Development of Proteolytic Targeting Chimeras to Target Lck in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy for which novel therapies are much needed especially in patients with relapsed diseases. By combining large-scale ex vivo pharmacotype profiling with network-based systems biology analyses, our group recently identified LCK dependency as a therapeutic vulnerability in 44% of T-ALL in children (Nat Cancer 2, 284-299, 2021). LCK inhibitors such as dasatinib exhibit striking anti-leukemia effects in this T-ALL subset. However, the transient LCK inhibition by dasatinib only resulted in incomplete response to monotherapy unless the drug was delivered continuously at a high level. Therefore, it is imperative to develop novel agents that produce sustained suppression of LCK signaling in T-ALL. To this end, we synthesized a set of proteolytic targeting chimeras (PROTACs) that target LCK and cereblon (CRBN) E3 ligase. These PROTACs bind and recruit LCK to CRBN E3 ligase, rendering LCK susceptible to ubiquitination and ultimately proteasomal degradation. Cell viability assay was performed in an LCK-dependent T-ALL cell line KOPT-K1 to determine its sensitivity to this panel of PROTACs. PROTACs showed up to 6.9-fold improvement in cytotoxicity relative to dasatinib. To validate PROTAC-induced LCK degradation, KOPT-K1 cells were treated with PROTACs or dasatinib at 100 nM for 24 hours and LCK protein was quantified by western blotting. LCK degradation occurred rapidly in the presence of PROTAC agents whereas dasatinib treatment did not affect LCK abundance. Furthermore, PROTAC-induced apoptosis of T-ALL cells was abolished by lenalidomide, a CRBN binder, suggesting that their cytotoxic effects were mediated by CRBN-driven LCK degradation. We also validated these findings in patient-derived T-ALL samples. In addition, we determined solubility, permeability, and stability of these PROTACs in vitro. Based on anti-leukemia effects as well as physical chemical properties, we prioritized PROTACs SJ001011646 and SJ001011447 as our top candidates for further evaluations. We hypothesized that the catalytic protein degradation by PROTACs will produce a more sustained suppression of the LCK signaling compared to transient LCK inhibition by dasatinib. To test this, we performed a wash-out assay comparing anti-leukemic effects of top PROTACs with dasatinib. KOPT-K1 cells were treated with vehicle, dasatinib, SJ001011646 and SJ001011447, respectively at 100 nM for 18 hours. Cells were then washed and placed in drug-free culture with viability monitored daily. The vehicle treated cells exhibited an exponential growth while drug treated groups showed dramatic growth inhibition within 2 days. Notably, dasatinib-treated cells continued to undergo apoptosis for 96 hours after drug removal before started to recover. By contrast, SJ001011447 treatment repressed cell growth for 144 hours post wash-out. Most impressively, there was no evidence of growth recovery in cells treated SJ001011646 even 240 hours after drug removal. In addition, we confirmed the formation of ternary complex of LCK, PROTAC, and E3 ligase, using the AlphaLISA assay. To systematically identify therapeutic targets of PROTACs, we performed proteomic profiling of KOPT-K1 cells before and after drug treatment in vitro. Cells were treated with vehicle, SJ001011447 or SJ001011646 at 100 nM for 24 hours before harvested for Tandem Mass Tag-based proteomic profiling. Overall, 126,670 unique peptides were identified and mapped to 10,158 proteins, of which 34 and 35 were significantly changed by SJ001011447 and SJ001011646, respectively (p<0.05, foldchange >2 or <-2). LCK was among proteins most significantly reduced after PROTAC treatment. Finally, we also developed formulation for PROTAC SJ001011646 for in vivo testing; and preclinical pharmacokinetic and pharmacodynamic characterization of this molecule is ongoing using T-ALL xenograft models. In conclusion, we developed LCK-targeting PROTACs with potent anti-leukemia effects. Highly effective in degrading LCK protein, these agents produced sustained LCK suppression superior to small molecule inhibitors, pointing to novel strategies to therapeutically target LCK in T-ALL. Disclosures No relevant conflicts of interest to declare.

Zinc clasp-based reversible toolset for selective metal-mediated protein heterodimerization

Zinc clasp motif derived from natural Zn(ii)-mediated interaction of CD4 co-receptor and Lck protein tyrosine kinase was used for specific and efficient protein heterodimerization. Optimized set of peptide tags forms highly stable complex in the selective heterodimer framework. Utility of obtained toolset demonstrates high specificity, Zn(ii)-dependent reversibility and remarkable kinetic properties.

ZAP-70 Protein Tyrosine Kinase Is Constitutively Targeted to the T Cell Cortex Independently of its SH2 Domains

ZAP-70 is a nonreceptor protein tyrosine kinase that is essential for signaling via the T cell antigen receptor (TCR). ZAP-70 becomes phosphorylated and activated by LCK protein tyrosine kinase after interaction of its two NH2-terminal SH2 domains with tyrosine-phosphorylated subunits of the activated TCR. In this study, the localization of ZAP-70 was investigated by immunofluorescence and confocal microscopy. ZAP-70 was found to be localized to the cell cortex in a diffuse band under the plasma membrane in unstimulated T cells, and this localization was not detectably altered by TCR stimulation. Analysis of mutants indicated that ZAP-70 targeting was independent of its SH2 domains but required its active kinase domain. The specific compartmentalization of ZAP-70 suggests that it may interact with an anchoring protein in the cell cortex via its hinge or kinase domains. It is likely that the maintenance of high concentrations of ZAP-70 at the cell cortex, that only has to move a short distance to interact with phophorylated TCR subunits, facilitates rapid initiation of signaling by the TCR. In addition, as the major increase in tyrosine phosphorylation induced by the TCR also occurs at the cell cortex (Ley, S.C., M. Marsh, C.R. Bebbington, K. Proudfoot, and P. Jordan. 1994. J. Cell. Biol. 125:639–649), ZAP-70 may be localized close to its downstream targets.

Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif

Several members of the Src family of protein tyrosine kinases have a N-terminal dual acylation motif which specifies their myristoylation and S-acylation. These lipid modifications are necessary for correct intracellular localisation to the plasma membrane and to detergent-resistant glycolipid-enriched membrane domains (GEMs). Using chimaeras of the Lck dual acylation motif with two normally cytosolic proteins (chloramphenicol acetyl transferase and galectin-3), we show here that this motif is sufficient to encode correct lipid modification and to target these chimaeras to the plasma membrane, as demonstrated by subcellular fractionation and confocal immunofluorescence microscopy of transiently transfected COS cells. In addition, the chimaeras are resistant to extraction with cold non-ionic detergent, indicating targeting to GEM subdomains in the plasma membrane. The dual acylation motif has potential for targeting proteins to specific plasma membrane subdomains involved in signalling.