benzofurans and abexinostat

Histone deacetylase (HDAC) inhibitor abexinostat is under investigation for the treatment of various cancers. Epigenetic changes including aberrant HDAC activity are associated with cancers, including myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL). In this phase 1 dose-escalation study, 17 patients with relapsed/refractory higher-risk MDS, AML, or ALL received oral abexinostat (60, 80 [starting dose], 100, or 120 mg) twice daily (bid) on Days 1-14 of 21-day cycles. The most common treatment-related grade ≥3 adverse events were thrombocytopenia (29%) and neutropenia (24%), none of which led to discontinuation. Maximum-tolerated dose was not reached. Of 12 evaluable patients, best response was stable disease in 1 patient. This study was closed due to limited clinical benefit. Future development of oral abexinostat 100 mg bid in patients with MDS, AML, or ALL should focus on combination regimens. ISRCTN registry: 99680465.

Topics: Adult; Aged; Benzofurans; Drug Resistance, Neoplasm; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Recurrence; Retreatment; Treatment Outcome; Young Adult

Histone deacetylase inhibitors are members of a class of epigenetic drugs that have proven activity in T-cell malignancies, but little is known about their efficacy in B-cell lymphomas. Abexinostat is an orally available hydroxamate-containing histone deacetylase inhibitor that differs from approved inhibitors; its unique pharmacokinetic profile and oral dosing schedule, twice daily four hours apart, allows for continuous exposure at concentrations required to efficiently kill tumor cells. In this phase II study, patients with relapsed/refractory non-Hodgkin lymphoma or chronic lymphocytic leukemia received oral abexinostat at 80 mg BID for 14 days of a 21-day cycle and continued until progressive disease or unacceptable toxicity. A total of 100 patients with B-cell malignancies and T-cell lymphomas were enrolled between October 2011 and July 2014. All patients received at least one dose of study drug. Primary reasons for discontinuation included progressive disease (56%) and adverse events (25%). Grade 3 or over adverse events and any serious adverse events were reported in 88% and 73% of patients, respectively. The most frequently reported grade 3 or over treatment-emergent related adverse events were thrombocytopenia (80%), neutropenia (27%), and anemia (12%). Among the 87 patients evaluable for efficacy, overall response rate was 28% (complete response 5%), with highest responses observed in patients with follicular lymphoma (overall response rate 56%), T-cell lymphoma (overall response rate 40%), and diffuse large B-cell lymphoma (overall response rate 31%). Further investigation of the safety and efficacy of abexinostat in follicular lymphoma, T-cell lymphoma, and diffuse large B-cell lymphoma implementing a less dose-intense week-on-week-off schedule is warranted. (

Topics: Adult; Aged; Aged, 80 and over; Benzofurans; Diarrhea; Disease-Free Survival; Drug Administration Schedule; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Male; Middle Aged; Remission Induction; Thrombocytopenia; Treatment Outcome

Topics: Acetylation; Adult; Aged; Alanine Transaminase; Angiogenesis Inhibitors; Antineoplastic Combined Chemotherapy Protocols; Aspartate Aminotransferases; Benzofurans; Carcinoma, Renal Cell; Disease Progression; Disease-Free Survival; Drug Resistance; Drug Resistance, Neoplasm; Epigenesis, Genetic; Fatigue; Female; Gene Expression; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Indazoles; Kidney Neoplasms; Male; Maximum Tolerated Dose; Middle Aged; Neutropenia; Pyrimidines; Sulfonamides; Thrombocytopenia; Treatment Outcome; Vascular Endothelial Growth Factor A; Young Adult

Additional targeted therapeutics are needed for the treatment of lymphoma. Abexinostat is an oral pan-histone deacetylase inhibitor (HDACi) displaying potent activity in preclinical models. We conducted a multicenter phase I/II study (N = 55) with single-agent abexinostat in relapsed/refractory lymphoma.. In phase I, 25 heavily pretreated patients with any lymphoma subtype received oral abexinostat ranging from 30 to 60 mg/m(2) twice daily 5 days/week for 3 weeks or 7 days/week given every other week. Phase II evaluated abexinostat at the maximum tolerated dose in 30 patients with relapsed/refractory follicular lymphoma or mantle cell lymphoma.. The recommended phase II dose was 45 mg/m(2) twice daily (90 mg/m(2) total), 7 days/week given every other week. Of the 30 follicular lymphoma and mantle cell lymphoma patients enrolled in phase II, 25 (14 follicular lymphoma, 11 mantle cell lymphoma) were response-evaluable. Tumor size was reduced in 86% of follicular lymphoma patients with an investigator-assessed ORR of 64.3% for evaluable patients [intent-to-treat (ITT) ORR 56.3%]. Median duration of response was not reached, and median progression-free survival (PFS) was 20.5 months (1.2-22.3+). Of responding follicular lymphoma patients, 89% were on study/drug >8 months. In mantle cell lymphoma, the ORR was 27.3% for evaluable patients (ITT ORR 21.4%), and median PFS was 3.9 months (range, 0.1-11.5). Grade 3-4 treatment-related adverse events (phase II) with ≥ 10% incidence were thrombocytopenia (20%), fatigue (16.7%), and neutropenia (13.3%) with rare QTc prolongation and no deaths.. The pan-HDACi, abexinostat, was overall well tolerated and had significant clinical activity in follicular lymphoma, including highly durable responses in this multiply relapsed patient population.

