Imatinib is the frontline treatment option in treating chronic myelogenous leukemia (CML). Hitherto, some patients relapse following treatment. Biochemical analysis of a panel of clonally derived imatinib-resistant cells revealed enhanced glucose uptake and ATP production, suggesting increased rates of glycolysis. Interestingly, increased lactate export was also observed in imatinib-resistant cell lines. Here, we show that metformin inhibits the growth of imatinib-resistant cell lines as well as peripheral blood mononuclear cells isolated from patients who relapsed following imatinib treatment. Metformin exerted these antiproliferative effects by inhibiting MCT1 and MCT4, leading to the inhibition of lactate export. Furthermore, glucose uptake and ATP production were also inhibited following metformin treatment due to the inhibition of GLUT1 and HK-II in an AMPK-dependent manner. Our results also confirmed that metformin-mediated inhibition of lactate export and glucose uptake occurs through the regulation of mTORC1 and HIF-1α. These results delineate the molecular mechanisms underlying metabolic reprogramming leading to secondary imatinib resistance and the potential of metformin as a therapeutic option in CML.

Imatinib resistance remains an unresolved problem in CML disease. Activation of JAK2/STAT3 pathway and increased expression of RUNX1 have become one reason for development of imatinib resistance in CML subjects. Metformin has gained attention as an antileukemic drug in recent times. However, the molecular mechanism remains elusive. The present study shows that RUNX1 is a novel substrate of AMP-activated kinase (AMPK), where AMPK phosphorylates RUNX1 at Ser 94 position. Activation of AMPK by metformin could lead to increased cytoplasmic retention of RUNX1 due to Ser 94 phosphorylation. RUNX1 Ser 94 phosphorylation resulted in increased interaction with STAT3, which was reflected in reduced transcriptional activity of both RUNX1 and STAT3 due to their cytoplasmic retention. The reduced transcriptional activity of STAT3 and RUNX1 resulted in the down-regulation of their signaling targets involved in proliferation and anti-apoptosis. Our cell proliferation assays using in vitro resistant cell line models and PBMCs isolated from CML clinical patients and normal subjects demonstrate that metformin treatment resulted in reduced growth and improved imatinib sensitivity of resistant subjects.

Genetic variants influencing the pharmacokinetics and/or pharmacodynamics of the chemotherapeutic drugs used in Acute Lymphoblastic Leukemia (ALL) therapy often contribute to the occurrence of treatment related toxicity (TRT). In this study, we explored the association of candidate genetic variants with early hematological TRT (grade 3–4) occurring within the first 100 days of low-dose methotrexate and 6-mercaptopurine based maintenance therapy (n = 73). Fourteen variants in the following candidate genes were genotyped using allele discrimination assay by real-time PCR: ABCB1, DHFR, GGH, FPGS, MTHFR, RFC1, SLCO1B1, TPMT, and NUDT15. Methotrexate polyglutamate (MTXPG3-5) levels in red blood cells were measured by LC-MS/MS. Early hematological TRT (grade 3–4) was seen in 54.9% of patients. The NUDT15c.415T allele was associated with early TRT occurrence [HR: 3.04 (95% CI: 1.5–6.1); p = 0.007]. Sensitivity of early TRT prediction improved (from 30.7% to 89.7%) by considering FPGS variant (rs1544105’T’) carrier status along with NUDT15c.415T allele [HR = 2.7 (1.5–4.7, p = 0.008)]. None of the considered genetic variants were associated with MTXPG3-5 levels, which in turn were not associated with early TRT. NUDT15c.415T allele carrier status could be used as a stratifying marker for Indian ALL patients to distinguish patients at high or low risk of developing early hematological TRT.

PURPOSE:The rationale of the current study was to develop 6-mercaptopurine (6-MP)-mediated hematological toxicity prediction model for acute lymphoblastic leukemia (ALL) therapeutic management.METHODS:A total of 96 children with ALL undergoing therapy with MCP-841 protocol were screened for all the ten exons of TPMT, exon 2, exon 3 and intron 2 of ITPA using bidirectional sequencing. This dataset was used to construct prediction models of leucopenia grade by constructing classification and regression trees (CART) followed by smart pruning.RESULTS:The developed CART model indicated TPMT*12 and TPMT*3C as the key determinants of toxicity. TPMT int3, int4 and int7 polymorphisms exert toxicity when co-segregated with one mutated allele of TPMT*12 or TPMT*3C or ITPA exon 3. The developed CART model exhibited 93.6% accuracy in predicting the toxicity. The area under the receiver operating characteristic curve was 0.9649.CONCLUSIONS:TPMT *3C and TPMT*12 are the key determinants of 6-MP-mediated hematological toxicity while other variants of TPMT (int3, int4 and int7) and ITPA ex2 interact synergistically with TPMT*3C or TPMT*12 variant alleles to enhance the toxicity. TPMT and ITPA variants cumulatively are excellent predictors of 6-MP-mediated toxicity.

