Valproic Acid Response Research Articles

Revisiting Concurrent Radiation Therapy, Temozolomide, and the Histone Deacetylase Inhibitor Valproic Acid for Patients with Glioblastoma-Proteomic Alteration and Comparison Analysis with the Standard-of-Care Chemoirradiation.

Glioblastoma (GBM) is the most common brain tumor with an overall survival (OS) of less than 30% at two years. Valproic acid (VPA) demonstrated survival benefits documented in retrospective and prospective trials, when used in combination with chemo-radiotherapy (CRT). The primary goal of this study was to examine if the differential alteration in proteomic expression pre vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA as compared to standard-of-care CRT. The second goal was to explore the associations between the proteomic alterations in response to VPA/RT/TMZ correlated to patient outcomes. The third goal was to use the proteomic profile to determine the mechanism of action of VPA in this setting. Serum obtained pre- and post-CRT was analyzed using an aptamer-based SOMAScan® proteomic assay. Twenty-nine patients received CRT plus VPA, and 53 patients received CRT alone. Clinical data were obtained via a database and chart review. Tests for differences in protein expression changes between radiation therapy (RT) with or without VPA were conducted for individual proteins using two-sided t-tests, considering p-values of <0.05 as significant. Adjustment for age, sex, and other clinical covariates and hierarchical clustering of significant differentially expressed proteins was carried out, and Gene Set Enrichment analyses were performed using the Hallmark gene sets. Univariate Cox proportional hazards models were used to test the individual protein expression changes for an association with survival. The lasso Cox regression method and 10-fold cross-validation were employed to test the combinations of expression changes of proteins that could predict survival. Predictiveness curves were plotted for significant proteins for VPA response (p-value < 0.005) to show the survival probability vs. the protein expression percentiles. A total of 124 proteins were identified pre- vs. post-CRT that were differentially expressed between the cohorts who received CRT plus VPA and those who received CRT alone. Clinical factors did not confound the results, and distinct proteomic clustering in the VPA-treated population was identified. Time-dependent ROC curves for OS and PFS for landmark times of 20 months and 6 months, respectively, revealed AUC of 0.531, 0.756, 0.774 for OS and 0.535, 0.723, 0.806 for PFS for protein expression, clinical factors, and the combination of protein expression and clinical factors, respectively, indicating that the proteome can provide additional survival risk discrimination to that already provided by the standard clinical factors with a greater impact on PFS. Several proteins of interest were identified. Alterations in GALNT14 (increased) and CCL17 (decreased) (p = 0.003 and 0.003, respectively, FDR 0.198 for both) were associated with an improvement in both OS and PFS. The pre-CRT protein expression revealed 480 proteins predictive for OS and 212 for PFS (p < 0.05), of which 112 overlapped between OS and PFS. However, FDR-adjusted p values were high, with OS (the smallest p value of 0.586) and PFS (the smallest p value of 0.998). The protein PLCD3 had the lowest p-value (p = 0.002 and 0.0004 for OS and PFS, respectively), and its elevation prior to CRT predicted superior OS and PFS with VPA administration. Cancer hallmark genesets associated with proteomic alteration observed with the administration of VPA aligned with known signal transduction pathways of this agent in malignancy and non-malignancy settings, and GBM signaling, and included epithelial-mesenchymal transition, hedgehog signaling, Il6/JAK/STAT3, coagulation, NOTCH, apical junction, xenobiotic metabolism, and complement signaling. Differential alteration in proteomic expression pre- vs. post-completion of concurrent chemoirradiation (CRT) is present with the addition of VPA. Using pre- vs. post-data, prognostic proteins emerged in the analysis. Using pre-CRT data, potentially predictive proteins were identified. The protein signals and hallmark gene sets associated with the alteration in the proteome identified between patients who received VPA and those who did not, align with known biological mechanisms of action of VPA and may allow for the identification of novel biomarkers associated with outcomes that can help advance the study of VPA in future prospective trials.

