Combination Of Vorinostat Research Articles

Vorinostat enhances gefitinib‑induced cell death through reactive oxygen species‑dependent cleavage of HSP90 and its clients in non‑small cell lung cancer with the EGFR mutation.

Although different mechanisms of acquired resistance to epidermal growth factor receptor (EGFR)‑tyrosine kinase inhibitors (TKIs) have been reported in non‑small cell lung cancers (NSCLCs), the optimal treatment for patients with acquired resistance has not been clearly defined. The purpose of this study was to investigate the antitumor effects of gefitinib in combination with vorinostat, a potent histone deacetylase inhibitor (HDACI), and their associated molecular mechanisms in relation to activating apoptosis in NSCLC. The treatment using a combination of vorinostat and gefitinib was more potent in promoting cell death by activating apoptosis than gefitinib alone in parental PC9 cells that harbor an EGFR‑activating mutation (EGFR exon 19 deletion) and gefitinib‑resistant PC9 cells (PC9GR) with an EGFR T790M mutation. This combination induced heat shock protein 90 (HSP90) cleavage and reduced the level of HSP90 client proteins, including EGFR, MET and AKT, in PC9 and PC9GR cells. The addition of 4‑(2‑aminoethyl) benzenesulfonyl fluoride hydrochloride, a scavenger of reactive oxygen species (ROS), inhibited the degradation of HSP90 client proteins and HSP90 cleavage that was induced by co‑treatment as well as the cleavage of caspase‑3, caspase‑8, and caspase‑9 and cell death. We also observed that cleavage of HSP90 and its clients were blocked when caspases were inhibited. These results revealed that co‑treatment with vorinostat and gefitinib induced ROS‑dependent caspase activation, leading to the downregulation of HSP90 clients through HSP90 cleavage. Collectively, our findings provide a new basis for strategies that combine vorinostat with an EGFR‑TKI to reverse EGFR‑TKI resistance in NSCLC.

Prior exposure of pancreatic tumors to [sorafenib + vorinostat] enhances the efficacy of an anti-PD-1 antibody

ABSTRACTCheckpoint immunotherapy antibodies have not shown efficacy in pancreatic adenocarcinoma. Pre-clinical studies and subsequently an on-going phase I trial have demonstrated the safety and efficacy of combinatorial radio-chemotherapy plus surgery in this malignancy, including the combination of sorafenib and vorinostat. The lethality of [sorafenib + vorinostat] was enhanced by gemcitabine. Exposure to [sorafenib + vorinostat] reduced the expression of β-catenin, ERBB1, BCL-XL and MCL-1, and the phosphorylation of AKT T308, AKT S473, GSK3 S9/21, mTORC1 and mTORC2. The drug combination increased the expression of Beclin1 and the phosphorylation of eIF2α S51. The drug combination rapidly reduced the levels of multiple HDAC proteins that was directly associated with the previously noted changes in tumor cell biology, as well as with alterations in the expression of biomarkers predictive for a response to checkpoint inhibitor antibodies. In vivo studies using the PAN02 model in its syngeneic mouse demonstrated that an anti-PD-1 antibody had no impact on tumor growth whereas a transient exposure to [sorafenib + vorinostat] significantly suppressed growth. The combination of [sorafenib + vorinostat] with an anti-PD-1 antibody caused a significant further reduction in tumor growth compared to the drug combination alone. Tumors transiently exposed three weeks earlier to [sorafenib + vorinostat] contained elevated levels of CD8+ cells, M1 macrophages and natural killer cells. Drug exposure plus an anti-PD-1 antibody further significantly enhanced the levels of these immune cells in the tumor. Our data argue for performing a new phase I trial in pancreatic cancer combining immunotherapy with [sorafenib + vorinostat].Abbreviations: ERK: extracellular regulated kinase; PI3K: phosphatidyl inositol 3 kinase; ca: constitutively active; dn: dominant negative; ER: endoplasmic reticulum; AIF: apoptosis inducing factor; AMPK: AMP-dependent protein kinase; mTOR: mammalian target of rapamycin; JAK: Janus Kinase; STAT: Signal Transducers and Activators of Transcription; MAPK: mitogen activated protein kinase; PTEN: phosphatase and tensin homologue on chromosome ten; ROS: reactive oxygen species; CMV: empty vector plasmid or virus; si: small interfering; SCR: scrambled; IP: immunoprecipitation; VEH: vehicle; HDAC: histone deacetylase.

