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.

Key Points MAPK pathway activation and Bim loss may represent a fundamental mechanism of resistance to histone deacetylase inhibitors. Combination of romidepsin with an MEK inhibitor may lead to greater responses in cancers in which the MAPK pathway is active.

Summary Romidepsin has shown promise in the treatment of T‐cell lymphomas, and so we evaluated molecular endpoints gathered from 61 patients enrolled on a phase II trial of romidepsin in cutaneous and peripheral T‐cell lymphoma at the National Institutes of Health. The endpoints included histone H3 acetylation and ABCB1 gene expression in peripheral blood mononuclear cells (PBMCs); ABCB1 gene expression in tumour biopsy samples; and blood fetal haemoglobin levels (HbF), all of which were increased following romidepsin treatment. The fold increase in histone acetylation in PBMCs at 24 h was weakly to moderately well correlated with the pharmacokinetic parameters Cmax and area under the curve (AUC)last (ρ = 0·37, P = 0·03 and ρ = 0·36, P = 0·03 respectively) and inversely associated with clearance (ρ = −0·44; P = 0·03). Histone acetylation in PBMCs at 24 h was associated with response (P = 0·026) as was the increase in fetal haemoglobin (P = 0·014); this latter association may be due to the longer on‐study duration for patients with disease response. Together, these results suggest that pharmacokinetics may be an important determinant of response to histone deacetylase inhibitors (HDIs) – the association with histone acetylation in PBMCs at 24 h is consistent with a hypothesis that potent HDIs are needed for a critical threshold of drug exposure and durable activity.

Romidepsin and vorinostat are histone deacetylase inhibitors (HDACis) that have activity in T-cell lymphomas, but have not gained traction in solid tumors. To gain deeper insight into mechanisms of HDACi efficacy, we systematically surveyed nineteen cell lines with different molecular phenotypes, comparing romidepsin and vorinostat at equipotent doses. Acetylation at H3K9 and H4K8 along with 22 other histone lysine acetylation and methylation modifications were measured by reverse phase proteomics array (RPPA), and compared with growth inhibition (IC50), and cell cycle arrest. These assays typically used to assess HDACi effect showed that acetylation and methylation of specific lysine residues in response to HDACis was consistent across cell lines, and not related to drug sensitivity. Using a treatment duration more reflective of the clinical exposure, cell death detected by annexin staining following a 6 hour drug exposure identified a subset of cell lines, including the T-cell lymphoma line, that was markedly more sensitive to HDAC inhibition. Kinetic parameters (Km values) were determined for lysine acetylation and for cell cycle data and were themselves correlated following HDACi exposure, but neither parameter correlated with cell death. The impact on cell survival signaling varied with the molecular phenotype. This study suggests that cellular response to HDACis can be viewed as two distinct effects: a chromatin effect and a cell death effect. All cells undergo acetylation, which is necessary but not sufficient for cell death. Cells not primed for apoptosis will not respond with cell death to the impact of altered histone acetylation. The divergent apoptotic responses observed reflect the variable clinical outcome of HDACi treatment. These observations should change our approach to the development of therapeutic strategies that exploit the dual activities of HDACis.

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Purpose Romidepsin is a potent histone deacetylase inhibitor (HDI) with activity in T-cell lymphoma. Given preclinical data demonstrating greater induction of gene expression with longer exposures to HDIs, a phase I study of a days 1, 3, and 5 romidepsin schedule was evaluated. A secondary objective was to assess the effect of romidepsin on radioactive iodine (RAI) uptake in thyroid cancers. Experimental Design Open label, single arm, phase I, 3 + 3 dose escalation study. Romidepsin was administered as a four-hour infusion on days 1, 3 and 5 of a 21-day cycle. Pharmacokinetics (PK) and pharmacodynamics (PD) were assessed, including histone acetylation in peripheral blood mononuclear cells (PBMCs); RAI uptake in refractory thyroid cancer; and HDI-related ECG changes. Results 28 patients with solid tumors, including eleven patients with thyroid cancer were enrolled. Six dose levels were explored and 7 mg/m2 on days 1, 3, and 5 was identified as tolerable. No RECIST-defined objective responses were recorded although nine patients had stable disease a median 30 weeks (range 21 – 112) including six with thyroid cancer a median of 33 weeks. PD studies detected acetylated histones in PBMCs and ECG changes beginning at low dose levels. Follow-up RAI scans in patients with RAI refractory thyroid cancer did not detect meaningful increases. Conclusions A romidepsin dose of 7 mg/m2 administered on days 1, 3, and 5 was found tolerable and resulted in histone acetylation in PBMCs. Although there were no objective responses with romidepsin alone, this schedule may be useful for developing combination studies in solid tumors.

