Kinase-Impaired BTK Mutations Are Susceptible to Clinical-Stage BTK and IKZF1/3 Degrader NX-2127

INTRODUCTION: Bruton’s tyrosine kinase (BTK) is a nonreceptor kinase in the B cell receptor (BCR) signaling cascade critical for B cell survival. As such, chronic lymphocytic leukemia (CLL) and other B cell cancers are sensitive to inhibition of BTK. Covalent and noncovalent inhibitors of BTK have revolutionized the treatment of these cancers. Therefore, understanding mechanisms by which acquired mutation in BTK confer drug resistance and developing new therapies to overcome resistance are critically important. RATIONALE: We recently discovered BTK mutations that confer resistance across covalent and noncovalent BTK inhibitors. In this study, we found that a group of these mutants impair BTK kinase activity despite still enabling downstream BCR signaling. We therefore set out to understand the nonenzymatic functions of BTK and explored targeted protein degradation to overcome the oncogenic scaffold function of mutant BTK. This effort included evaluation of BTK degradation in patients with CLL treated in a phase 1 clinical trial of NX-2127, a first-in-class BTK degrader (NCT04830137). RESULTS: BTK enzymatic activity assays revealed that drug resistance mutations in BTK fall into two distinct groups: kinase proficient and kinase impaired. Immunoprecipitation mass spectrometry of kinase-impaired BTK L528W (Leu528→Trp) revealed a scaffold function of BTK with downstream signaling and survival dependent on surrogate kinases that bind to kinase-impaired BTK proteoforms. To target the nonenzymatic functions of BTK, we developed NX-2127, a heterobifunctional molecule that engages the ubiquitin-proteasome system to simultaneously bind both BTK and the cereblon E3 ubiquitin ligase complex, inducing polyubiquitination and proteasome-dependent degradation of IKZF1/3 and all recurrent drug-resistant forms of mutant BTK. The activity of NX-2127 on BTK degradation was further demonstrated in patients with CLL treated in a phase 1 clinical trial of NX-2127, where \u3e80% BTK degradation was achieved and clinical responses were also seen in 79% of evaluable patients, independent of mutant BTK genotypes. CONCLUSION: We identified that BTK inhibitor resistance mutations fall into two distinct functional categories. Kinase-impaired BTK mutants disable BTK kinase activity while promoting physical interactions with other kinases to sustain downstream BCR signaling. This scaffold function of BTK was disrupted by NX-2127, a potent BTK degrader, which showed promising responses for patients with relapsed and refractory CLL, independently of mutant BTK functional category

Modulating serine palmitoyl transferase (SPT) expression and activity unveils a crucial role in lipid-induced insulin resistance in rat skeletal muscle cells

International audienceSaturated fatty acids, such as palmitate, promote accumulation of ceramide which impairs activation and signaling of protein kinase B (PKB/Akt) to important end-points such as glucose transport. Serine Palmitoyl Transferase (SPT) is a key enzyme regulating ceramide synthesis from palmitate and represents a potential molecular target in curbing lipid-induced insulin resistance. Here we explore effects of palmitate upon insulin action in L6 muscle cells in which SPT expression/activity have been reduced by shRNA or sustained incubation with myriocin, a SPT inhibitor. Incubation of L6 myotubes with palmitate (16h) increases intramyocellular ceramide and reduces insulin-stimulated PKB activation and glucose uptake. PKB inhibition was not associated with impaired IRS signaling and was ameliorated by short-term treatment with myriocin. Silencing SPT expression (~90%) by shRNA or chronic cell incubation with myriocin (7 days) markedly suppressed SPT activity and palmitate-driven ceramide synthesis. However, challenging these muscle cells with palmitate still inhibited the hormonal activation of PKB. This inhibition was associated with reduced IRS1/p85-PI3-kinase coupling that arises from diverting palmitate towards greater DAG synthesis, which elevates IRS1 serine phosphorylation via activation of DAG-sensitive PKCs. Treatment of SPT-shRNA cells or those treated chronically with myriocin with PKC inhibitors antagonised palmitate-induced loss in insulin signaling. The findings indicate that SPT plays a crucial role in desensitizing muscle cells to insulin in response to incubation with palmitate. Whilst short-term inhibition of SPT ameliorates palmitate/ceramide-induced insulin resistance, sustained loss/reduction in SPT expression/activity promotes greater partitioning of palmitate towards DAG synthesis, which impacts negatively upon IRS1-directed insulin signaling