Combination Of Romidepsin Research Articles

A phase I/II trial of the combination of romidepsin and lenalidomide in patients with relapsed/refractory lymphoma and myeloma: Activity in T-cell lymphoma.

8521 Background: Epigenetic manipulation and immunomodulation are therapeutic strategies in hematologic malignancies. Romidepsin (romi), a histone deacetylase inhibitor, and lenalidomide (len), an ...

Combination of romidepsin with cyclophosphamide, doxorubicin, vincristine, and prednisone in previously untreated patients with peripheral T-cell lymphoma: a non-randomised, phase 1b/2 study

Romidepsin is a histone deacetylase inhibitor approved in the USA for patients with recurrent or refractory peripheral T-cell lymphoma and has shown activity in this setting with mainly haematological and gastrointestinal toxicity. Although it has limited efficacy, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) therapy is widely used for treatment of de-novo peripheral T-cell lymphoma. We aimed to assess the safety, tolerability, and activity of romidepsin combined with CHOP in patients with previously untreated disease. We enrolled patients aged 18-80 years with histologically proven, previously untreated, peripheral T-cell lymphoma (Eastern Cooperative Oncology Group performance status ≤2) into a dose-escalation (phase 1b) and expansion (phase 2) study at nine Lymphoma Study Association centres in France. In the dose-escalation phase, we allocated consecutive blocks of three participants to receive eight 3 week cycles of CHOP (intravenous cyclophosphamide 750 mg/m(2), doxorubicin 50 mg/m(2), and vincristine 1.4 mg/m(2) [maximum 2 mg] on day 1 and oral prednisone 40 mg/m(2) on days 1-5) in association with varying doses of romidepsin. The starting dose was 10 mg/m(2) intravenously on days 1 and 8 of each cycle, and we used a 3 + 3 design. We assessed dose-limiting toxicities only during the first two cycles. The primary endpoint was to determine the recommended dose for the combination. For the phase 2 study, we aimed to increase the cohort of patients receiving the recommended dose to a total of 25 patients. Patients were assessed for safety outcomes at least twice per cycle according to the Common Terminology Criteria for Adverse Events, version 4.0. Safety analyses included all patients who received at least one dose of romidepsin and CHOP. This trial is registered at the European Clinical Trials Database (EudraCT), number 2010-020962-91 and ClinicalTrials.gov, number NCT01280526. Between Jan 13, 2011, and May 21, 2013, we enrolled 37 patients (18 treated in phase 1b and 19 patients in phase 2). Three of six patients initially treated at 10 mg/m(2) had a dose-limiting toxicity. The dose-escalation committee decided to modify the study protocol to redefine dose-limiting toxicities with regard to haematological toxicity. Three patients were treated with 8 mg/m(2) of romidepsin, an additional three at 10 mg/m(2) (one dose-limiting toxicity), and six patients at 12 mg/m(2) (three dose-limiting toxicities). We chose romidepsin 12 mg/m(2) as the recommended dose for phase 2. Of the 37 patients treated, three had early cardiac events (two myocardial infarctions and one acute cardiac failure). No deaths were attributable to toxicity. 25 (68%) of 37 patients had at least one serious adverse event. Overall, the most frequent serious adverse events were febrile neutropenia (five [14%] of 37 patients), physical health deterioration (five [14%]), lung infection (four [11%]), and vomiting (three [8%]). 33 (89%) of patients had grade 3-4 neutropenia, and 29 (78%) had grade 3-4 thrombocytopenia. Romidepsin can be combined with CHOP but this combination should now be tested in comparison to CHOP alone in a randomised trial. Celgene.

Romidepsin Synergizes with Lenalidomide in T Cell Lymphoma Cell Lines By Increasing Reactive Oxygen Species and Modulating PI3K/AKT and MAPK/ERK Signaling Pathways

