Assessment Of Drug Combinations Research Articles

The Significance of GADD45A Promoter DNA Hypermethylation in AML: Association with IDH1/2 and TET2 Mutation

BACKGROUNDWe have previously shown that one target of hyper-methylation in AML is the promoter of the tumour suppressor and stress-response mediator Growth Arrest and DNA Damage inducible 45A (GADD45A) (GADD45AmeHI; 42% of AML). In mice Gadd45a has recently been shown to play a critical role in HSC stress responses. Gadd45a deficiency leads to enhanced HSC self-renewal, DNA damage accumulation in HSC, increased susceptibility to leukemogenesis, and impairment in HSC apoptosis after genotoxic exposure (Chen et al, Blood 2014). These findings suggest that hypermethylation of the GADD45A gene may play an important role in the altered properties of HSC, leukaemic initiation and progression. Promoter hypermethylation of this gene defines a patient group with poor survival on standard therapy (Perugini et al, Leukaemia 2012). To explore further the molecular basis of the GADD45AmeHI patient group weperformed genetic profiling of diagnosis samples using a Sequenom multiplex mutation panel, or using whole exome sequencing for broader coverage (n=95 patients).Sequenom MassARRAY was used for quantitative detection of GADD45A promoter methylation in patient samples. For a cohort of matched diagnosis and relapse samples we used CpG methylation data for GADD45A determined by ERRBS (Akalin et al, PLoSGenetics 2012). Response to cytotoxic drugs and assessment of drug combinations with 5-Aza-deoxycytidine (decitabine, DAC) and anthracycline (Daunorubicin, DNR) was performed in AML cell lines, and with primary leukemic cell populations. RESULTSThe association of the GADD45AmeHI patient group with poor outcome was validated in an independent AML patient cohort of 48 patients from the Alfred Hospital, Melbourne, Australia (p=0.003; HR3.35). Whole exome sequencing and Sequenom multiplex analysis of 95 AML patients revealed a striking co-occurrence of the GADD45AmeHI phenotype with mutations in IDH1, IDH2, and TET2 (p<0.0001, Fisher’s exact test, Fig. 1). To test the prediction that GADD45A hypermethylation may be an important factor for relapse we investigated GADD45A promoter DNA methylation levels in paired diagnosis and relapse samples. In a paired analysis of 39 patients we show that relapse samples display a significant increase in GADD45A promoter CpG methylation (p=0.035, paired t-test). This is consistent with emergence in many patients following chemotherapy of a chemoresistant clone that has increased GADD45A methylation and reduced GADD45A activity.We next tested whether reactivation of GADD45A expression in GADD45AmeHI patient samples could be achieved through the use of hypo-methylation agents, and whether this is beneficial for response to chemotherapy. DAC treatment has been reported to induce DNA demethylation and GADD45A reactivation in primary AML samples (Klco et al, Blood 2013), and we observe reduced GADD45A promoter methylation and increased expression following DAC treatment of the GADD45AmeHI AML cell line (Mv4;11), consistent with DNA methylation-induced gene silencing of GADD45A. DAC pre-treatment of the GADD45AmeHI AML cell lines MOLM13 and Mv4;11, and three primary AML samples (GADD45AmeHI), resulted in increased GADD45A expression and increased DNR sensitivity. CONCLUSIONSDNA methylation of the GADD45A proximal promoter marks a large percentage of AML patients at diagnosis including the majority of those with IDH1/2 and TET2 mutations (collectively these occur in 28% of AML (Network CGAR, N Engl J Med, 2013)), and is an independent predictor of poor outcome in two independent patient cohorts. Our data shows that silencing of GADD45A through increased promoter CpG methylation maybe an important early event in leukaemogenesis associated with impaired TET2 activity. Based on recent studies describing the properties of Gadd45a-deficient murine HSC we suggest reduced GADD45A activity in this subset of patients may contribute to the properties of pre-leukaemic HSC that have been associated with IDH1/2 mutation and reported to display clonal expansion, resistance to chemotherapy, and ultimately a high risk of relapse.In vitro drug experiments suggest that a priming schedule of DAC followed by DNR may provide a successful tailored treatment strategy for GADD45AmeHI patients, in combination with GADD45A expression as a biomarker predicting increased DNR sensitivity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Improving existing tools for Mycobacterium xenopi treatment: assessment of drug combinations and characterization of mouse models of infection and chemotherapy

