The BTK Inhibitor ARQ 531 Targets Ibrutinib-Resistant CLL and Richter Transformation

Abstract

Targeted inhibition of Bruton tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has improved outcomes for patients with hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here, we describe preclinical investigations of ARQ 531, a potent, reversible inhibitor of BTK with additional activity against Src family kinases and kinases related to ERK signaling. We hypothesized that targeting additional kinases would improve global inhibition of signaling pathways, producing more robust responses. In vitro treatment of patient CLL cells with ARQ 531 decreases BTK-mediated functions including B-cell receptor (BCR) signaling, viability, migration, CD40 and CD86 expression, and NF-κB gene transcription. In vivo, ARQ 531 was found to increase survival over ibrutinib in a murine Eμ-TCL1 engraftment model of CLL and a murine Eμ-MYC/TCL1 engraftment model resembling Richter transformation. Additionally, ARQ 531 inhibits CLL cell survival and suppresses BCR-mediated activation of C481S BTK and PLCγ2 mutants, which facilitate clinical resistance to ibrutinib.Significance: This study characterizes a rationally designed kinase inhibitor with efficacy in models recapitulating the most common mechanisms of acquired resistance to ibrutinib. Reversible BTK inhibition is a promising strategy to combat progressive CLL, and multikinase inhibition demonstrates superior efficacy to targeted ibrutinib therapy in the setting of Richter transformation. Cancer Discov; 8(10); 1300-15. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195.

Figure 1.. Structure and inhibitory profile of ARQ 531.

A) Chemical structure of ARQ 531. B) 1.1 angstrom resolution crystal structure of ARQ 531 in complex with BTK. ARQ 531 occupies the ATP binding pocket and the solvent exposed side chain forms water mediated hydrogen bond network. C) Kinases demonstrating greater than 50% inhibition at 200 nM ARQ 531 were subjected to IC₅₀ determination at a physiological 1 mM ATP concentration. D) IC₅₀ values from Figure 1C were plotted in a representation of the human kinome. Representation was generated using KinMap. Illustration is reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

Figure 2.. Inhibition of BCR mediated signaling by ARQ 531.

A) Rosette-Sep isolated primary CLL cells were treated with escalating concentrations of ARQ 531 for 1 hour and then stimulated with anti-IgM for 15 minutes. Immunoblotting was then performed to show that ARQ 531 inhibits phosphorylation of BTK, AKT, and ERK. B) Immunoblot data from five CLL patients treated with ARQ 531 were quantitated using densitometry software. C) Primary CLL cells from three patients were treated in vitro with ARQ 531 and ibrutinib to contrast the effect on proximal BCR associated kinases including Src family members and Syk as well as downstream kinases including MEK and ERK. D) Rosette-Sep isolated primary CLL cells were assessed via immunoblot for transphosphorylation of Y551 following ARQ 531 treatment for one hour and anti-IgM stimulation for 15 minutes. For all experiments, differences were assessed using linear mixed-effects models. (**, 0.01≥p≥0.001; ***, p<0.001)

Figure 3.. ARQ 531 is cytotoxic to CLL cells, decreases NF-κB function, and inhibits migration.

A) Primary CLL cells were continuously treated with increasing concentrations of ARQ 531 for 48 hours to assess cytotoxicity via annexin V/PI staining and flow cytometry (n=9). B) Primary CLL cells were treated daily with ARQ 531 for two hours followed by drug washout and media replacement in order to simulate clearance of the reversible BTK inhibitor ARQ 531 (n=9). C) Transcriptional expression of MYC, CD40, and MCL-1 in CLL cells was assessed via real-time PCR following 72 hours of treatment with 1 μM ARQ 531 (n=7 for MYC, n=8 for MCL1 and CD40). D&E) The NF-κB dependent activation markers CD40 and CD86 were measured via flow cytometry 48 hours following treatment with 1 μM ARQ 531 and 3.2 μM CpG in CLL B cells (n=9). F&G) The number of CLL cells migrating through an 8.0 micron transwell insert towards the chemokines CXCL12 and CXCL13 were counted by flow cytometry (n=8 for CXCL12, n=9 for CXCL13). Differences were assessed using linear mixed-effects models. (*, 0.05≥p>0.01; **, 0.01≥p≥0.001; ***, p<0.001)

Figure 4.. ARQ 531 improves survival in the Eμ-TCL1 engraftment model compared to ibrutinib.

A) C57BL/6 mice engrafted with Eμ-TCL1 leukocytes via tail vein injection were treated with ARQ 531 for 60 days via daily oral gavage and monitored for survival and drug induced toxicity. Survival curves were estimated using the Kaplan-Meier method and curve differences among groups assessed using the log-rank test. B) White blood cell counts were measured weekly by microscopic examination. C) A cohort of mice were sacrificed after two weeks of treatment and spleens were weighed to measure disease progression (n=3 in each group). Analysis of variance (ANOVA) was used to compare spleen weight among groups. (*, 0.05≥p>0.01; **, 0.01≥p≥0.001; ***, p<0.001)

Figure 5.. ARQ 531 improves survival in the Eμ-MYC/TCL1 model compared to ibrutinib.

A) C57BL/6 mice engrafted via tail vein injection with Eμ-MYC/TCL1 leukocytes were treated with ARQ 531 via daily oral gavage starting 10 days after engraftment and monitored for survival. B) Percent CD19+ leukocytes collected from weekly bleeding were measured via flow cytometry. Survival curves were estimated using the Kaplan-Meier method and curve differences among groups assessed using the log-rank test.

Figure 6.. C481S BTK is inhibited by ARQ 531.

A) A HEK293T cell line stably transfected with WT or C481S BTK was treated with ARQ 531 or ibrutinib for one hour followed by SDS-PAGE to determine efficacy against C481S BTK. B) CLL cells isolated at baseline and time of progression from an ibrutinib resistant patient who acquired a C481S BTK mutation were treated with ARQ 531 for one hour followed by SDS-PAGE. C) Primary CLL cells isolated from ibrutinib resistant patients who all possessed BTK C481S mutations were treated with ARQ 531 for 72 hours to determine cytotoxicity (n=9). Differences were assessed using linear mixed-effects models. (**, 0.01≥p≥0.001)

Figure 7.. ARQ 531 inhibits BCR signaling in cells with ibrutinib-resistant PLCγ2 mutations.

A) A DT40 cell line stably transfected with WT, R665W, or L845F PLCγ2 and treated with ARQ 531 was assessed via SDS-PAGE to determine the effect on activation of the downstream kinases ERK and AKT. B) CLL cells isolated from an ibrutinib resistant patient harboring an R665W PLCγ2 mutation were treated with ARQ 531 followed by SDS-PAGE and assessed for distal BCR signaling. C) CLL cells isolated from an ibrutinib resistant patient harboring multiple PLCγ2 mutations were treated with ARQ 531 followed by SDS-PAGE and assessed for distal BCR signaling.