Abstract Background and purpose.T-cell lymphoma (TCL) is an heterogeneous and aggressive group of lymphoproliferative disorders which commonly relapse with current chemotherapy regimens and then difficult to treat. Therefore, novel treatment approach are urgently needed. Romidepsin is a potent and specific inhibitor of class I and II HDAC inhibitor that has shown remarkable activity in the treatment of TCL in preclinical studies and early-phase clinical trials. Lenalidomide (IMiD), is an immunomodulatory agent highly effective for the treatment of a wide range of hematological malignancies with a low toxicity profile. In this study, we sought to define the potential anticancer effect of romidepsin (Celgene) in combination with lenalidomide (Celgene) in TCL preclinical model. Methods . The TCL cell lines Hut-78 and Karpas-299 were treated with increasing concentrations of romidepsin (0,5-25 nM) and lenalidomide (1-100 µM) as single agents from 24-72h to identify the IC50 of each drug. The interaction between romidepsin (0,5nM, 1nM, 2,5nM) and lenalidomide (2µM, 4µM, 10µM) was evaluated using the Chou-Talalay method. The cell proliferation was determined using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit. The cell cytotoxicity was quantified by MTT. Clonogenic survival was studied with the methylcellulose clonogenic assay. Apoptosis, reactive oxygen species (ROS), cell cycle analysis and Bcl-2 protein expression were measured by flow cytometry, whereas caspase activation, AKT/PI3K and MAPK/ERK pathways were analyzed by Western blot. Results. Treatment with romidepsin alone resulted in time- and dose-dependent increase in cytotoxicity in Hut-78 and Karpas-299 cell lines with an IC50 at 24h of 5.87 nM and 6.36 nM for Hut-78 and Karpas-299, respectively. Lenalidomide alone did not inhibit cells viability up to72 h of treatment in the two TCL cell lines, as we previously described in other cell lines probably due to a more pleitropic (or cytostatic) effects. However, the combination of lenalidomide (10µM) with a low dose of romidepsin (2.5 nM) showed a strong synergistic interaction with combination index (CI) of 0.14 in Hut-78 cells and an additive effect in Karpas-299 cells, (CI of 1.08) after 24h. In Hut-78 cells sequential treatment with romidespin for 6h followed by washout and the next addition of lenalidomide for 24h enhanced the cytotoxic effect of romidepsin and confirmed its irreversible effect. Treatment with romidepsin and lenalidomide for 24 h reduced clonogenic survival to approximately 15 % in HUT-78 and to 28 % in Karpas-299. The combination did not trigger relevant decreased in the viability of normal peripheral blood mononuclear cells and suppressed cell proliferation of TCL cell lines when co-cultured with bone marrow mesenchymal stromal cells in a system that mimics the bone marrow microenvironment. Romidepsin and lenalidomide in combination induced apoptosis with little effect on cell cycle. Interestingly, the treatment with the caspase inhibitor z-VAD-fmk allowed detection of cell cycle arrest in G0/G1 phase and a decrease of S phase. The induction of apoptosis by drugs combination was associated with an increase in ROS production. The co-administration of the antioxidant N-acetyl cysteine (NAC) blocked the increased ROS generation and apoptosis observed in the drugs combination compared with control or single-agent treatments. Cell death was associated with the activation of caspases -3, -9 -8 and PARP and was mediated by Bim, Bax, Bad, Bcl-xL and Mcl-1 but did not modify Bcl-2 expression. The drugs combination induced up-regulation of cell cycle protein p21, p27 and a slight decrease of cyclin E and cyclin D. These events were associated with the dephosphorylation of PI3K/Akt and MAPK/ERK pathways. Finally, the induction of histone acetylation (H3) and acetylated alpha-tubulin was confirmed. Conclusion . The combination of romidepsin with lenalidomide triggered cell death through interacting mechanisms including ROS generation, caspase activation and down-regulation of PI3K/AKT and MAPK/ERK pathways. These results provided a rational basis for the clinical development of the combination of romidepsin and lenalidomide for the treatment of TCL Disclosures No relevant conflicts of interest to declare.

Final Analysis of the RO-CHOP Phase Ib/II Study: Romidepsin in Association with CHOP in Patients with Peripheral T-Cell Lymphoma (PTCL)

