diagnostic test in PDX mice models and patient samples in Phase II.