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Histone deacetylase inhibitors (HDACi) induce hyperacetylation of histones by blocking HDAC catalytic sites. Despite regulatory approvals in hematological malignancies, limited solid tumor clinical activity has constrained their potential, arguing for better understanding of mechanisms of action (MOA). Multiple activities of HDACis have been demonstrated, dependent on cell context, beyond the canonical induction of gene expression. Here, using a clinically relevant exposure duration, we established DNA damage as the dominant signature using the NCI-60 cell line database and then focused on the mechanism by which hyperacetylation induces DNA damage. We identified accumulation of DNA–RNA hybrids (R-loops) following romidepsin-induced histone hyperacetylation, with single-stranded DNA (ssDNA) breaks detected by single-cell electrophoresis. Our data suggest that transcription-coupled base excision repair (BER) is involved in resolving ssDNA breaks that, when overwhelmed, evolve to lethal dsDNA breaks. We show that inhibition of BER proteins such as PARP will increase dsDNA breaks in this context. These studies establish accumulation of R-loops as a consequence of romidepsin-mediated histone hyperacetylation. We believe that the insights provided will inform design of more effective combination therapy with HDACis for treatment of solid tumors. Implications: Key HDAC inhibitor mechanisms of action remain unknown; we identify accumulation of DNA–RNA hybrids (R-loops) due to chromatin hyperacetylation that provokes single-stranded DNA damage as a first step toward cell death., Part of this work was supported by the Center for Cancer Research, Intramural Research Program of the National Cancer Institute; (1ZIABC010621–12; to S.E. Bates) and (BC-006161 and BC-006150; to W.C. Reinhold and Y. Pommier), and the European Research Council (ERC2014 AdG669898 TARLOOP; to A. Aguilera).

// Julian C. Bahr 1, * , Robert W. Robey 1, 2, * , Victoria Luchenko 1 , Agnes Basseville 1 , Arup R. Chakraborty 1 , Hanna Kozlowski 2 , Gary T. Pauly 3 , Paresma Patel 3 , Joel P. Schneider 3 , Michael M. Gottesman 2 , Susan E. Bates 1, 4 1 Developmental Therapeutics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA 2 Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA 3 Chemical Biology Laboratory, National Cancer Institute, Frederick Cancer Research Center, Frederick, MD 21702, USA 4 Columbia University Medical Center, Division of Hematology/Oncology, New York, NY 10032, USA * These authors contributed equally to this work Correspondence to: Susan E. Bates, email: seb2227@cumc.columbia.edu Keywords: romidepsin, Ras mutation, apoptosis, MEK inhibitor, AKT inhibitor Received: May 27, 2016 Accepted: September 01, 2016 Published: September 13, 2016 ABSTRACT We previously demonstrated activation of the mitogen-activated protein kinase (MAPK) pathway in a series of romidepsin-selected T-cell lymphoma cell lines as a mechanism of resistance to the histone deacetylase inhibitor (HDI), romidepsin. As Ras mutation leads to activation of both the MAPK and the phosphoinositide 3-kinase (PI3K) pathway, we examined whether combining romidepsin with small molecule pathway inhibitors would lead to increased apoptosis in cancers harboring Ras mutations. We treated 18 Ras mutant or wild-type cell lines with romidepsin in the presence of a MEK inhibitor (PD-0325901) and/or an AKT inhibitor (MK-2206) and examined apoptosis by flow cytometry. A short-term treatment schedule of romidepsin (25 ng/ml for 6 h) was used to more closely model clinical administration. Romidepsin in combination with a MEK and an AKT inhibitor induced apoptosis preferentially in cells harboring mutant versus wild-type Ras (69.1% vs. 21.1%, p < 0.0001). Similar results were found in a subset of cell lines when belinostat was combined with the MEK and AKT inhibitors and when romidepsin was combined with the dual extracellular signaling-related kinase (ERK)/PI3K inhibitor, D-87503, which inhibited both the MAPK and PI3K pathways at 5–10 μM. The observed apoptosis was caspase-dependent and required Bak and Bax expression. Cells with wild-type or mutant Ras treated with romidepsin alone or in combination with the MEK inhibitor displayed increased expression of proapoptotic Bim. We thus conclude that cancers bearing Ras mutations, such as pancreatic cancer, can be targeted by the combination of an HDI and a dual inhibitor of the MAPK and PI3K pathways.