Background: Romidepsin is a histone deacetylase inhibitor approved by the FDA for patients with cutaneous T-cell lymphoma and PTCL who have received at least 1 prior therapy. In recurrent/refractory PTCL, it has been evaluated as a single agent in 2 phase II studies with overall response rates of 25-38%(Piekarz, Blood 2011 & Coiffier, J Clin Oncol 2012). Toxicity is mainly hematologic and digestive. We here present the final analysis of a phase Ib/II trial aiming to evaluate the safety, tolerability and efficacy of romidepsin in association with CHOP in patients with previously untreated PTCL.Patients & methods: Patients with PTCL were planned to receive 8 cycles of CHOP(cyclophosphamide 750 mg/m2 day 1, doxorubicin 50 mg/m2 day 1, vincristine 1,4 mg/m2 day 1, prednisone 40 mg/m2 days 1 – 5) in association with varying doses of romidepsin. Based on pharmacokinetic data and results of previous phase II studies, the starting dose of 10 mg/m2 days 1 & 8 was chosen. The dose-variation scheme followed a traditional “3+3” design. Dose-limiting toxicities (DLT) were considered during the first 2 cycles. DLT was initially defined as: Non hematological toxicity grade 3-4 or hematological toxicity grade 3 lasting more than 7 days or grade 4 lasting more than 3 days. The protocol was subsequently amended to tolerate if lasting less than 10 (grade 3) or 7 days (grade 4).Results: Eighteen patients have been treated in phase Ib of the study (Dupuis, Hematol Oncol 2013). Significant, albeit tolerable hematological toxicity having been observed in the first two cohorts, the definition of DLT was modified during the course of the study. The dose of 12 mg/m2 was chosen as the recommended dose for phase II. Nineteen patients were treated in the phase II part of the study. Two patients had an early cardiac event (myocardial infarction) and were excluded from the efficacy analysis. A third patient had a cardiac event (acute cardiac failure) after cycle 1 day one and continued on CHOP alone. There were no deaths attributable to toxicity. Late hematological toxicity was observed, thus some cycles after cycle 2 were delivered with only one administration of romidepsin at day 1: Among 509 planned romidepsin administrations, 93 (18%) were cancelled on decision of the investigator, mainly during the last 2 cycles and mainly because of hematological toxicity. Sixty-seven percent of patients had at least on serious adverse event (SAE). The most frequent SAEs were: Febrile neutropenia (13.5%), general physical health deterioration (13.5%), lung infection (10.8%), vomiting (8%). Thirty-eight percent of patients had Grade 3-4 neutropenia at any time of the study, 19% grade 3-4 thrombocytopenia, 8% grade 3-4 anemia. Observed response rates (IHP 2007 criteria) were (n=35): Complete response 51%, partial response 17%, and progressive disease 25%. With a median follow-up of 17.5 months, the estimated progression-free survival is 57% at 18 months with 5 progressions among the 24 responding patients. The overall survival rate at 18 months is 76,5%. Longer follow-up will be presented during the meeting.Conclusion: Romidepsin can be combined with CHOP at the price of foreseeable hematological toxicity. Some cardiovascular events have been observed but the relationship with romidepsin is questionable. The progression-free survival rates seem promising, and the combination of romidepsin and CHOP is actually compared with CHOP alone in the setting of a phase III randomized trial (phase III RO-CHOP study).References :1. Piekarz R, Blood. 2011;117(22):5827-34.2. Coiffier B, J Clin Oncol. 2012;30(6):631-63. Dupuis J, Hematological Oncology 2013;31(Suppl.1):96-150 DisclosuresOff Label Use: Romidepsin in association with CHOP for first line treatment of peripheral T-cell lymphoma. Morschhauser:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Tilly:Celgene: Research Funding. Coiffier:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

A Phase I Trial of Alisertib Plus Romidepsin for Relapsed/Refractory Aggressive B- and T-Cell Lymphomas

A Phase I Trial of Alisertib Plus Romidepsin for Relapsed/Refractory Aggressive B- and T-Cell Lymphomas

Abstract 1693: The combination of romidepsin and bendamustin is synergistically cytotoxic and reverses the malignant phenotype in preclinical models of T-cell lymphoma

Abstract Background: Peripheral T-cell lymphomas (PTCLs) represent approximately 10-15% of all non-Hodgkin lymphomas (NHL) in the Western world, and their incidence is increasing. Cases of PTCL tend to have an aggressive clinical course, with poor patient responses to conventional chemotherapy and poor long-term survival. So far, treatment approaches have mirrored diffuse large B-cell lymphoma (DLBCL) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) and CHOP-like chemotherapy are commonly used despite suboptimal results. Histone deacetylase inhibitors (HDACs) have been approved for the treatment of relapsed or refractory PTCLs given their single-agent activity in these diseases. To continue to improve responses in patients with PTCL, it is important to assess the utility of romidepsin as frontline therapy and as a component of combination therapies. Aim: Interactions between romidepsin (R) and bendamustine (B), a potent cytotoxic alkylating drug active against a panel of other lymphoproliferative disorders, was investigated in in preclinical models of T-cell Lymphoma. Experimental Design: Assays for cytotoxicity on 5 different T-cell lymphoma and leukemia cell lines (Jurkat, HD-MAR2, Karpas, Sup-T1, HH), mathematical analysis for synergism (Chou-Talalay equation), flow cytometry, and the Itk-Syk transgenic mouse model (K. Pechloff, 2010) were used to explore the in vitro and in vivo activities of R and B alone and in combination in T-cell lymphoid malignancies. Results: In vitro, romidepsin and bendamustine exhibited concentration- and time-dependent cytotoxicity against all the 5 different T-cell lymphoma and leukemia cell lines. Romidepsin showed synergism when combined with bendamustine in all cell lines studied. Romidepsin also induced potent apoptosis and caspase activation when combined with bendamustine across the panel. The impact of schedule on the activity of the combination was determined by assessing cell viability after treatment with B and R as follows: (1) simultaneous exposure; (2) B pretreatment followed by exposure to R; and (3) R pretreatment followed by exposure to B. In a new mouse model of PTCL in which a status of permanent T cell activation mediated by the Itk-Syk transcript induces highly malignant PTCLs with 100% penetrance that resemble the human disease, the combination of romidepsin and bendamustine enhanced efficacy compared with either drug alone. Conclusions: Collectively, these data strongly support the potential therapeutic role of romidepsin in combination with bendamustine for PTCLs and might constitute the basis for future phase I-II clinical trials. Citation Format: Cristiana Carniti, Silvia Gimondi, Antonio Vendramin, Sara Rizzitano, Paolo Corradini. The combination of romidepsin and bendamustin is synergistically cytotoxic and reverses the malignant phenotype in preclinical models of T-cell lymphoma. [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 1693. doi:10.1158/1538-7445.AM2014-1693

