Combination of Ibrutinib and BCL-2 or SYK Inhibitors in Ibrutinib Resistant ABC-Subtype of Diffuse Large B-Cell Lymphoma
Authors: Kuo Hsu-Ping , Crowley Richard , Xue Ling , Schweighofer Karl J. , Cheung Leo WK. , Hsieh Sidney , Eckert Karl , Versele Matthias , Chang Betty Y. 
Published: October 2019

Abstract
Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL), accounting for roughly 30% of newly diagnosed cases in the US. DLBCL can progress quickly, and in advanced cases is inconsistently cured with current therapies. Ibrutinib, a first-in-class Bruton's tyrosine kinase (BTK) inhibitor, is approved as a treatment for patients (pts) with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) who have had one prior treatment. The ABC subtype of DLBCL is considered especially high risk and characterized by chronic active B-cell receptor (BCR) signaling, which is blocked by ibrutinib. Recent phase II clinical trial results of ibrutinib as a single agent in DLBCL pts show an overall response rate of 41% in the ABC subtype (Wilson et al. ASH 2012). Responses of various cancers to single kinase targeted therapies are often limited by the cell's ability to bypass the target via alternative pathways or acquired mutations in the target or its pathway. It has been shown that a small number of CLL pts acquire resistance to ibrutinib through mutations in BTK and its substrate phospholipase C gamma 2 (PLCG2) in the B lymphoma cells following prolonged treatments (Woyach et al. NEJM 2014). Such mechanisms may be overcome by combinations of targeted agents. Through screening of wild-type and acquired ibrutinib-resistant ABC-DLBCL cell lines (e.g. expressing BTK C481S), we identify and report herein B-cell lymphoma-2 (BCL-2) and spleen tyrosine kinase (SYK) inhibitors that synergize with ibrutinib in vitro and in vivo.
Methods: Gene expression was analyzed by RT-qPCR using TaqMan Gene Expression Master Mix. Human DLBCL cell lines were treated with drugs for 3 days and the effect on cell growth was determined by CellTiter-Glo luminescent cell viability assay. SCID mice were treated when the TMD8 tumors reached 100-150 mm3. Annexin-V-positive and PI-negative population was detected as apoptotic cells in tumor cells at sacrifice. Cell adhesion and migration assays were performed as previously described (Chang et al. Blood 2013). Analysis of clinical samples used for BCL-2 gene expression profiling was performed using Affymetrix microarrays on FFPE specimens from the phase 2 PCYC-1106 trial (NCT01325701) and a rank based statistic (RankProd) was used to determine the significance of gene expression changes.
Results: DLBCL cell lines with higher BCL-2 expression were more sensitive to single agent ABT-199 than those with lower expression. Treatment of DLBCL cells with ibrutinib alone increased BCL-2 expression as well as their sensitivity to BCL-2 inhibitors. Combination treatment with BCL-2 inhibitors and ibrutinib completely inhibited tumor growth in murine models of ABC-DLBCL (Figure). Increased apoptotic cell populations were detected in the combination treated tumors compared to either treatment alone. Clinically, pretreatment tissue samples (n=28) from ABC-DLBCL pts who experienced objective responses to ibrutinib (CR+PR) had lower BCL-2 gene expression. A high BCL-2 mutation rate was observed in pts with poor response to ibrutinib (SD+PD). However, none of these mutations occurred in the BH3 or BH1 domains, both of which appear to interact with ABT-199 based on a 3-dimensional co-crystal structure of the inhibitor with BCL-2 (PDB code 4MAN) and further molecular simulation results. These findings suggest the potential benefit from combination therapy.
SYK is another downstream mediator of BCR signaling. Pretreatment of DLBCL cells with SYK inhibitors (e.g. R406) increased their sensitivity to ibrutinib. Ibrutinib resistant B-lymphoma cells with either C481S BTK or R665W PLCG2 mutations were re-sensitized to ibrutinib in combination with BCL-2 or SYK inhibitors, inhibiting cell growth, IgM-induced calcium flux, cell adhesion or migration in mutant containing cells.
Conclusions: Consistent with previous results from high-throughput combinatorial screenings of drugs interact favorably with ibrutinib (Mathews Griner, et al. PNAS 2013), we found BCL-2 and SYK may function in alternative survival pathways in DLBCL cells upon BTK inhibition. Human B lymphomas harboring ibrutinib-resistant C481S BTK or R665W PLCG2 may be re-sensitized by BCL-2 or SYK inhibitors, both of which provide a rationale for the design of combination therapies.
Figure 1 Combination of ibrutinib and ABT-199 on the effect of TMD-8 tumor growth. Figure 1. Combination of ibrutinib and ABT-199 on the effect of TMD-8 tumor growth.

Disclosures
Kuo: Pharmacyclics: Employment. Crowley:Pharmacyclics: Employment. Xue:Pharmacyclics: Employment. Schweighofer:Pharmacyclics: Employment. Cheung:Pharmacyclics: Employment. Hsieh:Pharmacyclics: Employment. Eckert:Pharmacyclics: Employment. Versele:Janssen Pharmaceutica: Employment. Chang:Pharmacyclics, Inc: Employment, Equity Ownership.