Abstract 1884: Dual targeting of cell cycle checkpoint and histone deacetylase overcomes bortezomib resistance in multiple myeloma

Abstract Although the introduction of novel agents, such as proteasome inhibitors, immunomodulatory drugs, and histone deacetylase inhibitors (HDACi) has improved the clinical outcomes of the patients with multiple myeloma (MM), most patients eventually relapse and develop drug resistance. Proteasome inhibitor, bortezomib (BTZ), is a key drug for the treatment of MM, and the prognosis of the patients with BTZ resistance is very poor. Therefore, novel therapeutic approaches to overcome BTZ resistance are urgently needed. WEE1 is a cell cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 induces cell cycle arrest, allowing for the damaged DNA to be repaired before the cell undergoes DNA replication, and prevents cells from mitotic lethality. WEE1 is overexpressed in various cancers, and relates to poor prognosis. In addition, the inhibition of WEE1 has been shown to induce cancer cells to premature mitotic entry leading to apoptotic cell death, suggesting that combining WEE1 inhibitor with DNA damaging agent is effective in cancer therapy. Resent research has shown that HDACi increase reactive oxygen species production resulting in increased DNA damage and impaired DNA repair. These data suggest that the combination of WEE1 inhibitor and HDACi might be an attractive therapeutic option against this incurable hematological malignancy. In the present study, we investigated the efficacy of WEE1 inhibitor MK-1775, a potent and highly-selective small molecule inhibitor of WEE1, in combination with HDACi in various MM cells. MTS assays showed that single agent MK-1775 inhibited the proliferation of various MM cell lines, including BTZ-resistant cell line, in a dose- (0 to 10 μM) and time- (0 to 72 h) dependent manner. In combination with vorinostat which is a pan-HDACi, MK-1775 inhibited the proliferation of BTZ-resistant MM cells more effectively than single agent. Furthermore, the combination of vorinostat and MK-1775 was able to re-sensitize BTZ-resistant MM cells to BTZ. Assays for apoptotic cell death demonstrated that MK-1775 induces both early and late apoptosis in MM cells. Western blotting analysis was performed to investigate the molecular mechanisms of MK-1775-induced cell death, showing that MK-1775 inhibited the expression of the target molecules of Bcl-2 and MCL1, and CDK1 phosphorylation. Moreover, the marked increase in PARP and Caspase 3 cleavage, and histone H2AX expression was observed following treatment with MK-1775 in combination with vorinostat. These findings suggest that the combination of MK-1775 and vorinostat induces synergistically DNA damage and promotes premature mitotic entry resulting in apoptotic cell death in MM cells. In conclusion, our data suggest that WEE1 in combination with HDACi might be a promising molecular target for the treatment of BTZ-resistant MM. Citation Format: Takayuki Tabayashi, Yuka Tanaka, Yasuyuki Takahashi, Yuta Kimura, Tatsuki Tomikawa, Tomoe Anan, Morihiko Sagawa, Reiko Watanabe, Michihide Tokuhira, Masahiro Kizaki. Dual targeting of cell cycle checkpoint and histone deacetylase overcomes bortezomib resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1884.

Abstract 5800: Vorinostat and indole-3-carbinol modulate microRNAs and histone deacetylase activity in subtypes of triple-negative breast cancer

Abstract Triple-negative breast cancer is a highly heterogeneous and aggressive subtype of breast cancer. Epigenetic drugs are continuing to emerge as promising therapies for triple-negative breast cancer (TNBC). These drugs, including the one used in this study, vorinostat, a histone deacetylase inhibitor (HDAC), are capable of multiple actions and are considered multifunctional drugs. Epigenetic mechanisms have been shown to play a role in the etiology of this cancer, through modulation of DNA methylation, histone modification or microRNAs. We have shown in this study that vorinostat and indole-3-carbinol (I3C) modulated the expression of microRNAs 221 and 222 differently in basal-like-2 (BL2), MSL, basal-like1 (BL1) and M subtypes of TNBC cells. Additional studies demonstrated that combination of tamoxifen and vorinostat significantly decreased expression of miRNA 221 in TNBC cell lines. MiRNAs are short noncoding RNAs that regulate the function of target genes post-transcriptionally. In addition, breast cancer microarrays further showed modulation of a number of other miRNAs that play a role in cell proliferation, drug resistance, cell invasion and metastasis. MicroRNAs are epigenetic targets for drug development. Using a global HDAC activity assay, it was also shown that subtypes of TNBC cells respond differently to vorinostat and I3C treatment, depending on their HDAC activity. HCC70, an African American cell line and a BL2 subtype, had the highest HDAC activity and its activity was significantly decreased by vorinostat treatment compared to BT549 (M), HCC 1806 (BL2), MCF7 (ER positive) and a basal nonmalignant cell line, MCF10A. Both vorinostat and I3C show great promise alone and in combination as effective agents in triple-negative breast cancer. Histone deacetylase inhibitors and dietary agents are emerging as therapy for triple-negative breast cancer and are currently in clinical trials. Citation Format: Beverly Lyn-Cook, Fatemeh NouriEmamzaden, Anfernee Hawkins, George Hammons, Beverly Word. Vorinostat and indole-3-carbinol modulate microRNAs and histone deacetylase activity in subtypes of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5800.