Effects of SCN1A and SCN2A polymorphisms on responsiveness to valproic acid monotherapy in epileptic children: A protocol for systematic review and meta-analysis.

Background: The gene mutation of coding sodium channel is one of the most important mechanisms in the pathogenesis of epilepsy. There exists a large inter-individual variation in the efficacy of valproic acid (VPA) against epilepsy. What are the genetic polymorphism influences of sodium channels on VPA response is still under discussion. In this study, a meta-analysis was used to further explore the effects of SCN1A and SCN2A gene polymorphism on VPA response in children with epilepsy.Methods: The PubMed, EMBASE, Web of Science, Chinese National Knowledge Infrastructure, Chinese Science and Technique Journals Database, China Biology Medicine disc, and Wan Fang Database were searched up to April 2021 for appropriate studies regarding the association between SCN1A and SCN2A gene polymorphism on VPA response in children suffering from epilepsy. The meta-analysis was conducted by Review Manager 5.3 software.Results: The results of this meta-analysis will be submitted to a peer-reviewed journal for publication.Conclusion: This meta-analysis will summarize the effects of SCN1A and SCN2A gene polymorphisms on VPA response in children with epilepsy.OSF Registration Number:DOI 10.17605/OSF.IO/N2786.

CYP3A5 genetic variants and their associations with carbamazepine and valproic acid response in Malaysian epileptic patients.

Epilepsy is a common chronic neurological condition characterized by recurrent seizures. Approximately 30 - 40% of epileptic patients do not respond to antiepileptic drugs. Previous studies suggest that CYP3A5 polymorphisms affect carbamazepine metabolism. To examine this hypothesis, in the present study, the associations between CYP3A5 variants (rs776746 and rs1419745) and response to carbamazepine and valproic acid monotherapy in Malaysian epileptic patients were evaluated. A total of 288 Malaysian epileptic patients were recruited and further reviewed, of whom 63 patients were on carbamazepine monotherapy, and 85 patients were on valproic acid monotherapy. There was no patient with drug hypersensitivity syndrome within the population. Subjects were genotyped by using Sequenom MassARRAY platform. This study found a significant association of CYP3A5 rs776746 with the carbamazepine treatment response in total patients (p=0.026) and Malay ethnic subgroup (p=0.006). In addition, a marginal significant association of CYP3A5 rs1419745 with carbamazepine treatment response was reported in the Malays. Similarly, CYP3A5 rs776746 was associated with valproic acid response in total patients (p=0.037) and Malays (marginal p=0.05). Our findings suggest that CYP3A5 polymorphisms affect carbamazepine and valproic acid response in Malaysian epileptic patients.

Association of UGT2B7 and CaMK4 with response of valproic acid in Chinese children with epilepsy

Valproic acid (VPA) is a widely used antiepileptic drug for epilepsy. However, approximately 30% of patients with epilepsy do not respond to this therapy even when it was appropriately used. In order to explore the potential genetic factors related to the VPA response, this pharmacogenetics study was conducted. A total of one hundred and fifty-seven Chinese children with epilepsy who were administered with by VPA for at least one year were enrolled. Thirteen single-nucleotide polymorphisms (SNPs) located in eight genes involving targets and metabolic enzymes of VPA were genotyped. The frequencies of these polymorphisms and the effect of genotypes on the efficacy of VPA were analyzed. The frequencies of two SNPs, rs7668258 (uridine diphosphate glucuronosyltransferase-2B7, UGT2B7) and rs306104 (calmodulin-kinase 4, CaMK4) were associated with VPA responses. However, no association was found for the other SNPs. Furthermore, the polymorphism of UGT2B7 influenced the adjusted concentration (AC) in the responders rather than in the non-responders. Two SNPs (UGT2B7 and CaMK4) were associated with VPA response, which may explain the pharmacological mechanism of VPA resistance to some extent.

