CG-806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broad Signaling Inhibition in Chronic Lymphocytic Leukemia Cells

Introduction: Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have fundamentally changed treatment of chronic lymphocytic leukemia (CLL). However, ibrutinib is not curative and used as a long-term therapy with associated toxicities and the risk of developing drug resistance, which are the most common reasons for treatment discontinuation. Ibrutinib resistance is related to acquired ibrutinib-resistant BTK or PLCG2 mutations. CG-806 is an oral small molecule non-covalent pan-BTK/pan-FLT3 inhibitor. CG-806 was structurally designed to target BTK without binding to C481 residual, and target other oncogenic kinases. It is being developed for treatment of CLL/SLL, non-Hodgkin's lymphomas and myeloid malignancies.

Methods: Peripheral blood mononuclear cells from patients with CLL were cultured for 14 days until outgrowth of nurselike cells (NLC). Before treatment with inhibitors, non-adherent cells were harvested and re-plated on autologous NLC at a concentration of 1x10⁷ cells/mL. MEC-1 CLL cell line was incubated with escalating doses of CG-806 or ibrutinib for 72 hours. The IC₅₀ of CG-806 and ibrutinib were measured using TACS® XTT cell proliferation/viability assay. The percentage and absolute number of viable MEC-1 and primary CLL cells were determined by flow cytometry with counting beads after staining with DiOC6/propidium iodide. For immunoblotting, CLL cells were pre-treated with CG-806 or ibrutinib for 2 hours and then stimulated with anti-IgM for 15 minutes. CLL cells co-cultured with NLC were treated with inhibitors for 24 hours before assessing B cell receptor (BCR) signaling by immunoblot and CCL3 and CCL4 concentration in the supernatants by ELISA.

Results: CG-806 inhibited the proliferation of MEC-1 CLL cells with an IC₅₀ of 32.0 nM (95% confidence interval (CI): 18.7 to 52.8 nM) compared to 3773 nM for ibrutinib (95% CI, 2427 to 6381 nM). CG-806 induced MEC-1 cell death in a dose- and time-dependent manner, with 0.1 µM CG-806 completely abrogating cell viability such that the total number of viable cells never rose over baseline, whereas ibrutinib was only mildly cytotoxic even at the highest test concentration of 10 µM. Separately, the activity and therapeutic potential of CG-806 were evaluated on primary CLL cells in co-culture with NLC, an in vitro model that resembles the lymph node microenvironment. After 120 hours, the viability of CLL cells treated with 0.5, 1, 5, or 10 µM of CG-806 significantly decreased by 14.0% (N=12, P=0.0024), 13.1% (P=0.0053), 27.9% (P<0.0001), and 48.5%(P<0.0001), respectively. Additionally, CG-806 caused a dose-dependent decrease in numbers of adherent NLC where treatment with 0.5 µM CG-806 resulted in 75.5% reduction in adherent NLC (N=12, P=0.0112). When primary CLL cells were stimulated with anti-IgM to trigger the BCR pathway, CG-806 inhibited BCR signaling-induced phosphorylation of BTK, PLCg2, AKT, ERK1/2, and S6 ribosomal protein, to the degree that was comparable with ibrutinib. Besides, CG-806 strongly suppressed SYK phosphorylation, which was not observed in ibrutinib-treated CLL cells. In primary CLL cells co-cultured with NLC, CG-806 treatment resulted in similar BCR signaling inhibition as in anti-IgM stimulated primary CLL cells, down-regulation of MYC, PARP cleavage, and reduced secretion of CCL3 and CCL4 by the CLL cells, a biomarker for BCR signaling activation.

Conclusion: Collectively, these data demonstrate that CG-806 broadly inhibits BCR signaling in CLL cells, resulting in CLL cell apoptosis and reduced proliferation. In addition, GC-806 targets elements of the CLL microenvironment, i.e. NLC, and thereby potentially targets pro-survival signals from the microenvironment. These findings support further development of CG-806 in B cell malignancies, including CLL patients who are intolerant, refractory or resistant to ibrutinib or other covalent or non-covalent BTK inhibitors.