Topics: Adult; Aged; Aged, 80 and over; Benzofurans; Disease-Free Survival; Drug Administration Schedule; Drug-Related Side Effects and Adverse Reactions; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lymphoma, Follicular; Lymphoma, Mantle-Cell; Male; Maximum Tolerated Dose; Middle Aged; Neoplasm Recurrence, Local; Recurrence

It has been demonstrated that several inhibitors of histone deacetylase (HDAC) can enhance chemotherapy-induced apoptosis and reduce sarcoma tumor volume in preclinical models. The authors sought to determine the maximum tolerated dose, pharmacokinetics/pharmacodynamics, safety, and toxicity of the HDAC inhibitor abexinostat (PCI-24781) when administered with doxorubicin to patients with metastatic sarcomas.. Participants were enrolled in a standard, phase 1, 3 + 3, dose-escalation study design. Abexinostat was administered on days 1 through 5 with 75 mg/m(2) of doxorubicin administered on day 4 of every 21-day cycle until patients developed disease progression or drug intolerance or reached a cumulative lifetime doxorubicin dose of 450 mg/m(2). Granulocyte-colony-stimulating factor (G-CSF) support was provided at physician discretion on arm A and was provided to all participants in arm B. From 3 to 6 participants initially received abexinostat 30 mg/m(2) twice daily, and subsequent cohorts were administered doses of 15 mg/m(2), 45 mg/m(2), or 60 mg/m(2) twice daily. All patients without progressive disease after receiving a cumulative lifetime doxorubicin dose of 450 mg/m(2) were given the option to continue with abexinostat as a single agent until they developed disease progression.. In total, 22 participants (10 who had previously experienced tumor growth after doxorubicin therapy) were enrolled (6 in arm A, 14 in arm B), 20 were evaluable for dose-limiting toxicity (DLT), and 17 were evaluable for radiologic response. In arm A, participants received abexinostat 15 mg/m(2) or 30 mg/m(2) twice daily. DLTs of grade 3 and 4 neutropenia were observed in 2 of 3 participants who received abexinostat 30 mg/m(2) twice daily. Neither of those patients received G-CSF prophylaxis. In arm B, participants received abexinostat at doses of 30 mg/m(2), 45 mg/m(2), or 60 mg/m(2) twice daily, all with mandated G-CSF support. Two DLTs were observed at the 60 mg/m(2) twice-daily dose (grade 3 infection, grade 4 thrombocytopenia). The pharmacokinetics of abexinostat were not affected by doxorubicin. HDAC activity, as measured by histone acetylation in peripheral blood mononuclear cells, was maximally inhibited at the abexinostat 30 mg/m(2) twice-daily dose. Of the 17 participants who were evaluable for radiologic response, 1 patient had a partial response, 9 patients had stable disease, and 7 patients had progressive disease as their best response; and 8 patients completed ≥ 5 cycles. Three of those participants had stable disease as their most recent disease status when the current report was written. Four participants who continued on monotherapy remained in stable disease for a median of 9.8 weeks after completing doxorubicin. The most common toxicities were fatigue, thrombocytopenia, and anemia. No study-related deaths were observed.. The maximum tolerated dose for abexinostat was 45 mg/m(2) twice daily administered on days 1 through 5 when patients received doxorubicin 75 mg/m(2) on day 4 of a 3-week cycle and G-CSF support was mandated. Toxicities were manageable, and tumor responses were observed. Additional studies are needed to further define the specific contributions of HDAC inhibition in patients who receive doxorubicin for the treatment of metastatic sarcoma.

Topics: Administration, Oral; Adult; Aged; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Benzofurans; Doxorubicin; Drug Administration Schedule; Female; Granulocyte Colony-Stimulating Factor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Maximum Tolerated Dose; Middle Aged; Sarcoma; Treatment Outcome; Young Adult

Background We determined the safety, pharmacokinetics, pharmacodynamics, and antitumour activity of abexinostat in B-cell lymphoma or chronic lymphocytic leukaemia. Patients and methods Thirty-five patients received oral abexinostat 30, 45, or 60 mg/m(2) bid in a 3 + 3 design in three 21-day schedules: 14 days on treatment in schedule 1 (D1-14); 10 days in schedule 2 (D1-5 and D8-12); and 12 days in schedule 3 (D1-4, D8-11, and D15-18). Safety, tumour response, plasma concentration, and histone H3 acetylation were measured. Results Two dose-limiting toxicities occurred in each schedule (one grade 3 febrile neutropenia; five grade 4 thrombocytopenia) at 60 mg/m(2) bid (maximal tolerated dose). The recommended dose was 45 mg/m(2) bid; schedule 1 was considered optimal. Non-haematological drug-related toxicities included grade 1 or 2 diarrhoea (43%), nausea (23%), and vomiting (11%); haematological toxicities included thrombocytopenia (31% grade 3, and 26% grade 4), which remained manageable and reversible on withdrawal. Of 29 evaluable patients, there were 2 complete and 6 partial responses; median duration of response was 14.6 months (range 3-16.5 months) (1 cycle is equivalent to 0.75 months). There was no evidence for nonlinear pharmacokinetics. There was a correlation between dose and histone acetylation. Conclusion Abexinostat has manageable toxicity and induced some durable complete and partial responses in B-cell lymphoma or chronic lymphocytic leukaemia. Our results suggest most favourable responses in patients with follicular lymphoma, though further research would be needed to confirm this finding.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Benzofurans; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Histone Deacetylase Inhibitors; Hodgkin Disease; Humans; Hydroxamic Acids; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Lymphoproliferative Disorders; Male; Maximum Tolerated Dose; Middle Aged

In the clinical development of oncology drugs, the recommended dose is usually determined using a 3 + 3 dose-escalation study design. However, this phase I design does not always adequately describe dose-toxicity relationships.. 125 patients, with either solid tumours or lymphoma, were included in the study and 1217 platelet counts were available over three treatment cycles. The data was used to build a population pharmacokinetic/pharmacodynamic (PKPD) model using a sequential modeling approach. Model-derived Recommended Doses (MDRD) of abexinostat (a Histone Deacetylase Inhibitor) were determined from simulations of different administration schedules, and the higher bound for the probability of reaching these MDRD with a 3 + 3 design were obtained.. The PKPD model developed adequately described platelet kinetics in both patient populations with the inclusion of two platelet baseline counts and a disease progression component for patients with lymphoma. Simulation results demonstrated that abexinostat administration during the first 4 days of each week in a 3-week cycle led to a higher MDRD compared to the other administration schedules tested, with a maximum probability of 40 % of reaching these MDRDs using a 3 + 3 design.. The PKPD model was able to predict thrombocytopenia following abexinostat administration in both patient populations. A model-based approach to determine the recommended dose in phase I trials is preferable due to the imprecision of the 3 + 3 design.