Abstract 5536: 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 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<0.001). Combined treatment with romidepsin and selumetinib, PD0325901 or vemurafenib resulted in a significantly higher percent of annexin positive cells (p<0.05) in 9 to 11 of the 11 BRAF mutant cell lines examined compared to treatment with romidepsin alone; expression of dephosphorylated Bim was observed. Immunoblot analysis demonstrated inhibition of the MAPK pathway at the concentrations used; apoptosis was associated with increased PARP cleavage. Using mitochondrial separation techniques, we also observed increased levels of Bim in the mitochondrial membrane fraction and reduced expression of mitochondrial hexokinase 2 in cells treated with combined romidepsin/MEK inhibitor compared to either compound alone. Our results suggest a critical role for apoptotic pathways in cell death due to romidepsin, that MAPK signaling promotes resistance via Bim degradation, and that combined treatment with romidpesin and BRAF or MEK inhibitors may be useful in the treatment of cancers harboring BRAF mutations. 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

A phase I/II trial of the combination of romidepsin and lenalidomide in patients with relapsed/refractory lymphoma and myeloma: Phase I results.

8582 Background: Epigenetic manipulation and immunomodulation are therapeutic strategies in hematologic malignancies. Romidepsin (Romi), a histone deacetylase inhibitor, and lenalidomide (Len), an ...

Romidepsin induces cell cycle arrest, apoptosis, histone hyperacetylation and reduces matrix metalloproteinases 2 and 9 expression in bortezomib sensitized non-small cell lung cancer cells

Histone deacetylase (HDAC) inhibitors have been proven to be effective therapeutic agents to kill cancer cells through inhibiting HDAC activity or altering the structure of chromatin. We recently reported that chemotherapy by the HDAC inhibitor, romidepsin activates the anti- apoptotic transcription factor NF-κB in A549 non-small cell lung cancer (NSCLC) cells and fails to induce significant levels of apoptosis. We also demonstrated that NF-κB inhibition with proteasome inhibitor bortezomib enhanced HDAC inhibitor induced mitochondrial injury and sensitize A549 NSCLC cells to apoptosis through the generation of reactive oxygen species. In this study, we investigate whether combined treatment with romidepsin and bortezomib would induce apoptosis in A549 NSCLC cells by activating cell cycle arrest, enhanced generation of p21 and p53, down-regulation of matrix metalloproteinases (MMPs) 2,9 also altering the acetylation status of histone proteins. Our data show that combination of romidepsin and bortezomib caused cell cycle arrest at Sub G0-G1 transition, up-regulation of cell cycle protein p21 and tumour suppressor protein p53. In addition, romidepsin down-regulated the expression of MMP-2,9 and hyperacetylation of histone H3 and H4 in bortezomib sensitised A549 NSCLC cells. From this study we concluded that romidepsin and bortezomib cooperatively inhibit A549 NSCLC cell proliferation by altering the histone acetylation status, expression of cell cycle regulators and MMPs. Romidepsin along with bortezomib might be an effective treatment approach for A549 NSCLC cells.