Abstract 1253: Dissecting the signaling cascades involved in growth inhibition and apoptosis of prostate cancer cells exposed to a novel bis-aspirinyl selenazolidine AS-10

Abstract Aspirin has been recommended for lowering risk of colorectal cancer, in addition to its cardiovascular health benefit at low dose (baby Aspirin) and over-the-counter pain medication. Through an innovative research program focused on enhancing its anticancer activity by incorporation of selenium, we identified bis-aspirinyl selenazolidine (AS-10) as a novel promising lead compound against different cancer cell types in the NCI-60 cancer cell line panel, including prostate cancer (PCa) (e.g., EC50 in range of 1.7 to 2.5 μM for PCa cells compared with aspirin in the millimolar range). We have earlier found that AS-10 inhibited androgen receptor (AR) signaling in LNCaP cells and induced caspase-mediated apoptosis along with activation of DNA damage response (DDR) proteins such as P53 and its canonical target P21Cip1 and the DNA double strand break marker protein phospho-H2A.X. The objective of the present work is to analyze the signaling pathways underlying the growth arrest and apoptotic effects of AS-10. In time course studies, AS-10 treatment decreased the expression of AR and its downstream target prostate specific antigen (PSA) as early as 3 h in LNCaP cells. AS-10 inhibited the progression of LNCaP cells from G1 into S phase, far ahead of an increase in apoptotic sub-G0/G1 cells. At the protein level, AS-10 treatment decreased the expression of proliferative oncoprotein c-Myc and anti-apoptotic molecules such as Mcl-1, Bcl-xL and survivin. Temporally, AS-10 increased P53 and P21Cip1 expression and PARP cleavage ahead of increased phospho-H2A.X which might simply mark apoptotically fragmented DNA. By si-RNA transfection, knocking down of P53 or P21Cip1 in LNCaP cells increased cell death induced by AS-10, indicating an anti-apoptotic role of this regulator-target pair. These data suggest that suppression of AR signaling and prosurvival proteins by AS-10 may contribute to cell cycle arrest and apoptosis, subject to P53 status. Additionally, aspirin has been known to acetylate histones and other proteins. Because AS-10 contains two aspirin moieties, we hypothesize that the combination of AS-10 with a histone deacetylates (HDAC) inhibitor (Vorinostat) may further increase overall acetylation and synergize to induce PCa cell death. Our results demonstrate that the combination of AS-10 and Vorinostat induced more than additive cell death response. In summary, AS-10 represents a promising potential chemopreventive agent for prostate carcinogenesis, alone or in combination with other drugs, through multiple molecular targets and cellular pathways and merits efficacy assessment in animal models. Citation Format: Deepkamal N. Karelia, Sangyub Kim, Srinivasa Ramisetti, Cheng Jiang, Shantu Amin, Arun K. Sharma, Junxuan Lu. Dissecting the signaling cascades involved in growth inhibition and apoptosis of prostate cancer cells exposed to a novel bis-aspirinyl selenazolidine AS-10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1253.

MutT homolog 1 counteracts the effect of anti-neoplastic treatments in adult and pediatric glioblastoma cells

Glioblastoma, a fatal disease in both adult and pediatric patients, currently has limited treatment options that offer no more than temporary relief. Our experiments with adult and pediatric glioblastoma cell lines showed that radiation induces a dose-dependent increase in the level of MutT homolog 1 (MTH1) - an enzyme that hydrolyzes oxidized purine nucleoside triphosphates. Similarly, the combination of vorinostat, which is a histone deacetylase inhibitor, and ABT-888, which is a PARP-1 inhibitor, enhanced clonogenic death and increased the MTH1 level, relative to each treatment alone. This result suggests that the MTH1 level is directly related to the damage that is inflicted upon the cells, and its activity protects them against anti-neoplastic therapy. Indeed, the MTH1 inhibitor TH588 and MTH1 siRNA increased glioblastoma's response to both radiation and the combination of vorinostat and ABT-888. TH588 also inhibited glioblastoma's capacity for migration and invasion. In normal fibroblasts, low radiation doses and the combination of vorinostat and ABT-888 decreased the level of the enzyme. TH588 did not alter the fibroblasts’ response to radiation and only mildly affected their response to the combination of vorinostat and ABT-888.In summary, the inhibition of MTH1 is required to better realize the therapeutic potential of anti-neoplastic treatments in glioblastoma.

A Phase II Trial of Bortezomib and Vorinostat in Mantle Cell Lymphoma and Diffuse Large B-cell Lymphoma

BackgroundThe proteasome inhibitor bortezomib has demonstrated marked preclinical activity when combined with the histone deacetylase inhibitor vorinostat in leukemia, multiple myeloma, and mantle cell lymphoma (MCL) cells. The present study evaluated the efficacy and safety of the combination in patients with relapsed or refractory MCL and diffuse large B-cell lymphoma (DLBCL). Patients and MethodsThe present multicenter, nonrandomized phase II trial used a Simon 2-stage design with 3 cohorts: cohort A, MCL with no previous bortezomib (including untreated MCL); cohort B, MCL with previous bortezomib; and cohort C, relapsed or refractory DLBCL with no previous bortezomib. Vorinostat (400 mg) was administered orally on days 1 to 5 and 8 to 12 before bortezomib (1.3 mg/m2), which was administered intravenously on days 1, 4, 8, and 11 of each 21-day cycle. ResultsFor the 65 treated patients (22 in cohort A, 4 in cohort B, and 39 in cohort C), the overall response rate was 31.8%, 0%, and 7.7%, respectively. The median progression-free survival was 7.6 months for cohort A and 1.8 months for cohort C. In cohort A, 7 patients had a partial response (PRs), 5 had stable disease (SD), 7 had progressive disease (PD), 1 was not assessed, and 2 were not evaluable. In cohort B, 2 had SD and 2 had PD. In cohort C, 3 had a PR, 8 had SD, 23 had PD, and 5 were not assessed. Baseline NF-κB activation, measured as nuclear RelA by immunohistochemistry, did not correlate with clinical response. ConclusionThe combination of bortezomib and vorinostat is safe and has modest activity in MCL and limited activity in DLBCL.