Topics: Antineoplastic Agents; Benzofurans; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Maximum Tolerated Dose; Models, Biological; Neoplasms; Thrombocytopenia

Abexinostat, an oral pan-histone deacetylase inhibitor (HDACi), was evaluated in patients with advanced solid tumours in two single agent phase I studies (PCYC-402 and CL1-78454-002). In PCYC-402 study testing four different administration schedules, the maximum tolerated dose (MTD) was established at 75 mg/m(2) BID (twice daily) and the recommended dose at 60 mg/m(2) BID regardless of the schedule tested. The dose limiting toxicity (DLT), consistently observed across all these schedules, was reversible thrombocytopenia. The CL1-78454-002 study was initially investigating an additional schedule of 14 days on/7 days off. While testing two first cohorts, thrombocytopenia was observed without reaching DLT. To address this issue, a pharmacokinetic/pharmacodynamic (PK/PD) model was used to predict the optimal schedule allowing higher doses with minimal thrombocytopenia. Several administration schedules were simulated using this model. A 4 days on/3 days off schedule was associated with the smallest platelet decrease. Accordingly, the CL1-78454-002 study was amended. After reaching MTD1 (75 mg/m(2) BID) with the initial schedule, subsequent cohorts received abexinostat on a revised schedule of 4 days on/3 days off, starting at one dose level below MTD1 (60 mg/m(2) BID). As expected, the dose-escalation continued for two more dose levels beyond MTD1. The MTD2 reached for this optimised schedule was 105 mg/m(2) BID and the recommended dose 90 mg/m(2) BID. In conclusion, early understanding of toxicities and PK determination allowed us to build a PK/PD model of thrombocytopenia, which predicted the optimal administration schedule. This optimised schedule is currently used in the trials in solid tumours with abexinostat.

Topics: Administration, Intravenous; Administration, Oral; Benzofurans; Computer Simulation; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Dosage Calculations; France; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Maximum Tolerated Dose; Models, Biological; Neoplasms; Platelet Count; Thrombocytopenia

Histone deacetylases (HDACs) are involved in key cellular processes and have been implicated in cancer. As such, compounds that target HDACs or drugs that target epigenetic markers may be potential candidates for cancer therapy. This study was therefore aimed to identify a potential epidrug with low toxicity and high efficiency as anti-tumor agents.

Topics: Animals; Apoptosis; Benzofurans; Breast Neoplasms; Calcium; Cell Proliferation; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Mice, Inbred BALB C; Mice, Nude; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries.

Topics: Acetylation; Animals; Benzofurans; Cell Cycle; Cells, Cultured; Chromatin Immunoprecipitation; CRISPR-Cas Systems; DNA End-Joining Repair; DNA Repair; Gene Editing; HEK293 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ku Autoantigen; Poly (ADP-Ribose) Polymerase-1; Rad51 Recombinase; Rad52 DNA Repair and Recombination Protein; Recombinational DNA Repair; Swine

Topics: Benzimidazoles; Benzofurans; Brain-Derived Neurotrophic Factor; Cells, Cultured; HEK293 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neural Stem Cells; Neuronal Outgrowth

Topics: Angiogenesis Inhibitors; Benzofurans; Histone Deacetylases; Humans; Hydroxamic Acids; Indazoles; Kidney Neoplasms; Pyrimidines; Sulfonamides

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Benzofurans; Female; Fluorodeoxyglucose F18; Hematopoietic Stem Cell Transplantation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lymphoma, T-Cell, Peripheral; Positron Emission Tomography Computed Tomography; Recurrence; Transplantation Conditioning; Transplantation, Homologous; Treatment Outcome

Mantle cell lymphoma (MCL) is a rare lymphoma caused by the t(11:14) juxtaposing the cyclin D1 (CCND1) locus on chromosome 11 and the immunoglobulin heavy chain (IgH) locus on chromosome 14. Several new treatments are proposed for MCL, including histone deacetylase inhibitors (HDACi). We have studied gene expression and chromatin organization in the translocated 11q13 locus in MCL cells as compared to lymphoblastoid cell lines as well as the effect of HDACi abexinostat on chromatin organization and gene expression in the 11q13 locus. We have identified a cluster of genes overexpressed in the translocation region on chromosome 11 in MCL cells. Abexinostat provokes a genome-wide disaggregation of heterochromatin. The genes upregulated after the t(11;14) translocation react to the HDACi treatment by increasing their expression, but their gene promoters do not show significant alterations in H3K9Ac and H3K9me2 levels in abexinostat-treated cells.

Topics: B-Lymphocytes; Benzofurans; Cell Line, Tumor; Chromatin; Chromatin Assembly and Disassembly; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lymphoma, Mantle-Cell; Promoter Regions, Genetic; Transcription, Genetic; Translocation, Genetic

: The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-β (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering.. This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways controlling adipocyte and osteoblast differentiation of hMSCs. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies for tissue engineering, bone disease, obesity, and metabolic-disorders.