Romidepsin and Lenalidomide Show a Synergistic Effect In T–Cell Lymphoma Cell Lines

Background and purposeSuccessful treatment of T cell lymphoma (TCL) is still problematic and identification of new pharmacological targets in this malignancy is urgently needed. Histone deacetylase (HDAC) inhibitors are emerging as an exciting new therapeutic option for lymphoid malignancies. These drugs increase the acetylation status and modulate the activity of a wide range of non-histone proteins, and effects on both histone and non-histone proteins may contribute to their anti-cancer activity. Romidepsin (depsipeptide) is a potent and specific inhibitor of class 1 HDACs that has shown remarkable activity in the treatment of TCL in preclinical studies and early-phase clinical trials. Lenalidomide belongs to the immunomodulatory agents (IMID®) and it is has been demonstrated to be very active for the treatment of several types of hematological neoplasia. Purpose of the present study was to determine whether lenalidomide potentiates romidepsin activity in TCL cell lines and if so, by which mechanisms. MethodsTCL cell lines (Hut-78: cutaneous TCL cells and Karpas-299 anaplastic lymphoma cells) were treated with increasing concentrations of either romidepsin (0.5 - 25 nM) or lenalidomide (1 - 100 µM), and IC50at 24-48 and 72 hours was calculated. The interaction between romidepsin (0.5, 1, 2.5 nM) and lenalidomide (2, 4, 10 µM) was evaluated using the Chou-Talalay method to determine if the combination had additive or synergistic effects. The cell cytotoxicity was assessed by MTT assay and apoptosis was measured with annexin-V/propidium iodide (PI) by flow cytometry. Caspase activation was confirmed by Western blot analysis. The effect of the combination on AKT/PI3K and MAPK/ERK signaling pathways and cyclin D1 expression was evaluated by Western blot. ResultsTreatment with romidepsin alone resulted in time- and dose-dependent cytotoxicity in both cell lines. The IC50 of romidepsin at 24-hour was 5.87 nM and 6.36 nM in Hut-78 and Karpas-299 respectively. Lenalidomide alone did not induce a cytotoxic effect, and we were unable to reach IC50 even after 72 h of treatment. However, after 24 hours, the combination of romidepsin (2.5 nM) and lenalidomide (10µM) (ratio 1:4) showed a strong synergistic interaction with a CI (combination index) of 0.14 in Hut-78 cells, and an additive effect with a CI of 1.08 in Karpas-299 cells. In HUT-78 cells, the combination of romidepsin and lenalidomide enhanced apoptosis compared with each drug alone by the activation of caspases-3, -9 and -8. No change in the expression of Bcl-2 was observed with either treatment alone, or in combination. These events were associated with dephosphorylation of PI3K/Akt and MAPK/ERK pathways, decreased expression of cyclin D1 and Bcl-xL, and an accumulation of acetylated alpha-tubulin. The combination had relatively modest effect on cell cycle parameters. The analysis of the cell cycle showed an increase percentage of cells in sub G0/G1 and G2/M phase, and decrease of S phase. ConclusionThese preliminary results indicate that the combination of romidepsin with lenalidomide has a synergistic effect in TCL cell lines and induces apoptosis through signaling events involving pro-survival pathways PI3K/AKT and MAPK/ERK, down-regulation of cyclin D1 and accumulation of acetylated alpha-tubulin. Data look promising and further investigations are required to better define the molecular mechanisms of cell death induced by the combination of romidepsin and lenalidomide in T cell lymphoma. Disclosures:No relevant conflicts of interest to declare.

