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Noncovalent inhibitors reveal BTK gatekeeper and auto-inhibitory residues that control its transforming activity
Shenqiu Wang, … , Anas Younes, Hans-Guido Wendel
Shenqiu Wang, … , Anas Younes, Hans-Guido Wendel
Published June 20, 2019
Citation Information: JCI Insight. 2019;4(12):e127566. https://doi.org/10.1172/jci.insight.127566.
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Research Article Hematology Oncology

Noncovalent inhibitors reveal BTK gatekeeper and auto-inhibitory residues that control its transforming activity

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Abstract

Inhibition of Bruton tyrosine kinase (BTK) is a breakthrough therapy for certain B cell lymphomas and B cell chronic lymphatic leukemia. Covalent BTK inhibitors (e.g., ibrutinib) bind to cysteine C481, and mutations of this residue confer clinical resistance. This has led to the development of noncovalent BTK inhibitors that do not require binding to cysteine C481. These new compounds are now entering clinical trials. In a systematic BTK mutagenesis screen, we identify residues that are critical for the activity of noncovalent inhibitors. These include a gatekeeper residue (T474) and mutations in the kinase domain. Strikingly, co-occurrence of gatekeeper and kinase domain lesions (L512M, E513G, F517L, L547P) in cis results in a 10- to 15-fold gain of BTK kinase activity and de novo transforming potential in vitro and in vivo. Computational BTK structure analyses reveal how these lesions disrupt an intramolecular mechanism that attenuates BTK activation. Our findings anticipate clinical resistance mechanisms to a new class of noncovalent BTK inhibitors and reveal intramolecular mechanisms that constrain BTK’s transforming potential.

Authors

Shenqiu Wang, Sayan Mondal, Chunying Zhao, Marjan Berishaj, Phani Ghanakota, Connie Lee Batlevi, Ahmet Dogan, Venkatraman E. Seshan, Robert Abel, Michael R. Green, Anas Younes, Hans-Guido Wendel

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Figure 1

BTK mutations affecting noncovalent BTKi.

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BTK mutations affecting noncovalent BTKi.
(A) BTK mutations identified i...
(A) BTK mutations identified in 50 ibrutinib-naive lymphoma specimens from 1575 sequenced samples in this study. Detailed amino acid change and patient data are included in Supplemental Table 1. (B) FACS analysis showing GFP and mutant BTK enrichment under RN486 treatment. (C) Summary of BTK sequencing results from 281 enriched clones; notably BTK (T474M and E513G) occurs as a double mutant in cis. (D) Immunoblot analysis of BTK Y223 autophosphorylation in HEK293T cells that express the indicated BTK alleles and lack endogenous BTK, treated with RN486. Total BTK was used as a control and quantification was done with ImageJ (NIH).

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