Topics: Adipocytes; Adipogenesis; Benzofurans; Cell Differentiation; Cell Line; Cell Lineage; Chromatin Immunoprecipitation; Computational Biology; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Developmental; Gene Library; Genotype; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mesenchymal Stem Cells; Myoblasts, Skeletal; Oligonucleotide Array Sequence Analysis; Osteoblasts; Osteogenesis; Phenotype; Signal Transduction; Transcription Factors

To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12.. Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P < 0.05). Furthermore, PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed.. PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

Topics: Animals; Benzofurans; Embryo, Mammalian; Embryonic Development; Female; Histone Deacetylase Inhibitors; Hydroxamic Acids; Nuclear Transfer Techniques; Pregnancy; Swine

Bruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

Topics: Adenine; Agammaglobulinaemia Tyrosine Kinase; Animals; Benzofurans; Cell Survival; Clone Cells; Drug Resistance, Neoplasm; Drug Synergism; Epigenesis, Genetic; Gene Expression Profiling; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Lymphocytic, Chronic, B-Cell; Mice, Inbred C57BL; MicroRNAs; Molecular Targeted Therapy; Mutant Proteins; Neoplasm Proteins; Piperidines; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; RNA Interference; Signal Transduction; Up-Regulation

Several publications have suggested that histone deacetylase inhibitors (HDACis) could reverse the repression of estrogen receptor alpha (ERα) in triple-negative breast cancer (TNBC) cell lines, leading to the induction of a functional protein. Using different HDACis, vorinostat, panobinostat, and abexinostat, we therefore investigated this hypothesis in various human TNBC cell lines and patient-derived xenografts (PDXs). We used three human TNBC cell lines and three PDXs. We analyzed the in vitro toxicity of the compounds, their effects on the hormone receptors and hormone-related genes and protein expression both in vitro and in vivo models. We then explored intra-tumor histone H3 acetylation under abexinostat in xenograft models. Despite major cytotoxicity of all tested HDAC inhibitors and repression of deactylation-dependent CCND1 gene, neither ERα nor ERβ, ESR1 or ESR2 genes respectively, were re-expressed in vitro. In vivo, after administration of abexinostat for three consecutive days, we did not observe any induction of ESR1 or ESR1-related genes and ERα protein expression by RT-qPCR and immunohistochemical methods in PDXs. This observation was concomitant to the fact that in vivo administration of abexinostat increased intra-tumor histone H3 acetylation. These observations do not allow us to confirm previous studies which suggested that HDACis are able to convert ER-negative (ER-) tumors to ER-positive (ER+) tumors, and that a combination of HDAC inhibitors and hormone therapy could be proposed in the management of TNBC patients.

Topics: Benzofurans; Cell Proliferation; Cyclin D1; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Indoles; Panobinostat; Receptor, ErbB-2; Triple Negative Breast Neoplasms; Vorinostat; Xenograft Model Antitumor Assays

Topics: Antibiotics, Antineoplastic; Benzofurans; Doxorubicin; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Sarcoma

EBV-related nasopharyngeal carcinomas (NPCs) still raise serious therapeutic problems. The therapeutic potential of the histone-deacetylase (HDAC) inhibitor Abexinostat was investigated using 5 preclinical NPC models including 2 patient-derived xenografts (C15 and C17). The cytotoxicity of Abexinostat used either alone or in combination with cis-platin or irradiation was assessed in vitro by MTT and clonogenic assays using 2 EBV-negative (CNE1 and HONE1) and 3 EBV-positive NPC models (C15, C17 and C666-1). Subsequently, the 3 EBV-positive models were used under the form of xenografts to assess the impact of systemic treatments by Abexinostat or combinations of Abexinostat with cis-platin or irradiation. Several cell proteins known to be affected by HDAC inhibitors and the small viral non-coding RNA EBER1 were investigated in the treated tumors. Synergistic cytotoxic effects of Abexinostat combined with cis-platin or irradiation were demonstrated in vitro for each NPC model. When using xenografts, Abexinostat by itself (12.5 mg/kg, BID, 4 days a week for 3 weeks) had significant anti-tumor effects against C17. Cooperative effects with cis-platin (2 mg/kg, IP, at days 3, 10 and 17) and irradiation (1 Gy) were observed for the C15 and C17 xenografts. Simultaneously two types of biological alterations were induced in the tumor tissue, especially in the C17 model: a depletion of the DNA-repair protein RAD51 and a stronger in situ detection of the small viral RNA EBER1. Overall, these results support implementation of phase I/II clinical trials of Abexinostat for the treatment of NPC. A depletion of RAD51 is likely to contribute to the cooperation of Abexinostat with DNA damaging agents. Reduction of RAD51 combined to enhanced detection of EBER 1 might be helpful for early assessment of tumor response.

Topics: Adolescent; Adult; Animals; Antineoplastic Agents; Benzofurans; Carcinoma; Cell Line, Tumor; Cell Survival; Cisplatin; Disease Models, Animal; Drug Synergism; Female; Herpesvirus 4, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inhibitory Concentration 50; Male; Mice; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Rad51 Recombinase; RNA, Viral; Tumor Burden; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

To explore the efficacy of PCI-24781, a broad-spectrum, hydroxamic acid-derived histone deacetylase inhibitor, in the treatment of gastric cancer (GC).. With or without treatment of PCI-24781 and/or cis-diamminedichloroplatinum (CDDP), GC cell lines were subjected to functional analysis, including cell growth, apoptosis and clonogenic assays. Chromatin immunoprecipitation and luciferase reporter assays were used to determine the interacting molecules and the activity of the enzyme. An in vivo study was carried out in GC xenograft mice. Cell culture-based assays were represented as mean ± SD. ANOVA tests were used to assess differences across groups. All pairwise comparisons between tumor weights among treatment groups were made using the Tukey-Kramer method for multiple comparison adjustment to control experimental-wise type I error rates. Significance was set at P < 0.05.. PCI-24781 significantly reduced the growth of the GC cells, enhanced cell apoptosis and suppressed clonogenicity, and these effects synergized with the effects of CDDP. PCI-24781 modulated the cell cycle and significantly reduced the expression of RAD51, which is related to homologous recombination. Depletion of RAD51 augmented the biological functions of PCI-24781, CDDP and the combination treatment, whereas overexpressing RAD51 had the opposite effects. Increased binding of the transcription suppressor E2F4 on the RAD51 promoter appeared to play a major role in these processes. Furthermore, significant suppression of tumor growth and weight in vivo was obtained following PCI-24781 treatment, which synergized with the anticancer effect of CDDP.. These data suggest that RAD51 potentiates the synergistic effects of chemotherapy with PCI-24781 and CDDP on GC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzofurans; Binding Sites; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cisplatin; Dose-Response Relationship, Drug; Drug Synergism; E2F4 Transcription Factor; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice, SCID; Promoter Regions, Genetic; Rad51 Recombinase; Signal Transduction; Stomach Neoplasms; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