The HDACi Romidepsin Markedly Synergizes With Inhibition Of ATM By KU60019 In Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a disease characterized by gross cell cycle dysregulation driven by the constitutive overexpression of cyclin D1. The identification of a “proliferation signature” in MCL, underscores the necessity of new therapeutic approaches aimed at lowering the proliferative signature of the disease, theoretically shifting the prognostic features of the disease.Romidepsin, an HDAC inhibitor (HDACi) approved for the treatment of relapsed T-cell lymphoma, is thought to induce cell cycle arrest and apoptosis. Central to the block of cell proliferation is the up-regulation of the cdk inhibitor p21Cip1/Waf1. However up-regulation of p21Cip1/Waf1 has also been shown to reduce sensitivity to romidepsin. HDACi activates p21Cip1/Waf1 expression via ATM and KU60019, a specific ATM inhibitor, has been shown to decrease the p21Cip1/Waf1 protein levels in a concentration dependent manner. We sought to explore the effect of the combination of romidepsin and KU60019 in inducing cell death in MCL.Analysis of romidepsin treated Jeko-1 cell extracts showed a marked effect on the expression of proteins involved in cell cycle regulation. Decrease expression of Emi1, a mitotic regulator required for the accumulation of the APC/C substrates was observed. Emi1 is also responsible for the stability of the E3 ubiquitin ligase Skp2 that specifically recognizes and promotes the degradation of phosphorylated cdk inhibitor p27. However, decrease in Emi1 protein levels, upon addition of romidepsin, was not followed by an increased expression of the cdk inhibitor p27. On the other end, increased expression of the cdk inhibitor p21Cip1/Waf1, was a common feature of all romidepsin treated MCL lines analyzed. Cell cycle analysis via Fluorescent Activated Cell Sorting (FACS) of romidepsin treated Jeko-1 cells showed an accumulation of romidepsin treated cells in the G2/M phase when compared to the control suggesting a p21Cip1/Waf1 induced cell cycle arrest.For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-GloTM followed by acquisition on a Biotek Synergy HT and IC50s calculated using the Calcusyn software. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR). Synergy analyses were performed using Jeko-1, Maver-1 and Z-138 cells treated with different concentrations of romidepsin corresponding to IC10-20 in combination with KU60019 at a concentration of 2.5, 5.0, 7.5 and 15 umol/L for 24, 48 and 72 hours. A synergistic cytotoxic effect was observed in all MCL cell lines when the HDACi was combined with KU60019 throughout the range of all concentrations. The RRR analysis showed a strong synergism at 48 and 72 hours in virtually all combinations of HDACi and KU60019 in all three cell lines. The results of drug:drug combination in two of the three cell lines are shown below. Protein expression analysis of Jeko-1 and Maver-1cells treated with single agents or combinations for 48 hours revealed changes in a host of proteins known to be involved in cell cycle control and apoptosis. The increased p21 protein expression upon addition of romidepsin, was not observed when the romidepsin treatment was combined with the KU60019. Increased activation of the programmed cell death proteins Caspase 8, induced by Fas, and Caspase 3 was observed upon combinations of the single agents in all three cell lines, resulting in an increased cleavage of Poly (ADP-ribose) polymerase (PARP-1). Finally, the abundance of the anti-apoptotic proteins Bcl-XL and BCL-2 showed a significant decrease after treatment with romidepsin plus increase concentrations of KU60019 when compared with their abundance in the presence of the single agents. Cell cycle analysis of Jeko-1 cells treated for 24 hours with single agents and combination suggests that the increased apoptosis is the result of inhibition of the p21Cip1/Waf1 induced G2/M cell cycle arrest by KU60019. Overall, these data demonstrated that the combination of romidepsin and KU60019 was synergistically effective in inhibiting the in vitro growth of the mantle cell lymphoma lines. [Display omitted] Disclosures:O'Connor: Celgene: Consultancy, Research Funding.

MAPK pathway activation leads to Bim loss and histone deacetylase inhibitor resistance: rationale to combine romidepsin with an MEK inhibitor

To identify molecular determinants of histone deacetylase inhibitor (HDI) resistance, we selected HuT78 cutaneous T-cell lymphoma (CTCL) cells with romidepsin in the presence of P-glycoprotein inhibitors to prevent transporter upregulation. Resistant sublines were 250- to 385-fold resistant to romidepsin and were resistant to apoptosis induced by apicidin, entinostat, panobinostat, belinostat, and vorinostat. A custom TaqMan array identified increased insulin receptor (INSR) gene expression; immunoblot analysis confirmed increased protein expression and a four- to eightfold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation in resistant cells compared with parental cells. Resistant cells were exquisitely sensitive to MEK inhibitors, and apoptosis correlated with restoration of proapoptotic Bim. Romidepsin combined with MEK inhibitors yielded greater apoptosis in cells expressing mutant KRAS compared with romidepsin treatment alone. Gene expression analysis of samples obtained from patients with CTCL enrolled on the NCI1312 phase 2 study of romidepsin in T-cell lymphoma suggested perturbation of the MAPK pathway by romidepsin. Immunohistochemical analysis of Bim expression demonstrated decreased expression in some skin biopsies at disease progression. These findings implicate increased activation of MEK and decreased Bim expression as a resistance mechanism to HDIs, supporting combination of romidepsin with MEK inhibitors in clinical trials.

Abstract 3399: Resistance to the histone deacetylase inhibitor romidepsin is associated with degradation of Bim following MAPK pathway activation.