SCN1A and SCN2A polymorphisms are associated with response to valproic acid in Chinese epilepsy patients.

There is a large inter-individual variation in the efficacy of valproic acid (VPA) against epilepsy. The genetic polymorphism influence of sodium channels on VPA response remains a matter of debate. The aim of the study was to explore the effect of SCN1A and SCN2A gene polymorphisms on VPA response in the treatment of epilepsy among Chinese patients. A total of 354 epileptic patients with VPA treatment were genotyped for five single nucleotide polymorphisms (SNP), including SCN1A rs10188577 T>C, rs2298771 T>C, rs3812718 G>A, and SCN2A rs2304016 A>G, rs17183814 G>A. A binary logistic regression analysis was performed to evaluate the association of genotype with VPA antiepileptic effects, adjusting the influence of confounding factors. Genotype distributions of all selected SNPs were consistent with the Hardy-Weinberg equilibrium in epilepsy patients. SCN1A rs3812718 and SCN2A rs2304016 were found to be significantly associated with VPA response, both in monotherapy and in VPA-based polytherapy. Patients with the rs3812718 A allele were more frequently seen in the VPA-responsive group (P < 0.05), and the rs2304016 G allele was related to an increased risk of resistance to VPA therapy (P < 0.05). Our study revealed that SCN1A rs3812718 and SCN2A rs2304016 polymorphisms might be markers of VPA response in Chinese epilepsy patients. ChiCTR-1800016477.

Lack of association between valproic acid response and polymorphisms of its metabolism, transport, and receptor genes in children with focal seizures.

This study aims to describe the associations between genetic polymorphisms and therapeutic effect of valproic acid (VPA) in children with focal seizures. Eighty-nine children with focal seizures on VPA therapy were enrolled. Patients' basic information, dosage regimens, and plasma concentrations were recorded. A 1-year follow-up was performed to evaluate the treatment response. Sixty-six single nucleotide polymorphisms involved in the metabolism, transport, and target receptor of VPA were identified, and their associations with VPA response were analyzed using logistic regression adjusted by various influence factors. Selected polymorphisms involved in the metabolism, transport, and target receptor of VPA were not associated with treatment effect in children with focal seizures. Three variants, rs9313892 (GABRA6, G > A, OR = 2.73, 95% CI 1.00 to 7.48, P = 0.051), rs4921195 (GABRA6, T > C, OR = 2.71, 95% CI 0.99 to 7.42, P = 0.053), and rs424740 (GABRG2, A > T, OR = 0.39, 95% CI 0.15 to 1.01, P = 0.053) had the potential to be associated with the VPA response. Selected genetic polymorphisms were not significantly associated with VPA response in children with focal seizures. However, three GABR variants showed potential to be associated with the response to VPA. Further and larger studies are warranted to confirm the results.

Correlation of MCT1 and ABCC2 gene polymorphisms with valproic acid resistance in patients with epilepsy on valproic acid monotherapy

Valproic acid (VPA) is used as one of the first-line antiepileptic drugs to control seizure in epilepsy patients. However, one third of patients do not respond to VPA. This study is to investigate the influence of single nucleotide polymorphisms (SNPs) in multidrug transporters on VPA responses in Han Chinese epilepsy patients on VPA monotherapy. Twelve SNPs involved in VPA transport pathways, including ABCC2, ABCC4, ABCG2, MCT1, MCT2 and OATP2B1 were genotyped in 153 Han Chinese epilepsy patients. We found that among all the patients, MCT1 rs60844753 CC carriers have higher incidence of VPA-resistance than CG carriers (P = 0.05), and in subgroup of generalized seizure, ABCC2 rs3740066 CC carriers had higher frequency of VPA resistance than TC + TT carriers (P = 0.03). Although other SNPs were not correlated with VPA resistance, significant ethnic difference was found in minor allele frequency of these SNPs, indicating that the influence of these SNPs on VPA efficacy should be broadly investigated in other ethnic populations. This study provides nominal evidence that SNPs of genes involved in the transport of VPA contribute to interpatient variation in VPA response. Although the associations were abolished after Bonferroni correction, the results provide an incentive for further research in sufficiently large samples.