MSH3 is a DNA mismatch repair (MMR) gene that undergoes frequent somatic mutation in colorectal cancers (CRCs) with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown.. We utilized isogenic HCT116 (MLH1-/MSH3-) cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3) and also MSH3 by chromosome 5 (HCT116+3+5). We generated HCT116+3+5, SW480 (MLH1+/MSH3+) and SW48 (MLH1-/MSH3+) cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU), SN-38, oxaliplatin, or the histone deacetylase (HDAC) inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed.. MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB) repair. We then utilized PCI-24781 that interferes with homologous recombination (HR) indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone.. MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1 status. PCI-24781-mediated impairment of HR enhanced oxaliplatin sensitivity, suggesting that reduced DSB repair capacity may be contributory.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Benzofurans; Camptothecin; Cell Death; Cell Nucleus; Cell Survival; Colonic Neoplasms; DNA Damage; DNA Mismatch Repair; DNA-Binding Proteins; Drug Screening Assays, Antitumor; Fluorescent Antibody Technique; Fluorouracil; Gene Knockdown Techniques; HCT116 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Irinotecan; MutL Protein Homolog 1; MutS Homolog 3 Protein; Nuclear Proteins; Organoplatinum Compounds; Oxaliplatin; Rad51 Recombinase

A population pharmacokinetic/pharmacodynamic (PK/PD) model was developed to describe the thrombocytopenia (dose-limiting toxicity) of abexinostat, a new histone deacetylase inhibitor. An optimal administration schedule of the drug was determined using a simulation-based approach.. Early PK and PK/PD data were analysed using a sequential population modeling approach (NONMEM 7), allowing for the description of a PK profile and platelet-count decrease after abexinostat administration with various administration schedules. Simulations of platelet count with several administration schedules over 3-week treatment cycles (ASC) and over a day (ASD) were computed to define the optimal schedule that limits the depth of thrombocytopenia.. An intermediate PK/PD model accurately described the data. The administration of abexinostat during the first 4 days of each week in a 3-week cycle resulted in fewer adverse events (with no influence of ASD on platelet count profiles), and corresponded to the optimal treatment schedule. This administration schedule was clinically evaluated in a phase I clinical trial and allowed for the definition of a new maximum tolerated dose (MTD), leading to a nearly 30% higher dose-intensity than that of another previously tested schedule. Lastly, a final model was built using all of the available data.. The final model, characterizing the dose-effect and the dose-toxicity relationships, provides a useful modeling tool for clinical drug development.

Topics: Benzofurans; Clinical Trials, Phase I as Topic; Computer Simulation; Dose-Response Relationship, Drug; Drug Administration Schedule; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Models, Biological; Platelet Count; Thrombocytopenia

Hormonal therapy resistance remains a considerable barrier in the treatment of breast cancer. Activation of the Akt-PI3K-mTOR pathway plays an important role in hormonal therapy resistance. Our recent preclinical and clinical studies showed that the addition of a histone deacetylase inhibitor re-sensitized hormonal therapy resistant breast cancer to tamoxifen. As histone deacetylases are key regulators of Akt, we evaluated the effect of combined treatment with the histone deacetylase inhibitor PCI-24781 and tamoxifen on Akt in breast cancer cells. We demonstrate that while both histone deacetylase and estrogen receptor inhibition down regulate AKT mRNA and protein, their concerted effort results in down regulation of AKT activity with induction of cell death. Histone deacetylase inhibition exerts its effect on AKT mRNA through an estrogen receptor-dependent mechanism, primarily down regulating the most abundant isoform AKT1. Although siRNA depletion of AKT modestly induces cell death, when combined with an anti-estrogen, cytotoxicity is significantly enhanced. Thus, histone deacetylase regulation of AKT mRNA is a key mediator of this therapeutic combination and may represent a novel biomarker for predicting response to this regimen.

Topics: Apoptosis; Benzofurans; Breast Neoplasms; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mitochondria; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; RNA, Messenger; Selective Estrogen Receptor Modulators; Tamoxifen

Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34(+) cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.

Topics: Acetylation; Benzofurans; Cell Growth Processes; DNA Repair; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Phosphorylation; Signal Transduction; Thrombocytopenia; Tumor Suppressor Protein p53

Neuroblastoma is the second most common solid tumor diagnosed during infancy. The survival rate among children with high-risk neuroblastoma is less than 40%, highlighting the urgent needs for new treatment strategies. PCI-24781 is a novel hydroxamic acid-based histone deacetylase (HDAC) inhibitor that has high efficacy and safety for cancer treatment. However, the underlying mechanisms of PCI-24781 are not clearly elucidated in neuroblastoma cells. In the present study, we demonstrated that PCI-24781 treatment significantly inhibited tumor growth at very low doses in neuroblastoma cells SK-N-DZ, not in normal cell line HS-68. However, PCI-24781 caused the accumulation of acetylated histone H3 both in SK-N-DZ and HS-68 cell line. Treatment of SK-N-DZ with PCI-24781 also induced cell cycle arrest in G2/M phase and activated apoptosis signaling pathways via the up-regulation of DR4, p21, p53 and caspase 3. Further proteomic analysis revealed differential protein expression profiles between non-treated and PCI-24781 treated SK-N-DZ cells. Totally 42 differentially expressed proteins were identified by MALDI-TOF MS system. Western blotting confirmed the expression level of five candidate proteins including prohibitin, hHR23a, RuvBL2, TRAP1 and PDCD6IP. Selective knockdown of RuvBL2 rescued cells from PCI-24781-induced cell death, implying that RuvBL2 might play an important role in anti-tumor activity of PCI-24781 in SK-N-DZ cells. The present results provide a new insight into the potential mechanism of PCI-24781 in SK-N-DZ cell line.

