Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127

Abstract

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Fig. 1.. Drug-resistant mutations in BTK occur in distinct enzymatic classes, and kinase-impaired forms of BTK have sustained B cell receptor signaling.

(A) Western blot analysis of downstream signaling in TMD8 cells with WT or knock-in of mutant BTK V416L, T474I, and L528W in the absence or presence of IgM stimulation. (B) Michaelis-Menten plots of the kinase activities of BTK WT and mutants. Kinase activities were measured using signal from tyrosine phosphorylation of a peptide substrate at a range of ATP concentrations in a FRET assay. For BTK M437R, V416L, and L528W, higher ATP concentrations were tested (500 μM). (C) Table showing apparent Michaelis-Menten constant of ATP required to yield a signal (K_m,_ATP) and relative catalytic efficiency (k_cat/K_m) of each BTK mutant purified protein within a 60-min range from the FRET-based kinase activity assay. Each value is the mean ± SD of three experiments. (D) Diagram of drug-resistant mutations in BTK and their impact on BTK enzymatic activity. (E) Measurements of intracellular calcium (Ca²⁺) release upon IgM-mediated cross-linking of the BCR over time in TMD8 cells overexpressing WT or mutant BTK. (F) Heatmap of phosphorylation of BTK, SYK, and LYN at the indicated amino acid residues in BTK WT, T474I, and L528W mutant cells in the presence or absence of pirtobrutinib treatment (1 μM for 1 hour). (G) Volcano plots of differentially phosphorylated peptides in BTK L528W versus WT BTK (top) or BTK T474I versus WT BTK (bottom) cells. Samples were prepared in triplicate and analyzed after phosphotyrosine enrichment using TMT mass spectrometry with an FDR adjusted significance threshold of P < 0.05. (H) Kinase substrate enrichment analysis showing predicted alterations in protein kinase–mediated signaling on the basis of hypo- or hyperphosphorylated substrates from (G), highlighting significantly active (red) and significantly inactive (blue) genes in BTK L528W versus WT BTK (top) or BTK T474I versus WT BTK (bottom) expressing cells. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 2.. Kinase-impaired BTK mutations enhance interaction with additional signaling proteins downstream of the BCR and discovery of the immunomodulatory drug-based BTK degrader NX-2127.

(A) Heat-map of signaling proteins identified from IP mass spectrometry interacting with 3X FLAG-tagged WT or mutant BTK in TMD8 cells in the absence or presence of IgM stimulation. (B) Stacked bar chart illustrating results from 2D DIGE gel analysis and protein identification after BTK immunoprecipitation from knock-in TMD8 cell lines. Proteins significantly up-regulated in L528W mutant highlighted in dot plot with proteins also seen in IP mass spectrometry experiment in red text. (C) Western blot analysis in knock-in TMD8 cells after immunoprecipitation of endogenous WT or mutant BTK in the presence or absence of IgM stimulation. (D) Relative GFP expression over a 6-day period in TMD8 BTK WT or BTK L528W knock-in cells after doxycycline induction of tetracycline-inducible HCK knockdown (shHCK 1, shHCK 2, or shHCK 3), ILK knockdown (shILK 1, shILK 2, or shILK 3) or nontargeted control vector. (E) Measurements of intracellular calcium (Ca²⁺) release upon IgM-mediated cross-linking of the BCR over time in TMD8 BTK WT or BTK L528W knock-in cells after 48-hour doxycycline induction of HCK or ILK knockdown. All Western blots are representative of three independent experiments. Nonlinear mixed model was used to analyze data in (D). Data in (D) represent data + SEM from n = 3 replicates. ****P < 0.0001.

Fig. 3.. NX-2127 binds to all classes of drug-resistant BTK mutant proteins and induces stable ternary complex formation with CRBN-DDB1 E2 ubiquitin ligase and BTK.

