67 8 mg Introduction As a compound class histone deacetylase
As a compound class, histone deacetylase inhibitors (HDIs) have been remarkably successful in the treatment of T-cell lymphoma . Vorinostat (suberanilohydroxamic 67 8 mg or SAHA) was approved by the FDA in 2006 for the treatment of cutaneous T-cell lymphoma , ; romidepsin (RD) was approved for this indication as well in 2009 ,  and later for peripheral T-cell lymphoma in 2011 , ; belinostat was approved for this latter indication in 2014 . Despite their success in treating T-cell lymphoma, the HDIs have not proven to be effective for the treatment of solid tumors . Nearly all clinical trials for solid tumors in which HDIs were used as single agents were unsuccessful; clinical trials with HDIs combined with other chemotherapeutic agents to treat solid tumors have also been largely disappointing . This points to the need for a better understanding of the mechanisms of HDI-mediated cell death to facilitate more effective combinations with HDIs.
Given the emergence of three HDIs approved for the treatment of T-cell lymphoma, it is important to define mechanisms of resistance to them. One recent study found increased levels of tissue transglutaminase, commonly induced by HDI treatment, in MCF7 cells selected for resistance to vorinostat and co-treatment of the resistant cells with transglutaminase inhibitors restored sensitivity to vorinostat . Another group found that the transcription factor GLI1 was overexpressed in HCT-116 colon carcinoma cells that were selected with vorinostat . In the case of romidepsin, it was reported early on that romidepsin was a substrate for the ATP-binding cassette transporter P-glycoprotein (P-gp)  and romidepsin-resistant cell lines were found to have increased levels of P-gp expression , , , . However, increased expression of P-gp does not appear to explain the resistance of solid tumors to romidepsin and clinical tumor samples taken from patients with resistant disease do not appear to have increased levels of P-gp .
To identify alternative mechanisms of resistance to romidepsin, we selected the HuT78 T-cell lymphoma cell line with romidepsin in the presence of P-gp inhibitors to exclude P-gp as a mechanism of resistance. The resulting sublines were found to have increased activation of the mitogen activated protein kinase (MAPK) pathway and increased sensitivity to MEK inhibitors . Activation of the MAPK pathway led to increased phosphorylation and degradation of the proapoptotic protein Bim and MEK inhibitor treatment led to rapid increases in Bim and increased cell death . Thus, the activity of romidepsin is highly dependent on mitochondrial engagement and activation of the intrinsic apoptotic pathway.
The effector proteins Bak and Bax are crucial components of the intrinsic apoptotic pathway; these proteins are members of the Bcl-2 family and effect release of cytochrome c, subsequently leading to formation of the apoptosome . Proapoptotic Bcl-2 family members include Bim, Puma, and Noxa, and push the balance towards cell death while the antiapoptotic members Bcl-2, Mcl-1 and Bcl-XL prevent Bak- or Bax-mediated release of cytochrome c . However, other proteins that are not members of the Bcl-2 family have been shown to affect the ability of cells to undergo apoptosis. Hexokinase 1 and hexokinase 2 (HK1 and HK2), proteins are primarily located in mitochondria, and have been shown to prevent apoptosis mediated by either Bak or Bax . In light of the fact that apoptosis induced by romidepsin and other HDIs is dependent on Bak and/or Bax expression , , we explored whether targeting mitochondrial hexokinases might improve the efficacy of HDIs.
Materials and methods
Discussion Hexokinases convert glucose to glucose-6-phosphate—the first step in glycolysis—and HK2 has been linked to the “Warburg effect”, the observation that cancer cells mainly generate ATP by glycolysis, as opposed to oxidative phosphorylation , . HK2 expression is higher in cancer cells compared to normal cells  and it has been linked to cancer metastasis, progression and tumor maintenance. In primary pancreatic ductal carcinoma tumor samples, HK2 was most highly expressed in metastases and stable knockdown of HK2 in cell line xenografts was associated with decreased metastatic potential in mice . Knockdown of HK2 in neuroblastoma cell line models also led to decreased tumor establishment and lung metastases in mice injected with knockdown cells versus control cells . HK2 was shown to be required for tumor establishment and maintenance in mouse models of KRAS-mutant lung cancer and ErbB2-driven breast cancer . Knockdown of HK2 or inhibiton of HK2 by 2-deoxyglucose was found to inhibit growth of KRAS mutant human and murine lung cancer cell line models . Thus, HK2 is an attractive target in cancer.