Topics: Acetylation; Apoptosis; ATPases Associated with Diverse Cellular Activities; Benzofurans; Blotting, Western; Carrier Proteins; Cell Line, Tumor; DNA Helicases; G2 Phase Cell Cycle Checkpoints; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Male; Mitosis; Models, Biological; Neoplasm Proteins; Neuroblastoma; Proteomics; Reproducibility of Results; Signal Transduction

Cancer stem cells (CSC) are the tumorigenic cell population that has been shown to sustain tumor growth and to resist conventional therapies. The purpose of this study was to evaluate the potential of histone deacetylase inhibitors (HDACi) as anti-CSC therapies.. We evaluated the effect of the HDACi compound abexinostat on CSCs from 16 breast cancer cell lines (BCL) using ALDEFLUOR assay and tumorsphere formation. We performed gene expression profiling to identify biomarkers predicting drug response to abexinostat. Then, we used patient-derived xenograft (PDX) to confirm, in vivo, abexinostat treatment effect on breast CSCs according to the identified biomarkers.. We identified two drug-response profiles to abexinostat in BCLs. Abexinostat induced CSC differentiation in low-dose sensitive BCLs, whereas it did not have any effect on the CSC population from high-dose sensitive BCLs. Using gene expression profiling, we identified the long noncoding RNA Xist (X-inactive specific transcript) as a biomarker predicting BCL response to HDACi. We validated that low Xist expression predicts drug response in PDXs associated with a significant reduction of the breast CSC population.. Our study opens promising perspectives for the use of HDACi as a differentiation therapy targeting the breast CSCs and identified a biomarker to select patients with breast cancer susceptible to responding to this treatment.

Topics: Animals; Antineoplastic Agents; Benzofurans; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inhibitory Concentration 50; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; RNA, Long Noncoding; Tumor Burden; Xenograft Model Antitumor Assays

Hypoxia inducible factor (HIF) is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI), enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells.

Topics: AMP-Activated Protein Kinases; Autophagy; Benzofurans; Cell Survival; Drug Synergism; Enzyme Activation; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphoma, Large B-Cell, Diffuse; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Gallbladder carcinoma (GBCa), a type of biliary tract cancer (BTC), has proven challenging to treat, demonstrating the need for more effective therapeutic strategies. In our current study, we examined the therapeutic effects of the histone deacetylase (HDAC) inhibitor PCI-24781 against GBCa that developed in BK5.erbB2 mice.. PCI-24781 [50 mg/kg/day] and control solutions were administered to BK5.erbB2 mice for 4 weeks. The therapeutic effect of PCI-24781 was evaluated by ultrasound biomicroscopy (USBM) throughout the experiment and histological analyses at the end of the experiment. To investigate potential mechanisms underlining the therapeutic effects of PCI-24781 on GBCa in BK5.erbB2 mice, PCI-24781-treated gallbladders were subjected to Western blot and RT-PCR analysis. The inhibitory effect of PCI-24781 on the growth of BTC cells was compared to the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and gemcitabine. To study the role of miRNAs in GBCa tumorigenesis, the expression profile of 368 miRNAs in GBCas from BK5.erbB2 (both treated and untreated) and wild type mice was analyzed.. Treatment of BK5.erbB2 mice with PCI-24781 for 1 month prevented 79% of GBCa cases from progression and showed a clinical effect in 47% of cases. We also confirmed a potent inhibitory effect on tumor cell growth in human BTC cell lines treated with PCI-24781. This effect was associated with downregulation of ErbB2 mRNA and ErbB2 protein/activity and upregulation of acetylated histone and acetylated tubulin. Treatment with PCI-24781 resulted in decreased expression of Muc4, an intramembrane ligand for ErbB2, in BTC cells. PCI-24781 had more effects on growth inhibition of BTC cells than SAHA. In addition, PCI-24781 effectively inhibited the growth of gemcitabine-resistant cells. miRNA profiling revealed that the expression of several miRNAs was significantly altered in GBCa in the BK5.erbB2 mouse compared to normal gallbladder, including upregulated miR21, which was downregulated by PCI-24781.. These results indicate that PCI-24781 potently inhibits the growth of BTC cells by decreasing ErbB2 expression and activity as well as regulating altered miRNA expression. PCI-24781 may have a potential value as a novel chemotherapeutic agent against human BTC in which ErbB2 is overexpressed.

Topics: Animals; Antimetabolites, Antineoplastic; Benzofurans; Carcinoma; Carrier Proteins; Cell Division; Cell Line, Tumor; Deoxycytidine; Disease Models, Animal; Gallbladder; Gallbladder Neoplasms; Gemcitabine; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Mice; Mice, Mutant Strains; Mucin-4; Phosphorylation; RNA, Messenger

To better understand the mechanisms of cytotoxicity and cell death induced by HDACI PCI-24781 in bone sarcoma cells.. Four bone sarcoma cell lines were treated with PCI-24781, and the cytotoxicity was investigated. Further, accumulation of acetylated histones, p21, and PARP cleavage were evaluated in PCI-24781-treated cells. The synergistic effect of PCI-24781 to doxorubicin and its mechanism was investigated in bone sarcoma cells.. MTT assay demonstrated that the growth of bone sarcoma cells was inhibited after treatment with PCI-24781. Accumulation of acetylated histones, p21, and PARP cleavage were found in PCI-24781-treated cells. Expression of DNA repair protein RAD51 was inhibited, and the expression of apoptosis protein GADD45α was induced by PCI-24781 in bone sarcoma cells. Bone sarcoma cells treated with PCI-24781 become more sensitive to doxorubicin. The caspase-3/7 activity was increased with doxorubicin and PCI-24781 treatment in these cells.. HDACI PCI-24781 has a synergistic effect on doxorubicin-induced apoptosis in bone sarcoma cells.