Comparative oncology approach to drug repurposing in osteosarcoma.

BackgroundOsteosarcoma is an orphan disease for which little improvement in survival has been made since the late 1980s. New drug discovery for orphan diseases is limited by the cost and time it takes to develop new drugs. Repurposing already approved FDA-drugs can help overcome this limitation. Another limitation of cancer drug discovery is the lack of preclinical models that accurately recapitulate what occurs in humans. For OS using dogs as a model can minimize this limitation as OS in canines develops spontaneously, is locally invasive and metastasizes to the lungs as it does in humans.MethodsIn our present work we used high-throughput screens to identify drugs from a library of 2,286 FDA-approved drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. The identified lead compound was then tested for synergy with 7 other drugs that have demonstrated activity against OS. These results were confirmed with in vitro assays and an in vivo murine model of OS.ResultsWe identified 13 drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. Auranofin was selected for further in vitro combination drug screens. Auranofin showed synergistic effects with vorinostat and rapamycin on OS viability and apoptosis induction. Auranofin demonstrated single-agent growth inhibition in both human and canine OS xenografts, and cooperative growth inhibition was observed in combination with rapamycin or vorinostat. There was a significant decrease in Ki67-positive cells and an increase in cleaved caspase-3 levels in tumor tissues treated with a combination of auranofin and vorinostat or rapamycin.ConclusionsAuranofin, alone or in combination with rapamycin or vorinostat, may be useful new treatment strategies for OS. Future studies may evaluate the efficacy of auranofin in dogs with OS as a prelude to human clinical evaluation.

Current Opportunities for Treatment Optimization of Mycosis Fungoides and Sezary Syndrome

Aim. To estimate vorinostat efficacy in patients with relapsed/refractory mycosis fungoides and Sezary syndrome. Materials & Methods. The total of 21 patients with refractory and progressive mycosis fungoides and Sezary syndrome receiving vorinostat 400 mg once daily were followed up from 2014 to 2017. The median age was 62 years (range 3479). The male to female ratio was 10/11. The median number of various regimens of pre-study systemic treatment was 4 (range 1-7). Progressive disease were observed in 85,7 % of patients before administration of vorinostat and after mono-and polychemotherapy. Results. The study group included 15 patients with mycosis fungoides and 6 patients with Sezary syndrome. Early stages of primary cutaneous T-cell lymphoma were diagnosed in 4 patients, the advanced stages in 17 patients. Seventeen patients received vorinostat treatment in monoregime; 4 patients were administered with vorinostat in combination with methotrexate or а-interferon. The median duration of vorinostat therapy was 6 months (range 1-38). Stabilization of the disease was observed in 47.6 % of cases, response to treatment in 38.1 % of cases (with 5 cases of complete response and 3 cases of partial response), and 14.3 % of patients had progression of the disease. The decrease of skin itching was reported in 38.1 % of patients; skin itching completely resolved in 28.6 % of cases. The adverse events required the vorinostat dose adjustment in 3 cases and treatment discontinuation in 3 cases. The total of 9 patients continue to receive vorinostat. Conclusion. Vorinostat treatment was shown to be effective in patients with refractory and advanced mycosis fungoides and Sezary syndrome not responding to various types of external, mono- and polychemotherapy. The therapy with vorinostat was associated with higher life expectancy and improved quality of life.

Vorinostat enhances the anticancer effect of oxaliplatin on hepatocellular carcinoma cells.

Oxaliplatin‐based systemic chemotherapy has been proposed to have efficacy in hepatocellular carcinoma (HCC). We investigated the combination of vorinostat and oxaliplatin for possible synergism in HCC cells. SMMC7721, BEL7402, and HepG2 cells were treated with vorinostat and oxaliplatin. Cytotoxicity assay, tumorigenicity assay in vitro, cell cycle analysis, apoptosis analysis, western blot analysis, animal model study, immunohistochemistry, and quantitative PCR were performed. We found that vorinostat and oxaliplatin inhibited the proliferation of SMMC7721, BEL7402, and HepG2 cells. The combination index (CI) values were all <1, and the dose‐reduction index values were all greater than 1 in the three cell lines, indicating a synergistic effect of combination of the two agents. Coadministration of vorinostat and oxaliplatin induced G2/M phase arrest, triggered caspase‐dependent apoptosis, and decreased tumorigenicity both in vitro and in vivo. Vorinostat suppressed the expression of BRCA1 induced by oxaliplatin. In conclusion, cotreatment with vorinostat and oxaliplatin exhibited synergism in HCC cells. The combination inhibited cell proliferation and tumorigenicity both in vitro and in vivo through induction of cell cycle arrest and apoptosis. Our results predict that a combination of vorinostat and oxaliplatin may be useful in the treatment of advanced HCC.

Replication Stress Leading to Apoptosis within the S-phase Contributes to Synergism between Vorinostat and AZD1775 in HNSCC Harboring High-Risk TP53 Mutation.