Group-I metabotropic glutamate receptors have been often implicated in various models of neuronal toxicity, however, the role played by the individual receptors and their putative mechanisms of action contributing to neurotoxicity or neuroprotection remain unclear. Here, using primary cultures of rat cerebellar granule cells and mouse cortical neurons, we show that conditions of trophic deprivation increased mGlu1 expression which correlated with the developing cell death. The inhibition of mGlu1 expression by specific siRNA attenuated toxicity, while adenovirus-mediated overexpression of mGlu1 resulted in increased cell death, indicating a causal relationship between the level of receptor expression and neuronal survival. In pharmacological experiments selective mGlu1 antagonists failed to protect from mGlu1-induced cell death, instead, neuronal survival was promoted by glutamate acting at mGlu1 receptors. Such properties are characteristic of a novel heterogeneous family of dependence receptors which control neuronal apoptosis. Our findings indicate that increased expression of mGlu1 in neurons creates a state of cellular dependence on the presence of its endogenous agonist glutamate. We propose a new role and a new mechanism for mGlu1 action. This receptor may play a crucial role in determining the fate of individual neurons during the development of the nervous system.

Background: This pharmacodynamic trial evaluated the effect of CBT-1 on efflux by the ATP binding cassette (ABC) multidrug transporter P-glycoprotein (Pgp/MDR1/ABCB1) in normal human cells and tissues. CBT-1 is an orally administered bisbenzylisoquinoline Pgp inhibitor being evaluated clinically. Laboratory studies showed potent and durable inhibition of Pgp, and in phase I studies CBT-1 did not alter the pharmacokinetics of paclitaxel or doxorubicin.Methods: CBT-1 was dosed at 500 mg/m 2 for 7 days; a 3-hour infusion of paclitaxel at 135 mg/m 2 was administered on day 6. Peripheral blood mononuclear cells (PBMCs) were obtained prior to CBT-1 administration and on day 6 prior to the paclitaxel infusion. Tc-sestamibi imaging was performed on the same schedule. The area under the concentration-time curve from 0 -3 hours (AUC 0 -3 ) was determined for 99m Tc-sestamibi. Results: Twelve patients were planned and enrolled. Toxicities were minimal and related to paclitaxel (grade 3 or 4 neutropenia in 18% of cycles). Rhodamine efflux from CD56ϩ PBMCs was a statistically significant 51%-100% lower (p Ͻ .0001) with CBT-1. Among 10 patients who completed imaging, the 99m Tc-sestamibi AUC 0 -3 for liver (normalized to the AUC 0 -3 of the heart) increased from 34.7% to 100.8% (median, 71.9%; p Ͻ .0001) after CBT-1 administration. Lung uptake was not changed.Conclusion: CBT-1 is able to inhibit Pgp-mediated efflux from PBMCs and normal liver to a degree observed with Pgp inhibitors studied in earlier clinical trials. Combined with its ease of administration and lack of toxicity, the data showing inhibition of normal tissue Pgp support further studies with CBT-1 to evaluate its ability to modulate drug uptake in tumor tissue.Discussion: Although overexpression of ABCB1 and other ABC transporters has been linked with poor outcome following chemotherapy efforts to negate that through pharmacologic inhibition have generally failed. This is thought to be a result of several factors, including (a) failure to select patients with tumors in which ABCB1 is a dominant resistance mechanism; (b) inhibitors that were not potent, or that impaired drug clearance; and (c) the existence of other mechanisms of drug resistance, including other ABC transporters. Although an animal model for Pgp has been lacking, recent studies have exploited a Brca1Ϫ/Ϫ mouse model of hereditary breast cancer that develops sporadic tumors similar to cancers in women harboring BRCA1 mutations. Treatment with doxorubicin, docetaxel, or the poly(ADP-ribose) polymerase inhibitor olaparib brings about shrinkage, but resistance eventually emerges. Overexpression of the Abcb1a gene, the mouse ortholog of human ABCB1, has been shown to be a mechanism of resistance in a subset of these tumors. Treating mice with resistant tumors with olaparib plus the Pgp inhibitor tariquidar resensitized the tumors to olaparib. Although results in this animal model support a new look at Pgp as a target, in this era of "targeted therapies," trial designs that directly assess modu...