The stromal cell-derived factor-1α SDF-1α (CXCL12)/CXCR4 axis has been linked to poor prognosis in some cancers. As histone deacetylase inhibitors (HDIs) exert antitumor effects by targeting proteins affecting cell migration, we sought to evaluate the effects of the HDIs apicidin, vorinostat, entinostat (MS-275) and romidepsin on the expression and function of CXCR4 in human cancer cell lines. After treatment with romidepsin, CXCR4 mRNA expression increased 12-fold in UOK121 renal cancer cells, 16-fold in H460 non-small cell cancer cells and 4-fold in SF295 glioma cells; treatment with other HDIs yielded similar effects. CXCR4 induction was not observed in MCF7 breast cancer cells or SW620 colon cancer cells. To evaluate the corresponding functional increase, the effect of CXCR4 ligand, CXCL12, on ERK1/2, STAT3 and c-SRC activation and cell migration was examined in UOK121, SF295 and H460 cells. Alone, the HDIs increased pERK1/2, while reducing pSTAT-3 and pSRC. Following CXCL12 exposure, pERK1/2 induction was maintained, but STAT3 and SRC phosphorylation was impaired. These findings resulted in reduced basal and CXCL12-mediated cell migration. In conclusion, HDIs upregulated CXCR4 mRNA expression but impaired CXCL12-dependent signaling cascades through STAT3 and c-SRC, suggesting a potential role for HDIs in delaying or preventing metastatic processes in solid tumors.

The histone deacetylase inhibitors (HDIs) have shown promise in the treatment of a number of hematologic malignancies, leading to the approval of vorinostat and romidepsin for the treatment of cutaneous T-cell lymphoma and romidepsin for the treatment of peripheral T-cell lymphoma by the U. S. Food and Drug Administration. Despite these promising results, clinical trials with the HDIs in solid tumors have not met with success. Examining mechanisms of resistance to HDIs may lead to strategies that increase their therapeutic potential in solid tumors. However, relatively few examples of drug-selected cell lines exist, and mechanisms of resistance have not been studied in depth. Very few clinical translational studies have evaluated resistance mechanisms. In the current review, we summarize many of the purported mechanisms of action of the HDIs in clinical trials and examine some of the emerging resistance mechanisms.

