Preclinical evaluation of proteolytic targeting of LCK as a therapeutic approach in T cell acute lymphoblastic leukemia

Abstract

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and there is an unmet need for targeted therapies, especially for patients with relapsed disease. We have recently identified pre–T cell receptor and lymphocyte-specific protein tyrosine kinase (LCK) signaling as a common therapeutic vulnerability in T-ALL. LCK inhibitor dasatinib showed efficacy against T-ALL in preclinical studies and in patients with T-ALL; however, this is transient in most cases. Leveraging the proteolysis targeting chimera (PROTAC) approach, we developed a series of LCK degraders using dasatinib as an LCK ligand and phenyl-glutarimide as a cereblon-directing moiety. Our lead compound SJ11646 exhibited marked efficiency in cereblon-mediated LCK degradation in T-ALL cells. Relative to dasatinib, SJ11646 showed up to three orders of magnitude higher cytotoxicity in LCK-activated T-ALL cell lines and primary leukemia samples in vitro, with drastically prolonged suppression of LCK signaling. In vivo pharmacokinetic and pharmacodynamic profiling indicated a 630% increase in the duration of LCK suppression by SJ11646 over dasatinib in patient-derived xenograft models of T-ALL, which translated into its extended leukemia-free survival over dasatinib in vivo. Last, SJ11646 retained a high binding affinity to 51 human kinases, particularly ABL1, KIT, and DDR1, all of which are known drug targets in other cancers. Together, our dasatinib-based phenyl-glutarimide PROTACs are promising therapeutic agents in T-ALL and valuable tools for developing degradation-based therapeutics for other cancers.

Phenyl Dihydrouracil: An Alternative Cereblon Binder for PROTAC Design

“…Error represents SD. c CRBN-DDB1 binding determined by fluorescent polarization assay. Error represents SEM ( n = 3). d Data previously reported in Hu et al …”

“…However, IMiDs are well-known to be inherently unstable, readily undergoing hydrolysis even under relatively mild conditions, such as incubation in commonly utilized cell culture media . We recently reported that phenyl glutarimide (PG)-based PROTACs retained cereblon affinity while displaying improved chemical stability and, consequently, improved protein degradation efficacy and cellular potency. − We also found PG-based PROTACs displaying improved selectivity profile with respect to the recruitment of classical off-target neosubstrates such as GSPT1. , However, while PG- PROTACs proved more stable than the corresponding IMiD-based PROTACs, they are still liable to hydrolyze in cell culture media. Moreover, the C-3 carbon of the glutarimide ring in PG was found to readily racemize in cell culture media, with up to 24% inversion over 24 h .…”

“…We postulated that replacing the glutarimide C-3 carbon in the phenyl glutarimide moiety with a nitrogen atom would not only address the racemization but would also result in further improved chemical stability. Following our earlier finding that pre-T cell receptor and LCK signaling are a common therapeutic vulnerability in T-cell acute lymphoblastic leukemia (T-ALL), we recently reported the development of highly potent and selective LCK PROTACs . The PROTACs were designed using dasatinib as an LCK ligand and the PG as a cereblon-directing moiety.…”

“…We next evaluated the effect of PROTACs on KOPT-K1 cell viability. We have previously shown that this human T-ALL cell line is highly sensitive to LCK inhibition and degradation . Cells were treated with compounds over 72 h, and their viability was assessed using the CellTiter-Glo assay kit (Promega).…”

“…Following our earlier finding that pre-T cell receptor and LCK signaling are a common therapeutic vulnerability in T-cell acute lymphoblastic leukemia (T-ALL), 12 we recently reported the development of highly potent and selective LCK PROTACs. 10 The PROTACs were designed using dasatinib as an LCK ligand and the PG as a cereblon-directing moiety. Our lead PG-PROTAC 1 (SJ11646) showed potent degradation of LCK, and a superior cytotoxicity in LCK-activated T-ALL cell lines and primary leukemia samples compared with dasatinib itself.…”