Purpose: The cure rate for patients with advanced head and neck squamous cell carcinoma (HNSCC) remains poor due to resistance to standard therapy primarily consisting of chemoradiation. As mutation of TP53 in HNSCC occurs in 60% to 80% of non-HPV-associated cases and is in turn associated with resistance to these treatments, more effective therapies are needed. In this study, we evaluated the efficacy of a regimen combining vorinostat and AZD1775 in HNSCC cells with a variety of p53 mutations.Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine the in vitro and in vivo sensitivity of high-risk mutant p53 HNSCC cell lines to vorinostat in combination with AZD1775. Cell cycle, replication stress, homologous recombination (HR), live cell imaging, RNA sequencing, and apoptosis analyses were performed to dissect molecular mechanisms.Results: We found that vorinostat synergizes with AZD1775 in vitro to inhibit growth of HNSCC cells harboring high-risk mutp53. These drugs interact synergistically to induce DNA damage, replication stress associated with impaired Rad51-mediated HR through activation of CDK1, and inhibition of Chk1 phosphorylation, culminating in an early apoptotic cell death during the S-phase of the cell cycle. The combination of vorinostat and AZD1775 inhibits tumor growth and angiogenesis in vivo in an orthotopic mouse model of oral cancer and prolongs animal survival.Conclusions: Vorinostat synergizes with AZD1775 in HNSCC cells with mutant p53 in vitro and in vivo A strategy combining HDAC and WEE1 inhibition deserves further clinical investigation in patients with advanced HNSCC. Clin Cancer Res; 23(21); 6541-54. ©2017 AACR.

Ruxolitinib combined with vorinostat suppresses tumor growth and alters metabolic phenotype in hematological diseases

JAK-2 dysregulation plays an important role as an oncogenic driver, and is thus a promising therapeutic target in hematological malignancies. Ruxolitinib is a pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2, and minor activity against JAK3. Vorinostat is an HDAC inhibitor that reduces JAK-2 expression, thus affecting JAK-2 mRNA expression and increasing JAK-2 proteasomal deterioration. Here we hypothesized that the combination of ruxolitinib and vorinostat could have synergistic effects against hematological disease. We tested combinations of low doses of ruxolitinib and vorinostat in 12 cell lines, and observed highly synergistic cytotoxic action in six cell lines, which was maintained for up to 120 h in the presence of stromal cells. The sensitivity of the six cell lines may be explained by the broad effects of the drug combination, which can affect various targets. Treatment with the combination of ruxolitinib and vorinostat appeared to induce a possible reversal of the Warburg effect, with associated ROS production, apoptotic events, and growth inhibition. Decreased glucose metabolism may have markedly sensitized the six more susceptible cell lines to combined treatment. Therapeutic inhibition of the JAK/STAT pathway seems to offer substantial anti-tumor benefit, and combined therapy with ruxolitinib and vorinostat may represent a promising novel therapeutic modality for hematological neoplasms.

Co-delivery of Vorinostat and Etoposide Via Disulfide Cross-Linked Biodegradable Polymeric Nanogels: Synthesis, Characterization, Biodegradation, and Anticancer Activity.

Treatment regimens for cancer patients using single chemotherapeutic agents often lead to undesirable toxicity, drug resistance, reduced uptake etc. Combination of two or more drugs is therefore becoming an imperative strategy to overcome these limitations. A step forward can be taken through delivery of the drugs used in combination via nanoparticles. Co-administration of chemotherapeutic drugs encapsulated in nanoparticles has been shown to result in synergistic effects and enhanced therapeutic efficacy. In present study, we explored the combination treatment of histone deacetylase inhibitor vorinostat (VOR) and topoisomerase II inhibitor etoposide (ETOP). The concurrent combination treatment of VOR and ETOP resulted in synergistic effect on human cervical HeLa cancer cells. VOR and ETOP were encapsulated into poly(ethylene glycol) monomethacrylate (POEOMA)-based disulfide cross-linked nanogels. The nanogels were synthesized using atom transfer radical polymerization (ATRP) via cyclohexane/water inverse mini-emulsion and were degradable in presence of intracellular glutathione (GSH) concentration. Both the drugs were loaded into the nanogels by physical encapsulation method and characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), dynamic light scattering (DLS), and differential scanning calorimetry (DSC). Both VOR- and ETOP-loaded nanogels showed sustained release profile. Furthermore, combination treatment drugs encapsulated of POEOMA nanogel demonstrated enhanced synergistic cytotoxic effect compared with combination of free drugs. Enhanced synergistic cell killing efficiency of drug-loaded POEOMA nanogels was due to increased apoptosis via caspase 3/7 activation. Therefore, combination of VOR- and ETOP-loaded PEG-based biodegradable nanogels may provide a promising therapy with enhanced anticancer effect.