Valproic acid inhibits glioblastoma multiforme cell growth via paraoxonase 2 expression.

We studied the potential mechanisms of valproic acid (VPA) in the treatment of glioblastoma multiforme (GBM). Using the human U87, GBM8401, and DBTRG-05MG GBM-derived cell lines, VPA at concentrations of 5 to 20 mM induced G2/M cell cycle arrest and increased the production of reactive oxygen species (ROS). Stress-related molecules such as paraoxonase 2 (PON2), cyclin B1, cdc2, and Bcl-xL were downregulated, but p27, p21 and Bim were upregulated by VPA treatment. VPA response element on the PON2 promoter was localized at position -400/−1. PON2 protein expression was increased in GBM cells compared with normal brain tissue and there was a negative correlation between the expression of PON2 and Bim. These findings were confirmed by the public Bredel GBM microarray (Gene Expression Omnibus accession: GSE2223) and the Cancer Genome Atlas GBM microarray datasets. Overexpression of PON2 in GBM cells significantly decreased intracellular ROS levels, and PON2 expression was decreased after VPA stimulation compared with controls. Bim expression was significantly induced by VPA in GBM cells with PON2 silencing. These observations were further shown in the subcutaneous GBM8401 cell xenograft of BALB/c nude mice. Our results suggest that VPA reduces PON2 expression in GBM cells, which in turn increases ROS production and induces Bim production that inhibits cancer progression via the PON2–Bim cascade.

Polymorphisms of ABAT, SCN2A and ALDH5A1 may affect valproic acid responses in the treatment of epilepsy in Chinese.

The clinical efficacy of valproic acid (VPA) varies greatly among epileptic patients. To find the potential genetic factors related to VPA responses, the pharmacogenetics study was conducted. Two hundred and one Chinese Han epileptic patients who were treated by VPA for at least 1 year were recruited. Up to 24 SNPs in 11 candidate genes that correlate with the metabolism, transport or target of VPA were genotyped. Three SNPs, rs1731017 (ABAT), rs2304016 (SCN2A) and rs1054899 (ALDH5A1) were found associated with VPA responses with the p-values of 0.003, 0.007 and 0.048, respectively. Further interaction analysis showed that the interaction between rs17183814 (ABAT) and rs1641022 (SCN2A) was also correlated with the response of VPA (p = 0.006). This study found three SNPs and one interaction among ABAT, SCN2A and ALDH5A1 were significantly associated with VPA response, which indicated that these genes may play important roles in the pharmacological mechanism of VPA.

Molecular dissection of valproic acid effects in acute myeloid leukemia identifies predictive networks

ABSTRACTHistone deacetylase inhibitors (HDACIs) like valproic acid (VPA) display activity in leukemia models and induce tumor-selective cytotoxicity against acute myeloid leukemia (AML) blasts. As there are limited data on HDACIs effects, we aimed to dissect VPA effects in vitro using myeloid cell lines with the idea to integrate findings with in vivo data from AML patients treated with VPA additionally to intensive chemotherapy (n = 12). By gene expression profiling we identified an in vitro VPA response signature enriched for genes/pathways known to be implicated in cell cycle arrest, apoptosis, and DNA repair. Following VPA treatment in vivo, gene expression changes in AML patients showed concordant results with the in vitro VPA response despite concomitant intensive chemotherapy. Comparative miRNA profiling revealed VPA-associated miRNA expression changes likely contributing to a VPA-induced reversion of deregulated gene expression. In addition, we were able to define markers predicting VPA response in vivo such as CXCR4 and LBH. These could be validated in an independent cohort of VPA and intensive chemotherapy treated AML patients (n = 114) in which they were inversely correlated with relapse-free survival. In summary, our data provide new insights into the molecular mechanisms of VPA in myeloid blasts, which might be useful in further advancing HDAC inhibition based treatment approaches in AML.