Topics: Acetylation; Apoptosis; Benzofurans; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Chondrosarcoma; Doxorubicin; Drug Synergism; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Nuclear Proteins; Osteosarcoma; Rad51 Recombinase

The antitumor activity of histone deacetylase inhibitors (HDACI) on multidrug-resistant sarcoma cell lines has not been previously described. Treatment of multidrug-resistant sarcoma cell lines with HDACI PCI-24781 resulted in dose-dependent accumulation of acetylated histone, p21 and poly(ADP-ribose)polymerase (PARP) cleavage products. Growth of these cell lines was inhibited by PCI-24781 at IC(50) of 0.43 to 2.7. When we looked for synergy of PCI-24781 with chemotherapeutic agents, we found that PCI-24781 reverses drug resistance in all four multidrug-resistant sarcoma cell lines and synergizes with chemotherapeutic agents to enhance caspase-3/-7 activity. Expression of RAD51 (a marker for DNA double-strand break repair) was inhibited and the expression of GADD45α (a marker for growth arrest and DNA-damage) was induced by PCI-24781 in multidrug-resistant sarcoma cell lines. In conclusion, HDACI PCI-24781 synergizes with chemotherapeutic drugs to induce apoptosis and reverses drug resistance in multidrug-resistant sarcoma cell lines.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Benzofurans; Blotting, Western; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Nuclear Proteins; Poly(ADP-ribose) Polymerases; Sarcoma

The concept of cancer stem cells is generally accepted in different malignancies. We have previously shown that the MDA-MB231 breast cancer cells were more radiation resistant when sorted for the two stem cell markers CD24 and ESA. In this study, we examined a possible mechanism that might underlie this phenotype by looking at cell cycle profile and the effect this has on DNA repair pathways. The cell cycle profile showed that there were more CD24(-) ESA(+) sorted MDA-MB231 cells in the S- and G(2)-phases compared with the unsorted cells, 60 and 38% respectively. Cyclin D and E protein levels supported the cell cycle profile and highlighted the possible involvement of homologous recombination (HR) repair in the radioresistant phenotype. To further support this, CD24(-) ESA(+) sorted MDA-MB231 cells demonstrated statistically significant more RAD51 and less γ-H2AX foci 2 h post 4Gy ionising radiation, compared with the unsorted population. Inhibition of the HR pathway effectively sterilised the CD24(- ) ESA(+) sorted MDA-MB231 cells but had no effect on the unsorted cells or MDA468 control breast cancer cell line. Although the changes we saw were specific to MDA-MB231, these results merit further investigation and can be crucial in identifying a mechanism responsible for cancer stem cells treatment resistance in primary tumors.

Topics: Benzofurans; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplastic Stem Cells; Rad51 Recombinase; Radiation Tolerance; Recombination, Genetic

Histone deactylase inhibitors (HDACi) are a promising new class of anticancer therapeutics; however, little is known about HDACi activity in soft tissue sarcoma (STS), a heterogeneous cohort of mesenchymal origin malignancies. Consequently, we investigated the novel HDACi PCI-24781, alone/in combination with conventional chemotherapy, to determine its potential anti-STS-related effects and the underlying mechanisms involved.. Immunoblotting was used to evaluate the effects of PCI-24781 on histone and nonhistone protein acetylation and expression of potential downstream targets. Cell culture-based assays were utilized to assess the effects of PCI-24781 on STS cell growth, cell cycle progression, apoptosis, and chemosensitivity. Quantitative reverse transcription-PCR, chromatin immunoprecipitation, and reporter assays helped elucidate molecular mechanisms resulting in PCI-24781-induced Rad51 repression. The effect of PCI-24781, alone or with chemotherapy, on tumor and metastatic growth was tested in vivo using human STS xenograft models.. PCI-24781 exhibited significant anti-STS proliferative activity in vitro, inducing S phase depletion, G(2)/M cell cycle arrest, and increasing apoptosis. Superior effects were seen when combined with chemotherapy. A PCI-24781-induced reduction in Rad51, a major mediator of DNA double-strand break homologous recombination repair, was shown and may be a mechanism underlying PCI-24781 chemosensitization. We showed that PCI-24781 transcriptionally represses Rad51 through an E2F binding-site on the Rad51 proximal promoter. Although single-agent PCI-24781 had modest effects on STS growth and metastasis, marked inhibition was observed when combined with chemotherapy.. In light of these findings, this novel molecular-based combination may be applicable to multiple STS histologic subtypes, and potentially merits rigorous evaluation in human STS clinical trials.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzofurans; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cisplatin; Dose-Response Relationship, Drug; Doxorubicin; Exonucleases; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Immunohistochemistry; Luciferases; Luminescent Measurements; Mice; Mice, SCID; Reverse Transcriptase Polymerase Chain Reaction; S Phase; Sarcoma, Experimental; Tumor Burden; Xenograft Model Antitumor Assays

We investigated the cytotoxicity and mechanisms of cell death of the broad-spectrum histone deacetylase (HDAC) inhibitor PCI-24781, alone and combined with bortezomib in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines and primary lymphoproliferative (CLL/SLL) cells.. Apoptosis, mitochondrial membrane potential, cell cycle analysis, and reactive oxygen species (ROS) were measured by flow cytometry, whereas caspase activation was determined by Western blot. Nuclear factor kappaB (NF-kappaB)-related mRNAs were quantified by reverse transcription-PCR, NF-kappaB-related proteins by Western blotting, and NF-kappaB DNA-binding activity by electromobility shift assay. Finally, gene expression profiling was analyzed.. PCI-24781 induced concentration-dependent apoptosis that was associated with prominent G(0)/G(1) arrest, decreased S-phase, increased p21 protein, and increased ROS in Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Dose-dependent apoptosis with PCI-24781 was also seen among primary CLL/SLL cells. PCI-24781-induced apoptosis was shown to be ROS- and caspase-dependent. Combined PCI-24781/bortezomib treatment resulted in strong synergistic apoptosis in all non-Hodgkin lymphoma lines (combination indices, 0.19-0.6) and was additive in Hodgkin lymphoma and primary CLL/SLL cells. Further, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone acetylation compared with either agent alone. Gene expression profiling showed that PCI-24781 alone significantly down-regulated several antioxidant genes, proteasome components, and NF-kappaB pathway genes, effects that were enhanced further with bortezomib. Reverse transcription-PCR confirmed down-regulation of NF-kappaB1 (p105), c-Myc, and IkappaB-kinase subunits, where NF-kappaB DNA binding activity was decreased.. We show that PCI-24781 results in increased ROS and NF-kappaB inhibition, leading to caspase-dependent apoptosis. We also show that bortezomib is synergistic with PCI-24781. This combination or PCI-24781 alone has potential therapeutic value in lymphoma.

