Repurposing Itraconazole As an Anti-Cancer Agent Against Diffuse Large B-Cell Lymphoma

Introduction: Results from the Retrospective Non-Hodgkin Lymphoma Research (SCHOLAR-1) study demonstrated that relapsed/refractory Diffuse Large B cell Lymphoma (r/r DLBCL) have a low response rate to third-line therapy (20-30%) and an overall survival of 6 months. It stresses the need to develop novel therapeutic strategies for patients with r/r lymphoma. Itraconazole (ITZ) is a common widely clinical used orally bioavailable azole antifungal drug, with a more than thirty years well-documented safety record. Recently, scientific evidence has highlighted ITZ anti-tumor effects at clinically achievable doses in patients with variety of solid tumor malignancies. However, little is known about ITZ activity in B-cell lymphoma, especially in chemotherapy resistant lymphoma. Here we are the first group to report that potent anti-tumor activity of ITZ as a single agent or combined with other chemotherapeutic drugs in both sensitive and resistant lymphoma pre-clinical models. We also further explored the mechanism of action of ITZ to overcome drug resistance.

Methods: A panel of rituximab-chemotherapeutic therapy-sensitive (TSCL), -resistant (TRCL) cell lines, developed in our laboratory, and primary tumor cells isolated from B-cell lymphoma patients were exposed to escalating doses of ITZ (0-20uM). IC₅₀ values were calculated using the GraphPad Prism6 software. Changes in cell viability, cell number and cellular ATP levels were evaluated using the Presto Blue, Trypan Blue and Titer Glo assay, respectively. Mitochondrial respiration rate indicated as oxygen consumption rate (OCR) were measured by an XFe96 flux analyzer. Mitochondrial membrane potential (MMP) were quantified by DiOC6 staining and flow cytometry. Subsequently, TSCL and TRCL cells were exposed to ITZ alone or in combination with various chemotherapy agents or small inhibitors and differences in cell viability was determined. Coefficient of synergy was calculated using the CalcuSyn software. HKII-VDAC complex disruption by ITZ was identified by co-immunoprecipitation and western blot. HKII knockdown was generated by using ON-TARGET plus SMART pool siRNA containing a mixture of 4 siRNAs designed to specifically target HKII. Standard ⁵¹Cr release assays were conducted to study the effects of ITZ on the anti-tumor activity of monoclonal antiCD20 antibodies against TSCL and TRCL cell lines. For in vivo studies, severe combined immunodeficiency (SCID) mice were inoculated via tail vein injection (iv) with Raji cells (day 0) and assigned to observation, rituximab (at 10mg/kg/dose), obinotuzumab (at 10mg/kg/dose) with or without ITZ. Anti-tumor activity was evaluated by functional imaging and length of survival.

Results: We found that ITZ induced a dose-dependent cell death in TSCL, TRCL and primary patient lymphoma cells. Single agent of ITZ decreased cell growth, reduced ATP production, lower the mitochondrial respiration rate and MMP in both TSCL and TRCL. ITZ exhibited additive/synergistic effects when combined with chemotherapeutic drugs (dexamethasone and cisplatin), proteasome inhibitors (bortezomib, carfilzomib, or ixazomib), targeted inhibitors (ABT199 and A-1210477 [an MCL-1 inhibitor]). At the molecular level, we found that, ITZ disrupted HKII/VDAC complex formation. Silencing HKII in TRCL attenuated the killing effect of ITZ. More importantly, ITZ in combination with rituximab or obinutuzumab led to reduced tumor intensity by functional imaging than rituximab or obinotuzumab single agent in vivo.

Conclusion: Our findings support the potential benefits for using ITZ as an anti-cancer drug are: 1) reduce costs, 2) clear defined toxicity profile, 3) availability, and 4) pre-clinical activity in rituximab-chemotherapy resistant lymphomas. ITZ can be an inexpensive and effective agent to enhance standard chemotherapy in B-cell lymphoma. Repurposing of ITZ can result in novel therapeutic options for lymphoma patients. (Research supported by Roswell Park Cancer Institute Alliance Grant, National Natural Science Foundation of China (NSFC) 81573063)