Molecular Guided Therapy Provides Sustained Clinical Response in Refractory Choroid Plexus Carcinoma

Choroid plexus carcinomas (CPCs) are rare, aggressive pediatric brain tumors with no established curative therapy for relapsed disease, and poor survival rates. TP53 Mutation or dysfunction correlates with poor or no survival outcome in CPCs. Here, we report the case of a 4 month-old female who presented with disseminated CPC. After initial response to tumor resection and adjuvant-chemotherapy, the tumor recurred and metastasized with no response to aggressive relapse therapy suggesting genetic predisposition. This patient was then enrolled to a Molecular Guided Therapy Clinical Trial. Genomic profiling of patient tumor and normal sample identified a TP53 germline mutation with loss of heterozygosity, somatic mutations including IDH2, and aberrant activation of biological pathways. The mutations were not targetable for therapy. However, targeting the altered biological pathways (mTOR, PDGFRB, FGF2, HDAC) guided identification of possibly beneficial treatment with a combination of sirolimus, thalidomide, sunitinib, and vorinostat. This therapy led to 92% reduction in tumor size with no serious adverse events, excellent quality of life and long term survival.

Vorinostat and metformin sensitize EGFR-TKI resistant NSCLC cells via BIM-dependent apoptosis induction.

There is a close relationship between low expression of BIM and resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). Vorinostat is a pan-histone deacetylase inhibitor (HDACi) that augments BIM expression in various types of tumor cells, however, this effect is attenuated by the high expression of anti-apoptotic proteins in EGFR-TKI resistant non-small cell lung cancer (NSCLC) cells. Vorinostat in combination with metformin – a compound that can inhibit anti-apoptotic proteins expression, might cooperate to activate apoptotic signaling and overcome EGFR-TKI resistance. This study aimed to investigate the cooperative effect and evaluate possible molecular mechanisms. The results showed that vorinostat combined with gefitinib augmented BIM expression and increased the sensitivity of EGFR-TKI resistant NSCLC cells to gefitinib, adding metformin simultaneously could obviously inhibit the expression of anti-apoptotic proteins, and further increased expression levels of BIM and BAX, and as a result, further improved the sensitivity of gefitinib both on the NSCLC cells with intrinsic and acquired resistance to EGFR-TKI. In addition, autophagy induced by gefitinib and vorinostat could be significantly suppressed by metformin, which might also contribute to enhance apoptosis and improve sensitivity of gefitinib. These results suggested that the combination of vorinostat and metformin might represent a novel strategy to overcome EGFR-TKI resistance associated with BIM-dependent apoptosis in larger heterogeneous populations.

Abstract 4051: Combination efficacy of HDAC inhibitor vorinostat and CDK-4/6 dual inhibitor palbociclib against therapy-resistant mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma accounting for about 7% of all non-Hodgkins lymphoma. While multiple therapy regimens are available to treat MCL patients, ultimately relapse from therapy-resistant MCL, making MCL carry the worst prognosis of all B cell lymphomas. Emerging evidence suggest that, dysregulated histone deacetylases (HDACs) and the key molecules of cell cycle regulator cyclin-dependent kinases (CDKs) have been shown to be commonly associated with many lymphomas including MCL, and are considered as promising targets for relapsed lymphoma therapy. Several inhibitors of these target pathways/molecules are in clinical trials as monotherapies or in combination with other anticancer agents. Therefore, in this study, we investigated the single agent and combination efficacies of the HDACs inhibitor vorinostat, and CDK-4/6 dual inhibitor palbociclib on MCL cell growth/survival and underlying molecular mechanism(s) using different MCL cell lines (Granta 519, Jeko-1 and JVM-2) including therapy-resistant MCL cell lines derived from Granta-519 (GRL, GRK and GRR). We used MTT assay to measure the survival and proliferation, agar colony formation assay to determine the effects of these inhibitors on the anchorage independent growth, Annexin-V analyses to examine the cells undergoing apoptosis following treatments and western blot analyses to determine the expression levels of target molecules including pRb, histone acetylation, cyclin-D1 and Bcl-2. The concentration of these inhibitors used in this study were similar to the published literature including sub-IC50 levels determined in our laboratory. Our results showed that both inhibitors as single agents or combined, significantly suppressed the cell growth, and induced apoptosis in therapy-resistant and other MCL lines in vitro. The single agent and combined anti-lymphoma efficacies of these inhibitors were further confirmed with the colony formation ability of the MCL cells grown in 0.3% agar semisolid media. In addition, combination of vorinostat and palbociclib treatment significantly inhibited the activation of phosphorylated-retinoblastoma (pRb) protein, a key molecule of cell cycle and increased the expression of acetylated-Histone H3 (H3-Ac) as determined by western blot analyses. Subsequently, the expression of Cyclin D1 (proliferation, MCL hallmark) and anti-apoptotic Bcl-2 proteins were also downregulated by these inhibitors. Together, our findings suggest that combination of vorinostat and palbociclib showed a significant synergistic anti-MCL activity by targeting associated pathways/molecules. This targeted approach warrants further preclinical evaluation for translation to the clinic. Citation Format: Nagendra K. Chaturvedi, Nathan D. Hatch, Garrett L. Sutton, Matthew J. Kling, Julie M. Vose, Shantaram S. Joshi. Combination efficacy of HDAC inhibitor vorinostat and CDK-4/6 dual inhibitor palbociclib against therapy-resistant mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4051. doi:10.1158/1538-7445.AM2017-4051