Ras pathway mutations are very common in cancer, with the highest prevalence for RAS itself, found in about 30% of tumors. Despite multiple efforts, targeting the RAS pathway has not yet been amenable to therapeutic intervention. Taking a different approach, our laboratory has focused on combination treatment with romidepsin, an HDAC inhibitor approved by the FDA to treat T-cell lymphoma. Our group indeed demonstrated perturbation of RAS downstream pathways after romidepsin treatment, by analyzing gene expression in samples obtained from patients treated with romidepsin for T-cell lymphoma (Chakraborty et al, Blood 2013, 121: 4115). The NCI-60 panel was screened with romidepsin plus or minus RAS pathway downstream inhibitors (MEK and/or Akt inhibitors). We found that mutations in RTK/RAS/BRAF correlated with romidepsin sensitivity (R = 0.472), with cell death ranging from 40% to 95% after 48h (assayed by annexin V labeling). Three KRAS mutant and three KRAS WT cell lines were then chosen to investigate the mechanism of this selectivity. Romidepsin induced histone acetylation and DNA damage from 30min treatment, and an increase in reactive oxygen species (ROS) after 10h exposure, independent of KRAS status. Only KRAS mutant cells showed mitochondrial membrane depolarization, cytochrome c release, and apoptosis, all starting about 24h after the beginning of treatment. Interestingly, cytoplasmic - but not mitochondrial - ROS scavengers rescued KRAS mutant cells from death, suggesting that ROS is required but not sufficient for romidepsin-induced apoptosis. Since Son et al (Nature 2013, 496: 101) reported that glutamine metabolism was reprogrammed by oncogenic KRAS to maintain redox balance, we explored this pathway using the Seahorse XF96 analyzer. We observed that, while all cell lines utilized glutamine for oxidative phosphorylation (OXPHOS) before drug exposure, romidepsin impaired the ability of only KRAS mutant cells to use glutamine for OXPHOS. Metabolomic analysis confirmed reduced glutamine use in KRAS mutant cells after drug treatment, as well as an inability to replenish their reduced glutathione stock. Moreover, this analysis also highlighted a romidepsin-induced drop in nucleotide synthesis in KRAS mutant cells, potentially leading to impaired DNA repair. Accordingly, adding back nucleosides to media blunted the effect of romidepsin on cell growth in KRAS mutant, but not in KRAS WT cells. From these results, we conclude that romidepsin causes cell damage through DNA damage and subsequent ROS release. And while KRAS WT cells are able to scavenge ROS and repair DNA breaks, KRAS mutant cells cannot. We hypothesize that the reprogramming of glutamine metabolism, which supports redox maintenance and may support nucleoside synthesis, makes KRAS mutant cells particularly sensitive to romidepsin treatment. Citation Format: Agnes Basseville, Carole Sourbier, Robert W. Robey, Dan L. Sackett, W. Marston Linehan, Susan E. Bates. Metabolic reprogramming in KRAS mutant cancer cells may cause sensitivity to the histone deacetylase (HDAC) inhibitor romidepsin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1771. doi:10.1158/1538-7445.AM2015-1771

In ongoing efforts to increase the solid tumor efficacy of the histone deacetylase inhibitor romidepsin, we characterized romidepsin-resistant T-cell lymphoma cell lines that were found to have activation of the mitogen-activated protein kinase (MAPK) pathway, leading to subsequent phosphorylation and degradation of the proapoptotic protein Bim. This led us to examine the combination of romidepsin with MAPK pathway inhibitors in cell lines harboring the V600E BRAF mutation that leads to constitutive activation of the MAPK pathway. To more closely simulate clinical administration of romidepsin, 11 V600E BRAF positive cell lines (8 melanomas and 3 colorectal cancers) were exposed to 25 ng/ml romidepsin in the presence or absence of the MAPK pathway inhibitors AZD6244 (selumetinib, 250 nM), PD0325901 (250 nM) or PLX4032 (vemurafenib, 1 µM) for 6 h, after which the cells were placed in romidepsin-free medium containing the inhibitors and incubated for an additional 42 h. Apoptosis was subsequently measured by Annexin V and propidium iodide staining. Of the 11 cell lines, 10 exhibited significantly higher annexin staining after short-term romidepsin treatment alone (control, median annexin value 7.1%±3.8% vs. treated, median annexin value 34.7%±17.8%, p Citation Format: Susan E. Bates, Victoria Luchenko, Agnes Basseville, Julian Bahr, Arup R. Chakraborty, Andrew McDonald, Alexandra Zimmer, Richard L. Piekarz, Robert W. Robey. Short-term romidepsin treatment combined with MAPK pathway inhibition results in decreased mitochondrial hexokinase 2, increased mitochondrial Bim and apoptosis in BRAF mutant cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5536. doi:10.1158/1538-7445.AM2014-5536