Mycobacterium xenopi is a common agent of non-tuberculous mycobacterial lung diseases in Europe. However, an optimal treatment regimen for M. xenopi infection has not yet been established. Appropriate in vitro and in vivo model systems are needed for characterization of the activity of potential drugs and drug combinations against M. xenopi. We utilized three experimental platforms to analyse the anti-M. xenopi activity of single and combination drug regimens. First, we determined the bacteriostatic and bactericidal activities of drugs alone and in combination in vitro. Second, we used serum from treated mice to evaluate drug activities ex vivo. Third, we analysed M. xenopi growth in four strains of mice (BALB/c, C57BL/6, beige and athymic nude) and developed a mouse model of chemotherapy for this infection. Two-drug combinations of ethambutol with rifampicin, rifapentine or moxifloxacin, and of clarithromycin with moxifloxacin were bactericidal in vitro, and the combination of ethambutol and rifampicin with either clarithromycin or moxifloxacin showed significant bactericidal activity ex vivo. Nude mice were the most susceptible strain to M. xenopi infection, and in this model amikacin-containing regimens were the most effective against M. xenopi. No difference in activity was found between regimens containing clarithromycin and moxifloxacin in vivo. The ethambutol/rifampicin combination with clarithromycin or moxifloxacin had significant bactericidal activity against M. xenopi. The nude mouse, being highly susceptible to M. xenopi, can be utilized for in vivo chemotherapy studies for this infection.

Preclinical Assessment of Drug Combinations for the Treatment of Pain: Isobolographic and Dose-Addition Analysis of the Opioidergic System

Opioid analgesics are the most frequently prescribed medications for the treatment of moderate or severe pain; however, their use is constrained by unwanted side effects. One therapeutic approach used to improve the side effect profile of opioids is the administration of a second drug in an opioid-containing mixture. Preclinical studies designed to predict the therapeutic potential of novel opioid-containing drug combinations are currently underway, and must rely on quantitative methods to assess their interactive effects. In this manuscript, an overview of isobolographic analysis is presented along with recent advances in isobolographic theory pertaining to drugs that differ in efficacy and to the statistical analysis of dose-addition. Next, studies using these analyses to assess the interactive effects of opioids and novel adjunct drugs, including selective COX-2 inhibitors, α(2)-adrenergic receptor agonists, δ-opioids, glutamate receptor antagonists and cannabinoid receptor agonists, are reviewed. Finally, comments on the future assessment of drug combinations for the treatment of pain-related disorders are made.

Drug safety profiling for assessment of drug combinations in oncology

15037 Background: It is important to understand the safety profile (SP)/toxicity of new drug regimens in oncology. We compared SPs of gemcitabine (Gem) + carboplatin (Carbo) in NSCLC with Gem alone and in different combinations and also tested the methodology of drug safety profiling (DSP). Methods: Spontaneous cases for the period of 1995–2005 were reviewed on the Lilly Safety Database (LSD). DSP was used to evaluate differences in the SPs of several combinations: Gem+Carbo in NSCLC, Gem+Carbo in ovarian cancer, Gem+ cisplatin (Cis) in NSCLC, Gem+Carbo in all indications, and Gem regardless of treatment regimen, for all indications. Frequencies of adverse events (AEs) for all MedDRA System Organ Classes (SOCs) were used for each regimen. In addition, the MedDRA Preferred Terms (PTs) were reviewed to detect potential safety signals. The numbers of AEs in different SOCs were assessed as proportions of the total reports for the Gem combinations in the LSD. Results: With the exception of the Investigations SOC, the proportions of AEs for patients treated for NSCLC with Gem+Carbo were consistent with those for patients treated for NSCLC with Gem+Cis and with Gem for all indications. However, the frequency in the Investigations SOC was consistent with that reported for Gem+Carbo in all indications (14.2% v. 12.0%). A greater frequency of AEs was seen in the Gastrointestinal Disorders SOC for patients treated with Gem+Carbo for ovarian cancer compared to patients treated with Gem+Carbo for NSCLC. The review of individual PTs for Gem+Carbo did not reveal any safety signals. Conclusions: The SP of Gem+Carbo in NSCLC using DSP showed similar patterns to all other Gem combinations with only some differences due to the indication. DSP is a useful tool in assessing the new drug combination treatments in existing or new indications. [Table: see text]

Evaluation of in vitro chemosensitivity using human lung cancer cell lines.

The use of well-characterized human lung cancer cell lines has allowed for new opportunities in preclinical and clinical drug evaluation. Development of semiautomated tests of in vitro cytotoxicity such as the MTT assay, which utilizes the formazan salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), has allowed for preclinical evaluation of novel chemotherapeutic agents and drug combinations. In addition, techniques such as this make possible the testing of sufficient data sets to allow determination of true biochemical drug synergy. Assessment of drug combinations which possess in vitro synergy or supraadditive effects can suggest chemotherapeutic regimens for further clinical testing. Using the MTT assay in conjunction with isobolographic analysis, it is possible to test commonly used regimens which are based on presumed or apparent in vivo drug synergy, such as the combination of etoposide and cis-platinum. This frequently prescribed combination was found to lack in vitro biochemical synergy when tested with human lung cancer cell lines, indicating that the observed clinical benefits of this drug combination may be due to factors in the tumor microenvironment, drug metabolism, or non-overlapping toxicities. Finally, although it remains to be determined if a significant role for in vitro drug testing will be found in direct clinical applications, preclinical drug evaluation during the drug development process using cultured tumor cell lines may ultimately allow for disease or patient specific therapies for testing.