Abstract CT029: A phase I study of vorinostat and temsirolimus in children with newly diagnosed or progressive diffuse intrinsic pontine glioma (DIPG)

Abstract Background/rationale: Diffuse intrinsic pontine glioma (DIPG) in children remains essentially incurable with a median survival of 12 months. Radiation therapy is currently the standard treatment modality. To date chemotherapy or biologic agents have had no meaningful increase in survival, making it imperative to profile novel therapeutic combinations. Data from biopsied DIPG showed the PI3K/AKT/mTOR pathway as a major oncogenic player. At the gene level high PDGFR amplification and overexpression of the PI3K-Akt-mTOR pathway were noted in these tumors. Focal amplifications of genes within the PI3K-Akt-mTOR signaling pathway were found in 47% of DIPG. Thus targeting these pathways with mTOR inhibitors could hold promise. Elevated expression of DNA repair proteins in the NHEJ and HRR pathway are known to induce resistance to radiation. Preclinical data shows inhibition of these pathways potentiates the sensitivity of these tumors to radiation. Recent genomic and epigenetic insights demonstrates the role of histone deacetylase (HDAC) in these tumors. Vorinostat a pan HDAC inhibitor has been associated with decreased expression of key DNA repair proteins in both the NHEJ and HRR pathways and have been tested in clinical trial for children with DIPG. Both temsirolimus (an mTOR inhibitor) and vorinostat are well described in pediatric clinical trials with a determined maximum tolerated dose (MTD). The efficacy of the combination of temsirolimus and vorinostat has been well-established in other malignancies including prostate cancer, mantle lymphoma, and renal carcinoma. This is the first multi-targeted therapeutic trial in pediatric DIPG, using vorinostat and temsirolimus in a combinatorial approach, targeting the key oncogenic pathway and molecules along with enhancing the radiation sensitivity. Correlative study: Stratum I (chemoradiotherapy phase): Histone acetylation will be quantified from peripheral blood mononuclear cell (PBMC) collected prior to the first dose of vorinostat and radiation and at the end of radiation. Stratum I and II (maintenance phase): Evaluation of histone acetylation, phosphorylated 70S6K and Akt on day 1 and 8 (prior to chemotherapy with temsirolimus) during cycle 1 and cycle 2 will be performed. Methods: Patients with a performance score of 50 or greater with newly diagnosed (Stratum 1) or progressive DIPG (stratum II) as confirmed by gadolinium enhanced MRI, will be eligible. Biopsy is not mandatory. Stratum I: Patients will receive radiation therapy concurrently with vorinostat on the days of radiation at a dose of 230 mg/m2/dose orally, followed by maintenance phase with vorinostat and temsirolimus for 12 cycles if patients do not have progressive disease. Stratum I and Stratum II (maintenance phase): Patients will receive vorinostat and temsirolimus for 12 cycles (28 day cycle), if patient do not have progressive disease. Vorinostat will be given once daily orally at 230 mg/m2/dose orally from day 1 to day 8, along with temsirolimus at 25mg/m2 intravenously on day 1 and day 8. This will be dose level 1 with provisions for 2 higher dose levels and 1 lower dose level if needed. A standard 3+3 design will be used and a minimum of 9 patients and a maximum of 18 patients will be enrolled. Trial is continuing as planned and patient are on dose level 1. Trial: NCT02420613 (currently recruiting). Citation Format: Soumen Khatua, Vidya Gopalakrishnan, Joya Chandra, Leena Ketonen, David I. Sandberg, Jeffrey Weinberg, Fuller Gregory, Kenneth R. Hess, Natalie Slone, Heather Meador, Mirriam Morrell, Wafik Zaky. A phase I study of vorinostat and temsirolimus in children with newly diagnosed or progressive diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT029. doi:10.1158/1538-7445.AM2017-CT029

Tolerability of paclitaxel/carboplatin (PCb) in solid tumor patients (pts) infected with HIV.

e14077 Background: Although cancer has long been a recognized hallmark of the HIV epidemic, the preservation of immunologic health with modern antiretroviral therapy (ART) and aging has resulted in a population increasingly susceptible to cancers not traditionally associated with advancing immunosuppression. Several of these cancers (including lung, anal and head &amp; neck) are seen in excess compared to the background population. Defining tolerability of standard treatments and analyzing potential interactions between ART and chemotherapy provides evidence necessary to mitigate treatment disparities. Methods: We conducted a study to evaluate the tolerability of PCb in HIV+ cancer pts. AMC-078 (NCT01249443), originally designed as a phase I of vorinostat in combination with fixed doses of P (at 175mg/m2) and Cb (AUC 6) every 3 weeks, was amended to study pts treated with PCb alone after phase III testing in the background population was negative for the combination in lung cancer. Eligibility criteria: PS ≤ 2, advanced solid tumor and normal organ function, including CD4 count &gt; 100 cells/mcL on stable ART. Up to 6 cycles of PCb were permitted. Clinically significant adverse events (AE) in prior cycles were managed by dose reductions. Results: 17 pts (10M/7F; median CD4, 389/mcL) were accrued, including lung (9) and anal (3) cancers; 8 pts had ritonavir (potent CYP inhibitor)-containing ART. 65 PCb cycles were administered to 16 evaluable pts, for a mean of 4+ cycles/pt; only 2 pts were treated with vorinostat. AE of special interest included ≥G3 (febrile) neutropenia and ≥ G2 neuropathy, below. 4 pts had partial responses (3 confirmed). Pharmacokinetic analyses (7 pts) are pending. Conclusions: PCb has similar toxicity profile in fit pts with HIV infection. No signal for worse myelosuppression or neuropathy was observed by ART regimen. Routine use of GCSF or empiric dose reduction for presumed risk is unjustified. Results support standard cancer treatment for this underserved population. Clinical trial information: NCT01249443. [Table: see text]