(A) Chemical structure and properties of NX-2127. (B) Binding curves of NX-2127 (top), ibrutinib (middle), and pirtobrutinib (bottom) to full-length BTK WT and mutant proteins in a FRET probe displacement assay. For ibrutinib, binding to BTK proteins was measured kinetically without preincubating compound and protein. Curves and IC₅₀ values represent binding at steady state at 60 min. For NX-2127 and pirtobrutinib, compound and protein were incubated for 120 min, and binding curves and IC₅₀ values were determined using an end-point readout. (C) Dissociation constants (K_d) of NX-2127 determined by SPR and IC₅₀ of NX-2127, pirtobrutinib, and ibrutinib measured in the FRET probe displacement assay. Each value is the mean ± SD of three or more experiments. (D) Ternary complex formation in human embryonic kidney 293 cells coexpressing NanoLuc BTK WT or mutant and Halo-tag cereblon. The assay was performed with a range of NX-2127 concentrations. Data represent normalized NanoBRET signal to DMSO after 6-hour treatment in technical triplicate. (E) Binding of NX-2127 to BTK WT (left) and L528W (right) in the absence and presence of saturating CRBN/DDB1 (1 mM) complex to determine cooperativity using the FRET probe displacement assay. (F) Cooperativity values of different ternary complexes between full-length BTK (WT and mutants) and CRBN/DDB1 induced by NX-2127. Each IC₅₀ value is the mean ± SD of three experiments. Cooperativity was calculated from the ratio of averaged IC₅₀ without and with saturating CRBN/DDB1 complex. Data in (B) and (F) represent average values + SD from n = 3 or more independent experiments.

Fig. 4.. NX-2127 degrades BTK drug-resistant mutant proteins and abrogates BCR signaling to result in malignant cell death.

(A) Crystal structures of NX-2127 hook bound to WT (yellow, PDB ID 8GC7) and L528W mutant (blue, PDB ID 8GC8) BTK proteins aligned on their C-lobes. View into the BTK active site with the proteins shown as ribbons. The ligands and resistance mutation sites are shown as sticks and highlighted in light red for the WT and in purple for the L528W mutant structure. P-loop residues have been removed for visibility. (B) NX-2127 hook (pink) bound BTK L528W mutant shown as gray ribbons with highlighted C-spine (blue mesh and sticks) and R-spine (yellow mesh and sticks). Three mutation sites covered in this study are located in the C-spine and are indicated as magenta sticks. The R-spine is not fully assembled with M449 on the αC-helix not placed in the correct position. For reference, a WT BTK structure in active C-helix “in” conformation (PDB ID 3K54) is depicted in cyan ribbons, where the K430 and E445 side chains (shown as sticks) form the required salt bridge. Cβ atoms of the salt bridge residues from K430 and E445 are shown as green spheres for comparison with the same atoms in the L528W structure shown as red spheres. This salt bridge between K430 and E445 side chains was not observed in the L528W structure. (C) Dose curves of knock-in TMD8 cells expressing WT and mutant (C481S, V416L, T474I, and L528W) BTK treated with NX-2127 for 24 hours and 50% degradation concentration (DC₅₀) for BTK, IKZF1 (Ikaros), and IKZF3 (Aiolos) were determined. (D) Measurements of intracellular calcium (Ca²⁺) release upon IgM-mediated cross-linking of the BCR over time in knock-in TMD8 cells after 24-hour treatment with either vehicle (top panel) or 1 μM NX-2127 (bottom panel). (E) Heatmap of 50% growth inhibition (GI₅₀) of TMD8 cells expressing WT and mutant BTK after 72-hour treatment with NX-2127, BTK inhibitors, and immunomodulatory drugs. Data in (C) and (E) were averaged from n = 3 independent experiments, and mean ± SEM is displayed.

Fig. 5.. Effective BTK and IKZF1 degradation and clinical response in CLL patients treated with NX-2127.

At the time of the datacut (September 2022), 21 patients were sequenced by next-generation sequencing, 20 patients were evaluated for BTK degradation, and 14 patients were disease evaluable. (A) Intracellular BTK levels in CD19⁺ CD3⁻ cells from the peripheral blood of CLL patients with distinct categories of BTK mutations treated with NX-2127. Mean ± SEM values for each time point are shown. (B) Maximum percent change in sum of the product of the diameters (SPD) of tumors from baseline in patients from (A). (C) Hemoglobin values in a representative CLL BTK C481R mutant patient at the start of treatment with pirtobrutinib followed by transition to NX-2127 after development of resistance to pirtobrutinib due to acquisition of a new BTK L528W mutation. (D) Percent of BTK protein in CD19⁺ cells in the peripheral blood as well as IKZF1 protein levels in peripheral blood mononuclear cells (inset) after start of NX-2127 treatment of patient from (C).