Down-modulation of SEL1L, an Unfolded Protein Response and Endoplasmic Reticulum-associated Degradation Protein, Sensitizes Glioma Stem Cells to the Cytotoxic Effect of Valproic Acid

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments.

VPA response in SMA is suppressed by the fatty acid translocase CD36

Functional loss of SMN1 causes proximal spinal muscular atrophy (SMA), the most common genetic condition accounting for infant lethality. Hence, the hypomorphic copy gene SMN2 is the only resource of functional SMN protein in SMA patients and influences SMA severity in a dose-dependent manner. Consequently, current therapeutic approaches focus on SMN2. Histone deacetylase inhibitors (HDACi), such as the short chain fatty acid VPA (valproic acid), ameliorate the SMA phenotype by activating the SMN2 expression. By analyzing blood SMN2 expression in 16 VPA-treated SMA patients, about one-third of individuals were identified as positive responders presenting increased SMN2 transcript levels. In 66% of enrolled patients, a concordant response was detected in the respective fibroblasts. Most importantly, by taking the detour of reprograming SMA patients' fibroblasts, we showed that the VPA response was maintained even in GABAergic neurons derived from induced pluripotent stem cells (iPS) cells. Differential expression microarray analysis revealed a complete lack of response to VPA in non-responders, which was associated with an increased expression of the fatty acid translocase CD36. The pivotal role of CD36 as the cause of non-responsiveness was proven in various in vitro approaches. Most importantly, knockdown of CD36 in SMA fibroblasts converted non- into pos-responders. In summary, the concordant response from blood to the central nervous system (CNS) to VPA may allow selection of pos-responders prior to therapy. Increased CD36 expression accounts for VPA non-responsiveness. These findings may be essential not only for SMA but also for other diseases such as epilepsy or migraine frequently treated with VPA.

A genomic approach to predict synergistic combinations for breast cancer treatment

We leverage genomic and biochemical data to identify synergistic drug regimens for breast cancer. In order to study the mechanism of the histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) in breast cancer, we generated and validated genomic profiles of drug response using a series of breast cancer cell lines sensitive to each drug. These genomic profiles were then used to model drug response in human breast tumors and show significant correlation between VPA and SAHA response profiles in multiple breast tumor datasets, highlighting their similar mechanism of action. The genes deregulated by VPA and SAHA converge on the cell cycle pathway (Bayes Factor 5.21, and 5.94, respectively, p-value 10−8.6 and 10−9, respectively). In particular, VPA and SAHA upregulate key cyclin-dependent kinase (CDK) inhibitors. In two independent datasets, cancer cells treated with CDK inhibitors have similar gene expression profile changes to the cellular response to HDAC inhibitors. Together, these results led us to hypothesize that VPA and SAHA may interact synergistically with CDK inhibitors such as PD-033299. Experiments show that HDAC and CDK inhibitors have statistically significant synergy in both breast cancer cell lines and primary 3-dimensional cultures of cells from pleural effusions of patients. Therefore, synergistic relationships between HDAC and CDK inhibitors may provide an effective combinatorial regimen for breast cancer. Importantly, these studies provide an example of how genomic analysis of drug response profiles can be used to design rational drug combinations for cancer treatment.

