The exact mechanism by which

The exact mechanism by which mitochondrial hexokinases such as HK1 and HK2 prevent apoptosis is unclear. Mitochondrial hexokinases have been shown to bind with the voltage-dependent anion channel 1 (VDAC1), giving them direct access to ATP for use as an energy source [36]. Akt has been shown to promote the interaction between hexokinases and VDAC and therefore prevent apoptosis [37]. Some groups have suggested that HK1 acts to prevent the formation of Bax channels at the outer mitochondrial membrane [38]. HK2 is believed to interact with mitochondrial VDAC1 and prevent mitochondrial permeabilization by Bak and Bax through an as yet undefined mechanism [39]. While the interaction between hexokinases and VDAC proteins has been implicated in prevention of apoptosis, deletion of all VDAC isoforms has not been found to have an effect on apoptosis [40], casting doubt on this theory. Although our report appears to be the first to combine an HDI with compounds that target mitochondrial hexokinases, previous studies have suggested the possibility that HDIs exert their toxic effects via interference with glucose metabolism by hexokinases. Wardell et al. demonstrated that treating the multiple myeloma cell line OPM2 with valproic Aliskiren Hemifumarate receptor or SAHA resulted in decreased expression of GLUT1 protein as well as expression of its encoding SLC2A1 mRNA [41]. This was accompanied by an increase in HK1 expression. Despite this, hexokinase activity was apparently inhibited by HDI treatment [41]. They conclude that apoptosis induced by HDIs is due to their ability to affect metabolism. Amoedo et al. saw similar effects when they treated H460 lung cancer cells with sodium butyrate, noting decreased levels of SLC2A1 expression and increased mitochondrial HK1 expression [42]. However, they found that HDI treatment increased hexokinase activity. It is not clear if the observed differences between the two reports are due to model selection or the specific HDI used. However, effects of HDIs on cellular metabolic function do seem to contribute to their toxicity. It is interesting to note that half of the cell lines that showed sensitivity to the romidepsin and clotrimazole/bifonazole combination bear KRAS mutations, specifically the A549, HCT-116, and MDA-MB-231 lines. We previously demonstrated the selective activity of romidepsin in cells bearing KRAS mutation when downstream KRAS signaling pathways were inhibited [27]. This work targeting a hexokinase in combination with romidepsin offers a different strategy by which to exploit the KRAS-directed activity of HDIs, and perhaps a more direct one. The question is invariably raised as to whether targeting hexokinases will be too toxic for clinical translation. This can be answered in part by the in vivo studies noted above, in which knockout of hexokinases did not lead to excess toxicity. Preclinical studies with clotrimazole to date have been limited. Some reports have suggested that it might be useful alone or in combination with other drugs for the treatment of cancer [43]. Clotrimazole is effective as a single agent in breast, melanoma, lung and colon cancer models in vitro [44], [45], [46]. The combination of clotrimazole with cisplatin synergistically increased cisplatin efficacy in a glioblastoma cell line model [47] and the combination of clotrimazole with imatinib led to synergistic toxicity in the T47D breast cancer cell line [48]. Clotrimazole as a single agent was found to increase the survival of rats with intracranial gliomas [49] and decrease tumor growth in an oral squamous cell carcinoma xenograft model [50]. Despite these promising results, the clinical use of clotrimazole as a systemic therapy is limited by its low water solubility and poor bioavailability [43]. A number of interesting combinations of HDIs with targeted therapies have recently been suggested. The combination of the polo-like kinase inhibitor volasertib and the HDI belinostat has been shown to synergistically induce apoptosis in lymphoma cell line models [51]. Belinostat in combination with the CDK inhibitor seliciclib resulted in high levels of apoptosis in non-small cell lung cancer models and did not depend on p53 activity [52]. We recently demonstrated that either romidepsin or belinostat in combination with inhibitors of the MAPK and/or PI3K pathways induced apoptosis selectively in cancers harboring mutant Ras [27]. Those results mirrored those of Ischenko et al., who demonstrated increased efficacy of trichostatin A when combined with a MEK and a PI3K inhibitor [53]. Our data with clotrimazole point to an entirely new therapeutic target not yet exploited in cancer cells and suggest that HDIs sensitize cells to this novel target. We are pursuing medicinal chemistry strategies to develop a compound more suited for preclinical and clinical testing.