Itraconazole, an Oral Antifungal Drug, Inhibits Tumor Growth and Enhances Therapeutic Agent Activity in Double Hit Lymphoma
Authors: Gu Juan J , Yang Lianjuan , Jarmusz Benjamin , Cress Daniel , Mavis Cory , Barth Matthew J. , Hernandez-Ilizaliturri Francisco J. 
Published: October 2019

Abstract
Background: Among B-cell lymphoma, double hit lymphoma (DHL) is defined by a MYC/8q24 combined with another recurrent BCL/18q21 breakpoint. DHL most affects elderly patients (median age ranged from 51 to 65 years) and have a poor prognosis with a median overall survival of 0.2-1.5 year. R+CHOP or high-intensity treatment including high-dose chemotherapy followed stem cell transplant yield to suboptimal outcomes. Therefore, there is a need to discover alternative effective agents. Itraconazole, an oral antifungal drug, was reported had potent anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer by interfering with the apoptotic threshold of cancer cells. Its activity in DHL has yet to be defined.
Methods: Double hit lymphoma cell lines: DOHH2, VAL and ROS50 were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism 6 software. Loss of ATP, Caspase 3/7 activation and loss of mitochondrial membrane potential (∆ψm) following itraconazole exposure were assessed by Cell Titer Glo, Caspase3 /7 detection kit and DiOC6 staining, respectively. DHL cells were exposed to itraconazole or vehicle combined with various inhibitors, such as BCL-2 inhibitor ABT199, c-MYC inhibitor JQ1, bruton's kinase inhibitor ibrutinib and different generations of proteasome inhibitors (carfilzomib or MLN4924) for 48h and cell viability was determined by Presto Blue assay. Coefficient of synergy index was calculated using the CalcuSyn software.
Result: Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition in DHL. In vitro exposure cells to itraconazole resulted in a loss of ATP, loss of mitochondrial membrane potential and activation of caspase 3/7 activities. Itraconazole caused G1 cell cycle arrest and decrease S phase in DHL. Moreover, itraconazole had a strong synergistic anti-tumor effect combined with various inhibitors, including BCL-2 inhibitor ABT199, c-MYC inhibitor JQ1, bruton's kinase inhibitor ibrutinib and different generations of proteasome inhibitors (bortezomib, carfilzomib, Ixazomib or MLN4924).
Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against DHL. Disruption of mitochondrial membrane potential and activation of caspases may contribute its antitumor activity against DHL. It is worth noticing that combined itraconazole with DHL specific target inhibitors ABT199 or JQ1 had a strong synergistic efficacy. Our findings offered the first assessment of the efficacy of itraconazole as a novel anticancer agent in double hit lymphoma. The molecular mechanism of action of itraconazole need to be further investigated in the near further.

Disclosures
No relevant conflicts of interest to declare.