Seizure control in patients with idiopathic generalized epilepsies: EEG determinants of medication response

In a minority of patients with idiopathic generalized epilepsies (IGEs), seizures continue despite appropriate treatment. We sought to determine the clinical and EEG factors associated with medication response in these patients. All patients with IGEs evaluated by epilepsy specialists between 17 November 2008 and 16 November 2009 were included. We collected information on seizure freedom (dependent variable), EEG asymmetries, response to valproic acid (VPA), MRI characteristics, medication use, demographics, and seizure history (predictors). We identified 322 patients with IGEs; 45 (14%) were excluded from analyses because they had always had a normal EEG (N=26), there were no EEG data (N=3), or they were non-compliant with medication (N=26). Patients with juvenile myoclonic epilepsy were more likely to respond to VPA than were patients with other IGEs, and VPA response was associated with seizure freedom. When EEG characteristics were considered, presence of any focal EEG abnormalities (focal slowing, focal epileptiform discharges, or both) was associated with decreased odds of seizure freedom. These findings suggest that patients with IGEs with poor seizure control may have atypical IGEs with possibly focal, for example, frontal, rather than thalamic onset.

Risk factors for valproic acid resistance in childhood absence epilepsy

Aims Valproic acid (VPA) is reported to be effective for the control of absence seizures in 75% of children. The aim of this study was to determine the clinical and socio-demographic factors associated with VPA response in newly diagnosed childhood absence epilepsy (CAE) and to determine if these factors also influence the chances of achieving long-term seizure freedom. Methods Medical charts of 180 children with CAE were retrospectively reviewed. Clinical, electroencephalographic and imaging findings were recorded to correlate with complete VPA response and long-term epilepsy outcome. Factors associated with non-responsiveness were identified individually and in a multivariable logistic regression analysis. Results Treatment was successful in 112 (58.3%) children. More children that were non-responsive to VPA experienced generalized tonic clonic seizures (GTCS) (33.8% vs. 13.4% for responders; p = 0.001) and 52.9% had a pre-treatment seizure frequency greater than 10/day (vs. 27.0% for responders; p < 0.001). Finally, responders were older at time of diagnosis versus non-responders ( p = 0.001). Absence of long-term seizure freedom was linked to the presence of GTCS, the absence of initial response and the need for multiple AEDs to control seizures. Interpretation Our results suggest that clinical phenotypes are associated with reduced response rates to VPA. This should be taken into account when counselling families of children with newly diagnosed absence epilepsy.

Valproic Acid–Induced Deregulation In Vitro of Genes Associated In Vivo with Neural Tube Defects

The utility of an in vitro system to search for molecular targets and markers of developmental toxicity was explored, using microarrays to detect genes susceptible to deregulation by the teratogen valproic acid (VPA) in the pluripotent mouse embryonal carcinoma cell line P19. Total RNA extracted from P19 cells cultured in the absence or presence of 1, 2.5, or 10mM VPA for 1.5, 6, or 24 h was subjected to replicated microarray analysis, using CodeLink UniSet I Mouse 20K Expression Bioarrays. A moderated F-test revealed a significant VPA response for 2972 (p < 10(-3)) array probes (19.4% of the filtered gene list), 421 of which were significant across all time points. In a core subset of VPA target genes whose expression was downregulated (68 genes) or upregulated (125 genes) with high probability (p < 10(-7)) after both 1.5 and 6 h of VPA exposure, there was a significant enrichment of the biological process Gene Ontology term transcriptional regulation among downregulated genes, and apoptosis among upregulated, and two of the downregulated genes (Folr1 and Gtf2i) have a knockout phenotype comprising exencephaly, the major malformation induced by VPA in mice. The VPA-induced gene expression response in P19 cells indicated that approximately 30% of the approximately 200 genes known from genetic mouse models to be associated with neural tube defects may be potential VPA targets, suggestive of a combined deregulation of multiple genes as a possible mechanism of VPA teratogenicity. Gene expression responses related to other known effects of VPA (histone deacetylase inhibition, G(1)-phase cell cycle arrest, induction of apoptosis) were also identified. This study indicates that toxicogenomic responses to a teratogenic compound in vitro may correlate with known in vitro and in vivo effects, and that short-time (< or =6 h) exposures in such an in vitro system could provide a useful component in mechanistic studies and screening tests in developmental toxicology.

Valproic Acid Induces Apoptosis in Chronic Lymphocytic Leukemia Cells by Targeting Several Pro- and Anti-Apoptotic Genes.

