Kinase Dead BTK Mutations Confer Resistance to Covalent and Non-Covalent BTK Inhibitors, But Are Susceptible to Clinical Stage BTK Degrader NX-2127

Kinase Dead BTK Mutations Confer Resistance to Covalent and Non-Covalent BTK Inhibitors, But Are Susceptible to Clinical Stage BTK Degrader NX-2127

Justin Taylor ¹, Skye Montoya ¹, Jessie Bourcier ², Omar Abdel-Wahab ²

¹ Sylvester Comprehensive Cancer Center at the University of Miami, Miami, FL, USA

² Memorial Sloan Kettering Cancer Center, New York, NY, USA

Background: Increasing use of the covalent Bruton tyrosine kinase (BTK) inhibitors ibrutinib, acalabrutinib, and zanubrutinib as well as the noncovalent BTK inhibitors nemtabrutinib and pirtobrutinib, have elucidated a series of acquired BTK mutations, some of which can confer cross-resistance to other BTK inhibitors, in patients with B-cell malignancies.

Aims: Here we identify three distinct enzymatic classes of BTK inhibitor resistance BTK kinase domain mutations and show that all classes are susceptible to BTK degradation preclinically and in patients treated with the clinical stage BTK degrader NX-2127.

Methods: To identify the signaling mechanisms of kinase dead BTK mutants, we generated CRISPR-CAS9 knockin mutant cells and utilize several orthogonal proteomic approaches in BTK-dependent human B cell lymphoma cells expressing WT or kinase-dead mutations BTK V416L and L528W. We performed global phosphoproteomics, kinobead assays, BTK immunoprecipitation mass spectrometry studies, and 2D differential gel electrophoresis to unbiasedly elucidate a novel scaffolding function of BTK.

Results: Our biochemical and biological assays revealed that in addition to impeding drug binding, some BTK drug resistance mutations also appear to have diminished or absent kinase enzymatic function and reduced autophosphorylation of tyrosine 223 (Y223), a marker of BTK activation. Our data suggest that recurrent BTK mutations occur in distinct enzymatic groups: certain BTK mutations are kinase proficient (T474I/F and C481S mutations), while others greatly reduce enzymatic activity (M437R mutation), and finally some mutations completely render BTK kinase dead (V416L, C481Y/R/F and L528W mutations, see Figure). Despite their lack of BTK activation, upon B-cell receptor (BCR) stimulation kinase dead BTK mutants showed enhanced activation of downstream BCR signals and hyperactivated calcium release. Collectively our data revealed enhanced physical interactions of kinase dead BTK with protein kinases HCK and ILK as well as activation of multiple signaling moieties in malignant B cells.

Given the newly discovered scaffolding function of BTK, we decided to test strategies to eliminate, rather than inhibit mutant BTK proteins. While several PROTAC degraders of BTK have been demonstrated to target wild-type or C481S-mutant forms of BTK in preclinical studies, we now demonstrate the first clinical-stage (clinicaltrials.gov #NCT04830137) degrader of BTK, NX-2127, which binds to each drug resistant BTK mutant proteoform (as demonstrated by biophysical and structural data) and induces their proteasomal degradation causing inhibition of BCR signaling. Treatment of chronic lymphocytic leukemia (CLL) patients with NX-2127 achieves >80% degradation of BTK and we demonstrate proof-of-concept therapeutic benefit in patients with CLL.

Conclusion: These data reveal a distinct oncogenic scaffolding function of kinase dead BTK which confers resistance across FDA-approved BTK inhibitors. Importantly, regardless of enzymatic group or signaling mechanism, each recurrent BTK mutant can be overcome in patients with CLL treated in a Phase 1b dose expansion with NX-2127.

This research was funded by a Clinical Scientist Development Award to JT from the Doris Duke Charitable Foundation.

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