Abstract Inhibition of histone deacetylase (HDAC) enzymes represents a promising therapeutic approach in clinical oncology, as aberrant gene expression and alterations in histone acetylation due to HDACs have been implicated in tumor development and progression. Even though several histone deacetylase inhibitors (HDIs) are currently in clinical trials, so far only the HDIs romidepsin and vorinostat have been approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma (CTCL). During clinical trials with romidepsin in CTCL, disease progression was noted in some patients who initially responded to therapy, while the disease in other patients did not respond to therapy suggesting that both de novo and acquired resistance to romidepsin were observed. To identify molecular determinants of resistance, we selected HuT78 CTCL cells with romidepsin in the presence of inhibitors of P-glycoprotein (Pgp) to prevent upregulation of Pgp as a mechanism of resistance. Resistant sublines were approximately 250- to 385-fold resistant to romidepsin; the Pgp inhibitor tariquidar did not significantly reverse resistance. The sublines also exhibited resistance to apoptosis following treatment with the HDIs apicidin, belinostat, entinostat, panobinostat, and vorinostat. A custom gene-expression array detected elevated expression of insulin receptor (INSR) in romidepsin resistant cells compared to parental cells. Immunoblot analysis of downstream effectors of the IR pathway demonstrated a 4- to 8-fold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation. Even though resistant cells did not respond to 48 h treatment with inhibitors of the insulin receptor, they exhibited exquisite sensitivity to treatment with as little as 1 nM of the MEK inhibitor PD0325901. Sensitivity to MEK inhibition in resistant cells was associated with restoration of the pro-apoptotic protein Bim. Combined treatment of romidepsin with MEK inhibitors also significantly yielded greater apoptosis in resistant cells compared to romidepsin and MEK inhibitor treatment alone. Gene expression analysis of circulating tumor samples obtained from patients with CTCL enrolled on the NCI 1312 Phase II romidepsin study suggested interaction of romidepsin with the MAPK pathway, indicated by altered expression of genes demonstrated to be under its control. These findings implicate activation of MEK as a resistance mechanism to romidepsin, and suggest combination of romidepsin with MEK inhibitors in clinical trials. Citation Format: Arup R. Chakraborty, Rob Robey, Zhirong Zhan, Victoria Luchenko, Michael Gottesman, Nathan Collie, Jean-Pierre Gillet, Richard Piekarz, Andrew Kossenkov, Louise Showe, Susan Bates. Resistance to the histone deacetylase inhibitor romidepsin is associated with degradation of Bim following MAPK pathway activation. [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 3399. doi:10.1158/1538-7445.AM2013-3399

A high rate of durable responses with romidepsin, bortezomib, and dexamethasone in relapsed or refractory multiple myeloma

AbstractWe report results from a study exploring the combination of romidepsin, bortezomib, and dexamethasone for the treatment of patients with multiple myeloma (MM) previously treated with > 1 prior therapy. The primary objective was to determine the maximum tolerated dose (MTD) of the combination using a novel accelerated dose-escalation schedule in patients with relapsed or refractory MM. The secondary objective was to determine overall response (OR), time to progression (TTP), and overall survival (OS). The MTD identified was bortezomib 1.3 mg/m2 (days 1, 4, 8, and 11), dexamethasone 20 mg (days 1, 2, 4, 5, 8, 9, 11, and 12), and romidepsin 10 mg/m2 (days 1, 8, and 15) every 28 days. Thrombocytopenia (64%) was the most common ≥ grade 3 hematologic toxicity. Peripheral neuropathy occurred in 76% of patients (n = 19) (≥ grade 3, 8%; 95% confidence interval [CI] 1%-26%). Maintenance romidepsin 10 mg/m2 (on days 1 and 8 of a 28-day cycle) proved feasible, with 12 patients receiving a median of 7.5 cycles (range: 1-29). An OR (M-protein) of > minor response (MR) was seen in 18 of 25 patients (72%); 2 (8%) had complete remissions (CRs) and 13 (52%) had partial responses (PRs), including 7 (28%) with very good PRs (VGPRs). The median TTP was 7.2 (95% CI: 5.5-19.6) months, and the median OS was > 36 months. This regimen shows activity with manageable toxicity and warrants further evaluation. This trial was registered at www.clinicaltrials.gov as NCT00431990.

Abstract 2624: Elevated expression of phosphorylated mitogen activated protein kinase kinase (MEK) as a mechanism of resistance to the histone deacetylase inhibitor romidepsin in HUT 78 cutaneous T-cell lymphoma cells