Histone deacetylase inhibitors have been shown to modulate the cell cycle, to induce apoptosis and to sensitize cancer cells to other chemotherapeutics. Among these inhibitors, valproic acid (VPA), an antiepileptic drug, is being discussed as a promising novel anti-cancer drug. Chronic Lymphocytic Leukemia (CLL) is a clinically heterogeneous disease remaining incurable despite introducing new promising treatments. The effects of VPA and its mechanism of action were evaluated on mononuclear cells isolated from 40 CLL patients. Exposure of CLL cells to increased doses of VPA (0.5–5mM) leads to a dose-dependent cytotoxicity and apoptosis in all CLL patients. VPA treatment induced apoptotic changes in CLL cells including phosphatidylserine externalization and DNA fragmentation. The mean apoptotic rates were similar between IGHV mutated and unmutated patients, the latter presenting a more aggressive clinical course. VPA induced apoptosis via the extrinsic pathway involving engagement of the caspase-8-dependent cascade. Interestingly, VPA increased the sensitivity of leukemic cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) even among resistant patients. Moreover, VPA at physiological concentration of 1mM can significantly increase the in vitro cytotoxic effects of fludarabine, bortezomib and the natural product honokiol allowing the reduction of effective concentration 50% (EC50). In order to understand the early mechanism of action of VPA, we investigated gene expression profiles of 14 CLL-patient samples (7 with a good prognosis and 7 with a bad prognosis regarding IGHV mutational status and Zap-70 expression) treated in vitro during 4 hours with a physiological dose (1mM) of VPA and compared with their untreated counterpart using Affymetrix technology. No difference in gene modulation was observed between poor and good prognosis patients after VPA treatment. Modulation of several pro- and anti-apoptotic mRNA expression was confirmed by a real-time reverse transcription-PCR. The molecular analysis of the apoptotic machinery involved in VPA response revealed the up-regulation of APAF1 (5.5 fold, P<0.0001), BNIP3 (2.2 fold, P=0.0006), PTEN (1.9 fold, P=0.0002), CASP6 (2.5 fold, P<0.0001) and the down-regulation of CFLAR/FLIP (2.0 fold, P<0.0001), BCL2 (1.6 fold, P=0.0222), AVEN (1.9 fold, P<0.0001), BIRC4/XIAP (1.7 fold, P<0.0001) and BIRC1/NAIP (1.6 fold, P=0.0007). In conclusion, VPA induced apoptosis of CLL cells at clinically relevant concentration by selective activation of the caspase-8 (extrinsic) pathway and by targeting several pro- and anti-apoptotic genes. Therefore, the combined application of VPA with other drugs might be considered as a potential strategy for CLL treatment.

In Vitro and In Vivo Monitoring of Valproic Acid Effects on Gene Expression Signatures in Adult Acute Myeloid Leukemia.