Topics: Aged; Antineoplastic Agents; Apoptosis; Benzofurans; Blotting, Western; Boronic Acids; Bortezomib; Caspases; Cell Cycle; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Female; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lymphoma; Lymphoma, Non-Hodgkin; Male; Membrane Potential, Mitochondrial; Middle Aged; NF-kappa B; Pyrazines; Reactive Oxygen Species; Tumor Cells, Cultured

PCI-24781 is a novel broad spectrum histone deacetylase inhibitor that is currently in phase I clinical trials. The ability of PCI-24781 to act as a radiation sensitizer and the mechanisms of radiosensitization were examined.. Exponentially growing human SiHa cervical and WiDr colon carcinoma cells were exposed to 0.1 to 10 micromol/L PCI-24781 in vitro for 2 to 20 h before irradiation and 0 to 4 h after irradiation. Single cells and sorted populations were analyzed for histone acetylation, H2AX phosphorylation, cell cycle distribution, apoptotic fraction, and clonogenic survival.. PCI-24781 treatment for 4 h increased histone H3 acetylation and produced a modest increase in gammaH2AX but negligible cell killing or radiosensitization. Treatment for 24 h resulted in up to 80% cell kill and depletion of cells in S phase. Toxicity reached maximum levels at a drug concentration of approximately 1 micromol/L, and cells in G(1) phase at the end of treatment were preferentially spared. A similar dose-modifying factor (DMF(0.1) = 1.5) was observed for SiHa cells exposed for 24 h at 0.1 to 3 micromol/L, and more radioresistant WiDr cells showed less sensitization (DMF(0.1) = 1.2). Limited radiosensitization and less killing were observed in noncycling human fibroblasts. Cell sorting experiments confirmed that depletion of S-phase cells was not a major mechanism of radiosensitization and that inner noncycling cells of SiHa spheroids could be sensitized by nontoxic doses. PCI-24781 pretreatment increased the fraction of cells with gammaH2AX foci 24 h after irradiation but did not affect the initial rate of loss of radiation-induced gammaH2AX or the rate of rejoining of DNA double-strand breaks.. PCI-24781 shows promise as a radiosensitizing agent that may compromise the accuracy of repair of radiation damage.

Topics: Benzofurans; Cell Line, Tumor; DNA Breaks, Double-Stranded; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Radiation Tolerance; Radiation-Sensitizing Agents

Histone deacetylase (HDAC) inhibitors such as the phenyl hydroxamic acid PCI-24781 have emerged recently as a class of therapeutic agents for the treatment of cancer. Recent data showing synergy of HDAC inhibitors with ionizing radiation and other DNA-damaging agents have suggested that HDAC inhibitors may act, in part, by inhibiting DNA repair. Here we present evidence that HDAC enzymes are important for homologous recombinational repair of DNA double-strand breaks. Combination studies of PCI-24781 with the poly(ADP-ribose) polymerase inhibitor PJ34, an agent thought to produce lesions repaired by homologous recombination (HR), resulted in a synergistic effect on apoptosis. Immunofluorescence analysis demonstrated that HDAC inhibition caused a complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigations revealed that inhibition of HDAC enzymes by PCI-24781 led to a significant reduction in the transcription of genes specifically associated with HR, including RAD51. RAD51 protein levels were significantly decreased after 24 h of drug exposure both in vitro and in vivo. Consistent with inhibition of HR, treatment with PCI-24781 resulted in a decreased ability to perform homology directed repair of I-SceI-induced chromosome breaks in transfected CHO cells. In addition, an enhancement of cell killing was observed in Ku mutant cells lacking functional nonhomologous end joining compared with WT cells. Together these results demonstrate that HDAC enzymes are critically important to enable functional HR by controlling the expression of HR-related genes and promoting the proper assembly of HR-directed subnuclear foci.

Topics: Animals; Apoptosis; Benzofurans; Cell Line, Tumor; DNA Repair; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Rad51 Recombinase; Radiation Tolerance; Radiation-Sensitizing Agents; Recombination, Genetic; Transcription, Genetic; Up-Regulation

CRA-024781 is a novel, broad spectrum hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor activity in vitro and in vivo preclinically and is under evaluation in phase I clinical trials for cancer. CRA-024781 inhibited pure recombinant HDAC1 with a K(i) of 0.007 mumol/L, and also inhibited the other HDAC isozymes HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-024781 resulted in the accumulation of acetylated histone and acetylated tubulin, resulting in an inhibition of tumor cell growth and the induction of apoptosis. CRA-024781 parenterally administered to mice harboring HCT116 or DLD-1 colon tumor xenografts resulted in a statistically significant reduction in tumor growth at doses that were well tolerated as measured by body weight. Inhibition of tumor growth was accompanied by an increase in the acetylation of alpha-tubulin in peripheral blood mononuclear cells, and an alteration in the expression of many genes in the tumors, including several involved in apoptosis and cell growth. These results reveal CRA-024781 to be a novel HDAC inhibitor with potent antitumor activity.

Topics: Acetylation; Animals; Antineoplastic Agents; Benzofurans; Biomarkers, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Drug Design; Enzyme Inhibitors; Female; HCT116 Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; In Vitro Techniques; Mice; Mice, Inbred BALB C; Poly(ADP-ribose) Polymerases; Transcription, Genetic; Tumor Cells, Cultured