Abstract Elevated expression of phosphorylated mitogen activated protein kinase kinase (MEK) as a mechanism of resistance to the histone deacetylase inhibitor romidepsin in HUT 78 cutaneous T-cell lymphoma cells. Histone deacetylase inhibitors (HDIs) have shown promise in the treatment of T-cell lymphomas including cutaneous and peripheral T-cell lymphomas. However, resistance to romidepsin limits its activity in some patients. A detailed understanding of the mechanisms of resistance to HDIs may lead to strategies designed to increase clinical efficacy. To study mechanisms of resistance to the HDI romidepsin, the HUT78 cutaneous T-cell lymphoma cell line was exposed to increasing concentrations of romidepsin in the presence of the P-glycoprotein (P-gp) inhibitors verapamil or valspodar (PSC-833) to prevent the emergence of P-gp, a known resistance mechanism. The DpVp35 and DpVp50 sublines are maintained in 35 ng/ml and 50 ng/ml romidepsin, respectively, in the presence of 5 µg/ml verapamil while DpP75 is maintained in 75 ng/ml romidepsin and 3 µg/ml valspodar. In 4-day cytotoxicity assays, the sublines are approximately 55-fold resistant to romidepsin and are not cross resistant to the HDIs belinostat, panobinostat or vorinostat. Low but detectable levels of P-gp do not explain the resistance. We used a custom drug resistance gene expression array and found increased expression of insulin receptor (IR) in the resistant cells that was confirmed by immunoblot analysis. Elevated expression of phosphorylated mitogen activated protein kinase kinase (MEK), a downstream effector of the IR pathway, was also observed in the resistant cells compared to the parental cells. Interestingly, resistant cells were found to be exquisitely sensitive to MEK inhibition, as significant apoptosis was observed after 48 h in the presence of 5 nM of the MEK inhibitor PD0325901 and 10 nM of the MEK inhibitor AZD 6244 as measured by flow cytometry with annexin V and by immunoblot examining poly (ADP-ribose) polymerase (PARP) cleavage. No significant apoptosis was observed in parental cells at concentrations up to 500 nM. Resistant cells were not, however, sensitive to extracellular related kinase (ERK) inhibition or phosphatidylinositol 3-kinase (PI3K) inhibition in as determined by annexin V assay. In summary, we hypothesize that activated MEK can mediate resistance to romidepsin, but may also lead to collateral sensitivity to MEK inhibitors. The emerging role of activated MEK as a mechanism of resistance to romidepsin suggests combination of romidepsin with MEK inhibitors in future clinical trials. 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 2624. doi:10.1158/1538-7445.AM2011-2624

The Combination of Romidepsin and Bortezomib Results in Synergistic Induction of Apoptosis in Human B-Lymphoma Cell Lines.

Abstract 1689Poster Board I-715 IntroductionThe use of the proteasome inhibitor bortezomib has demonstrated activity in multiple myeloma and lymphomas. The HDAC inhibitor romidepsin is being evaluated in CTCL and PTCL, though its activity in B-cell lymphomas is less clear. We hypothesized that the combination of bortezomib and romidepsin would result in synergistic apoptosis in different B-cell NHL cell lines based upon the observed activity of this combination in more mature B-cell malignancies such as myeloma. Experimental DesignDaudi, HT, Ramos and SUDHL-4 cell lines were exposed to different concentrations of bortezomib and romidepsin, separately, concurrently, and sequentially. Cell viability was assessed using MTT-assay, induced apoptosis was evaluated using Annexin V and PI staining from 24-48 hours. Apoptosis was also evaluated using western blot analysis of caspases and PARP cleavage. LC3 and HDAC6 level expressions were performed to determine if the effect of the combination was a result of the aggresome or autophagy pathway. Cell cycle studies were also performed to study if there were any changes after treating cells with the combination. ResultsThe combination of bortezomib and romidepsin resulted in synergistic B-cell apoptosis as measured by MTT-assay with combination indices of < 0.5. This was associated with increased caspases and PARP cleavage as early as 24 hours after exposure. Order of addition experiments demonstrated definite sequence specificity. When romidepsin was added first, and 6 hours later followed by bortezomib, apoptosis was enhanced, compared to both agents being given concurrently or when bortezomib was administered first. Cell cycle analysis studies demonstrated that pretreatment of cells with romidepsin for 6 hours followed by the addition of bortezomib arrested the cells in G2M phase. HDAC6 expression was significantly reduced following combination therapy, and LC3-I was cleaved to LC3-II in treated cells suggesting that the combination affected aggresome formation and autophagy. ConclusionThe combination of romidepsin and bortezomib at low nanomolar concentrations suggests that this may be an important clinical combination to test in patients with relapsed or refractory B-cell malignancies. Sequence of administration data is currently being tested to determine if the effect is a result of autophagy inhibition as is seen in myeloma cell lines. Additional mechanistic studies will be presented with the goals of identifying predictors of response that can then be validated in prospective clinical trials. DisclosuresLechowicz:Gloucester: Consultancy. Kaufman:Millennium: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Gloucester: Research Funding; Novartis: Consultancy; BMS: Consultancy; Millennium: Consultancy, Research Funding; Celgene: Consultancy. Flowers:Millennium: Research Funding.