Inhibitors of histone deacetylases (HDACIs) like valproic acid (VPA) display activity in murine leukemia models, and induce tumor-selective cytoxicity against blasts from patients with acute myeloid leukemia (AML). However, despite of the existing knowledge of the potential function of HDACIs, there remain many unsolved questions especially regarding the factors that determine whether a cancer cell undergoes cell cycle arrest, differentiation, or death in response to HDACIs. Furthermore, there is still limited data on HDACIs effects in vivo, as well as HDACIs function in combination with standard induction chemotherapy, as most studies evaluated HDACIs as single agent in vitro. Thus, our first goal was to determine a VPA response signature in different myeloid leukemia cell lines in vitro, followed by an in vivo analysis of VPA effects in blasts from adult de novo AML patients entered within two randomized multicenter treatment trials of the German-Austrian AML Study Group. To define an VPA in vitro “response signature” we profiled gene expression in myeloid leukemia cell lines (HL-60, NB-4, HEL-1, CMK and K-562) following 48 hours of VPA treatment by using DNA Microarray technology. In accordance with previous studies in vitro VPA treatment of myeloid cell lines induced the expression of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2D coding for p21 and p19, respectively. Supervised analyses revealed many genes known to be associated with a G1 arrest. In all cell lines except for CMK we examined an up-regulation of TNFSF10 coding for TRAIL, as well as differential regulation of other genes involved in apoptosis. Furthermore, gene set enrichment analyses showed a significant down-regulation of genes involved in DNA metabolism and DNA repair. Next, we evaluated the VPA effects on gene expression in AML samples collected within the AMLSG 07-04 trial for younger (age<60yrs) and within the AMLSG 06-04 trial for older adults (age>60yrs), in which patients are randomized to receive standard induction chemotherapy (idarubicine, cytarabine, and etoposide = ICE) with or without concomitant VPA. We profiled gene expression in diagnostic AML blasts and following 48 hours of treatment with ICE or ICE/VPA. First results from our ongoing analysis of in vivo VPA treated samples are in accordance with our cell line experiments as e.g. we also see an induction of CDKN1A expression. However, the picture observed is less homogenous as concomitant administration of ICE, as well as other factors, like e.g. VPA serum levels, might substantially influence the in vivo VPA response. Nevertheless, our data are likely to provide new insights into the VPA effect in vivo, and this study may proof to be useful to predict AML patients likely to benefit from VPA treatment. To achieve this goal, we are currently analyzing additional samples, and we are planning to correlate gene expression findings with histone acetylation status, VPA serum levels, cytogenetic, and molecular genetic data.

Phase I trial of a sequence-specific combination of the HDAC inhibitor, valproic acid (VPA), and the topoisomerase II inhibitor, epirubicin, in advanced solid tumors: Clinical results and correlative studies

3068 Background: Multiple lines of evidence indicate that histone deacetylase inhibitors (HDACi) potentiate topoisomerase (topo) inhibitors. The HDACi-induced histone acetylation and chromatin modulation facilitates DNA access and target recruitment for topo II inhibitors. Methods: This Phase I trial explored a sequence-specific combination of VPA and epirubicin in solid tumors. A VPA loading dose and 6 oral doses (q12h) were given prior to epirubicin in 3-week cycles. Histone acetylation and topo II expression were evaluated in pre-and post-VPA peripheral blood mononuclear cells and tumor samples. Results: To date, 42 patients [median age 53 (39–78)] have been treated in 12 cohorts: IV VPA loading (mg/kg)/epirubicin (mg/m2): 15/75, 30/75, 45/75, 60/75, 75/75 and 75/100, oral loading: 75/100, 90/100, 100/100, 120/100, 140/100 and 160/100. Tumor types included: breast (10), melanoma (11), lung (6), sarcoma (2), GYN (2), GI (5) and others (6). Dose-limiting toxicities included somnolence (1) and neutropenia (1). No exacerbation of epirubicin-related toxicities was observed. Objective responses were seen across different tumor types in anthracycline-resistant and -refractory tumors, despite a median number of 3 (0–6) prior regimens: Partial response; 7/37 (19%), stable disease/minor response: 16/37 (43%). Patients received a median number of 4 (1–10) treatment cycles. Study treatment was stopped despite a clinical benefit or response in 4/33 patients after reaching maximal epirubicin doses (≤750 mg/m2). VPA peak and trough plasma concentrations increased linearly up to 120 mg/m2. MTD is being defined at 160/100 mg/kg/d VPA. H3 and H4 histone acetylation and topo II expression have been correlated with VPA dose, plasma concentration and response. Conclusion: A sequence-specific combination of VPA and epirubicin is active without exacerbation of epirubicin toxicity. VPA plasma peak and trough levels exceeding concentrations needed for biological effects and in vitro synergy are easily achievable with minimal toxicity. The noteworthy anti-tumor activity seen in this heavily pretreated Phase I population warrants further exploration. [Table: see text]