Unlocking the chromatin of adenoid cystic carcinomas using HDAC inhibitors sensitize cancer stem cells to cisplatin and induces tumor senescence

Adenoid cystic carcinoma (ACC) is an uncommon malignancy of the salivary glands that is characterized by local recurrence and distant metastasis due to its resistance to conventional therapy. Platinum-based therapies have been extensively explored as a treatment for ACC, but they show little effectiveness. Studies have shown that a specific group of tumor cells, harboring characteristics of cancer stem cells (CSCs), are involved in chemoresistance of myeloid leukemias, breast, colorectal and pancreatic carcinomas. Therapeutic strategies that target CSCs improve the survival of patients by decreasing the rates of tumor relapse, and epigenetic drugs, such as histone deacetylase inhibitors (HDACi), have shown promising results in targeting CSCs. In this study, we investigated the effect of the HDACi Suberoylanilide hydroxamic acid (Vorinostat), and cisplatin, alone or in combination, on CSCs and non-CSCs from ACC. We used CSCs as a biological marker for tumor resistance to therapy in patient-derived xenograft (PDX) samples and ACC primary cells. We found that cisplatin reduced tumor viability, but enriched the population of CSCs. Systemic administration of Vorinostat reduced the number of detectable CSCs in vivo and in vitro, and a low dose of Vorinostat decreased tumor cell viability. However, the combination of Vorinostat and cisplatin was extremely effective in depleting CSCs and reducing tumor viability in all ACC primary cells by activating cellular senescence. These observations suggest that HDACi and intercalating agents act more efficiently in combination to destroy tumor cells and their stem cells.

Treatment Effects of Vorinostat and Letrozole Combination on Breast Cancer Cell Survival and Peripheral Blood Mononuclear Cell Differentiation

HDAC inhibitors are new class of drugs that show promising impact on the cancer growth and metastatic process. Letrozole is one of the aromatase inhibitors that could also elicit strong anticancer effects on breast cancers through direct as well as indirect mechanisms. Our aim was to understand the intracellular mechanism whereby Vorinostat, in monotherapy and in combination with Letrozole, can induce cell cycle arrest and apoptosis in breast cancer cells. In addition, we also attempted to determine how Vorinostat and Letrazole can inhibit Peripheral Blood Mononuclear Cell (PBMC) differentiation into osteoclasts and cause programmed cell death. We performed assays to measure the anti‐proliferative, pro‐apoptotic effects of monotherapy (Vorinostat) and combination (Vorinostat + Letrozole) treatments in cancer cells (MCF‐7) as well as in PBMC, that was isolated from healthy individuals and undergoing osteoclast differentiation in a specialized medium that contained RANKL and M‐CSF (Macrophage – colony stimulating factor). Immediately after treatments, determination of cytotoxicity on MCF‐7 was performed using MTT assay and the pro‐apoptotic effects were assessed by immunoblotting and RT‐PCR analyses. Our results confirmed that both Vorinostat and Letrozole were able to induce cell death in MCF‐7 cells in a dose and time dependent manner. The IC50 value for Vorinostat were around 1.5 μM, and 1.2 mM respectively for MCF‐7 and PBMC. In our experiments, Letrozole showed much stronger effects and the IC50 values were 4 nM and 2.5 nM respectively for the above listed cells. Induction of apoptosis in these cells were evidenced by the elevation of p21, Bax, Caspase 3 and down regulation of Bcl2. The induction of apoptosis was further confirmed by DNA fragmentation in our in vitro experimental systems. Furthermore, our experiments with PBMC using both mono therapies and the combination treatments revealed that, they were able to block the osteoclast differentiation at reduced doses when combined together for treatments. It has been reported that the elevated levels of acetylated histones H3 and H4 and consequent intracellular signals are responsible for the cell cycle arrest and pro‐apoptotic events that are seen when HDAC inhibitors are used. Similar mechanisms may be responsible for the intracellular effects observed in our experimental systems. Thus, the results obtained from our in vitro experiments suggest that combination of Vorinostat and Letrozole is very dynamic and effective, not only in blocking the proliferation of MCF‐7 cells but also in inhibiting the differentiation of PBMC into osteoclasts and reducing their survival capacity. This suggests that, during clinical use, the above indicated combination might provide dual benefits, by blocking cancer cell proliferation as well as reduction of breast cancer associated osteoporosis caused by osteoclasts. In essence, the above discussed treatment outcomes may be achieved by minimizing the differentiation and formation of osteoclasts from PBMC in cancer patients, that appears to be further supported by the induction of programmed cell death.