ABCG2 is a member of the G subfamily of human ABC transporters. It is an ATP-dependent protein and is a half-transporter that must dimerize for function. In various tumor cell lines, ABCG2 was involved in resistance to chemotherapy, by transporting numerous anticancer agents including tyrosine kinase inhibitors. Studies show the transporter has an important role in normal tissue protection, including the blood-brain and maternal-fetal barriers. It has also been observed that ABCG2 may play a protective role with respect to exposure to smoke or dietary carcinogens. A common variant in human ABCG2, the polymorphism Q141K, impairs expression and function, thereby reducing drug clearance and increasing the toxicity of chemotherapy and other substrate drugs. Although the activity of the Q141K variant is regularly described, biochemical data remain rare. Q141K has an impaired trafficking to cell surface compared to the WT protein and is sequestered in the aggresome. Here we characterized the Q141K and WT protein in human cells, studying post-translational modification, biochemistry, dimerization status, half-life, and endocytosis. Glycosylation and phosphorylation are equivalent, as is ATP binding and hydrolysis, and substrate binding using IAAP competition assays. Cell treatment with romidepsin, an HDAC inhibitor approved by FDA as an anticancer agent, restored the impaired variant trafficking, leading to an increase in drug efflux. The restoration of ABCG2 Q141K function has potential applications in oncology and in medicine, in improving normal tissue protection and drug elimination, as well as in cancer prevention since several carcinogens are substrates for ABCG2. Citation Format: Agnes Basseville, Suneet Shukla, Akina Tamaki, Robert Robey, Suresh V. Ambudkar, Susan E. Bates. Characterization of the aggresome targeted Q141K ABCG2/BCRP variant. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2193. doi:10.1158/1538-7445.AM2013-2193

ABCG2 is an ATP-binding cassette half-transporter that has garnered interest in pharmacogenomics as a modulator of oral drug absorption, drug excretion, and drug distribution through expression at the blood-brain barrier and the maternal-fetal barrier. A single nucleotide polymorphism (SNP) C421A in ABCG2, resulting in a glutamic acid to lysine mutation at amino acid 141 (Q141K), confers impaired protein expression and function. Pharmacogenomic studies have linked the SNP to increased exposure to substrate drugs including irinotecan, topotecan, sunitinib and gefitinib. Moreover, it has been shown that 10 % of gout cases can be attributed to the Q141K variant. We have studied the biology underlying altered expression and function of the ABCG2 variant. Q141K protein was resistant to degradation by Endo H, suggesting that it was fully processed and folded. Q141K ABCG2 expression was increased by the lysosome inhibitor bafilomycin, indicating that some of the protein reached the cell surface, although at reduced levels. But the largest fraction of the Q141K variant was retained in an intracellular compartment where a part was eliminated through the proteasomal pathway while the undegraded ABCG2 accumulated in the aggresomes, as shown by colocalization of the transporter with the centrosome marker, γ-tubulin. The ability of several agents to impact ABCG2 trafficking to the cell surface was evaluated. Mitoxantrone, an ABCG2 substrate previously noted to act as a pharmacological chaperone, induced a 2-fold increase in Q141K ABCG2 expression at the cell surface but only slightly enhanced ABCG2-related efflux. Romidepsin and other histone deacetylase inhibitors (HDIs), known to increase levels of ABCG2 mRNA, were also assayed. A significant increase in total protein, surface expression, and function was seen in Q141K ABCG2 variant when exposed to various HDIs. Immunofluorescence analysis by confocal microscopy showed a dramatic shift to the plasma membrane following exposure to the HDIs, whereas the localization of α-tubulin, HDAC6 and vimentin, involved in aggresome formation and structure, were unchanged. These results support the notion that Q141K ABCG2 variant is trapped in the aggresome and that HDIs aid localization to the surface. Some supplementary experiments showed that the Q141K ABCG2 states of phosphorylation, acetylation and dimerization were unmodified after HDIs treatment, and that the chaperones BiP, Hsp70 or Hsp90 were not involved in rescue. Investigations into the HDI-mediated mechanisms are underway. In addition to non-oncologic indications, the restoration of ABCG2 function has potential applications in oncology, in improving normal tissue protection and drug elimination, as well as in cancer prevention since several carcinogens are substrates for ABCG2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2610. doi:10.1158/1538-7445.AM2011-2610