Abstract 423: Induced proximity and proteolytic targeting of LCK as a novel therapeutic approach in T-cell leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer, with a particularly dismal prognosis in patients with relapsed diseases. Our group recently discovered LCK dependency as a therapeutic vulnerability in 33% of T-ALL and LCK inhibitor dasatinib exhibited strong efficacy in vitro and in vivo (Nat Cancer 2, 284, 2021). However, the transient LCK inhibition by dasatinib resulted in only partial responses, and novel agents are needed to suppress LCK signaling in T-ALL in a sustained fashion to achieve long-term efficacy. We synthesized a set of proteolytic targeting chimeras (PROTACs) that bring LCK to cereblon (CRBN) E3 ligase for ubiquitination and degradation. LCK-PROTACs showed extremely high anti-leukemia sensitivity, i.e., up to 1,561.3 fold increase over dasatinib in LCK-activated T-ALL cell line. Complete LCK degradation was induced by most LCK-PROTACs. Based on solubility, permeability, stability, and anti-leukemia effects in vitro, we prioritized SJ001011646 for further evaluations. SJ001011646 induced 50% LCK degradation at the concentration at ~0.001nM, with superior cytotoxic effects in patient-derived primary T-ALL samples compared to dasatinib. In addition, we confirmed the formation of ternary complex of LCK, SJ001011646, and CRBN, using the AlphaLISA assay. In a wash-out experiment, LCK-dependent T-ALL cells were exposed to SJ001011646 or dasatinib for 18 hours and then monitored for viability recovery. Both dasatinib and SJ001011646 led to dramatic growth inhibition within 2 days. While dasatinib-treated cells started to recover 96 hours after drug removal, cells treated with SJ001011646 failed to recover even after 240 hours. To systematically identify therapeutic targets of SJ001011646, we performed proteomic profiling of T-ALL cells before and after drug treatment in vitro. Overall, 126,670 unique peptides were identified and mapped to 10,158 proteins, of which LCK was most significantly reduced by PROTAC. In parallel, kinome-binding profiling confirmed LCK as a primary target of SJ001011646. Finally, we explored pharmacokinetic and pharmacodynamic properties of SJ001011646 in vivo, using patient-derived xenograft models of T-ALL. Given at the same dosage level as a single injection, SJ001011646 showed 367% increase in the duration of LCK suppression compared to dasatinib. Taken together, we described the development and preclinical evaluation of LCK-targeting PROTACs in T-ALL. Inducing proximity to CRBN and therefore degr