Background and Objectives: Globally the survival of ALL has increased tremendously with the five-year overall survival (OS) reaching 90% in high-income countries (HICs) (1, 2). However, in India, the five-year OS has been reported to be 30-70 %( 3) and deaths due to treatment related toxicity (TRT) in ALL were ranged from 2-24 % (4), which is ten times higher compared to HICs suggesting increased susceptibility to the toxicity of the chemotherapeutic drugs. This could be due to frequent variants in candidate genes determining the pharmacodynamic response or pharmacokinetics of the chemotherapeutic drugs used during maintenance therapy of ALL. Variations in the patient management, supportive care therapy are the other possible reasons of this increased incidence of TRT. Many patients are lost to follow-up due to the TRT, and eventually will die of the disease progression. The present study aimed to explore the association of common genetic variants in the candidate genes with early treatment related hematological toxicities (grade 3-4) in patients with Acute Lymphoblastic Leukemia (ALL) receiving low-dose MTX (LDMTX) and 6-Mercaptopurine (6-MP) based maintenance therapy. Materials and Methods: This prospective study was conducted between August 2011 and May 2016 and approved by institutional scientific and ethics committees. A total of 71 patients (43 males and 28 females aged between 1-51 years) with ALL were enrolled in the study after obtaining written informed consent and in the case of children, from legally accepted guardians. Patients below 25 years of age were treated using protocol-841 (MCP) I2A and those older than 25 years were treated with modified GMALL-84 protocols. MTX and 6-MP doses did not differ across both the protocols during maintenance therapy. Germline DNA samples collected at the time of remission from peripheral mononuclear cells was used to genotype15 selected variants frequent in the following candidate genes : ABCB1, DHFR, GGH, FPGS, MTHFR, RFC1, SLCO1B1, TPMT, and NUDT15 using allele discrimination assay by real-time PCR. LC-MS/MS method was used to measure methotrexate polyglutamate (MTXPG3-5) levels in RBC's.Toxicities were graded according to the Common Terminology Criteria for Adverse Events (CTCAE-version 4.03). Early grade 3-4 hematological toxicities occurring within first 100 days of the maintenance therapy was studied for its association with the genetic variants and other risk factors. Cumulative incidence curves were plotted and a risk factor analysis was performed using multivariate Cox regression. Co-linearity between variables was assessed. We used a back-ward selection method, retaining those variables with P-values<0.05 in the final model. Data analysis was done using statistical software « R ». Results: The cumulative incidences of early hematological TRT (grade 3-4) and relapse were 54.9 %, and 38.0 %, respectively. The relapse free survival was 59.2 %. The median follow-up of all patients from the start of maintenance was 1018 days. In multivariate analysis including all genetic variants, age, and WBC counts at diagnosis, we observed a significant increase in the risk of TRT in carriers of NUDT15*3 allele(rs116855232 ; p=0.002 ; univariate Hazards ratio : 2.81 (95% CI : 1.41-5.59). 3435C>T variant in ABCB1 gene showed a trend of association with that of relapse free survival. Neither the genetic variants studied were associated with the methotrexate polyglutamate levels, nor were the levels associated with the clinical outcomes. Conclusion: The NUDT15*3 allele carriers status could be used as one of the stratifying markers in South Indian ALL patients at the time of diagnosis to distinguish high and low-risk patients to develop early hematological toxicity, especially related to 6-mercaptopurine based ALL maintenance therapy protocols. Disclosures No relevant conflicts of interest to declare.

Two chemotherapeutic agents, 6-mercaptopurine and methotrexate, which are part of consolidation therapy in leukemia, are found to be associated with hematological toxicity in some patients. S-adenosyl methionine, the metabolic byproduct of folate pathway, is shown to stabilize thiopurine methyl transferase activity and controls the toxic levels of the 6-thioguanine nucleotide by methylating most of the metabolic products of 6-mercaptopurine. Existing literature points to the association of the genetic variants of the folate pathway, such as glutamate carboxypeptidase II C1561T, reduced folate carrier 1 G80A and thymidylate synthase (TYMS) 5'-UTR 28bp tandem repeat, with hematological toxicity either alone or in conjunction with thiopurine methyl transferase variant alleles. Methotrexate, which inhibits two crucial enzymes of folate pathway i.e. dihydrofolate reductase and TYMS, is also found to induce toxicity in the presence of reduced folate carrier 1, G80A, TYMS 5'-UTR 28bp tandem repeat, methylene-tetrahydrofolate reductase (MTHFR) C677T, methionine synthase reductase A66G polymorphisms. Furthermore, survivors of childhood ALL are found to develop attention deficit hyperactivity disorder, when they harbor MTHFR C677T, MTHFR A1298C and methionine synthase A2756G polymorphisms. These cognitive defects are attributed to low levels of 5-methyl tetrahydrofolate and S-adenosyl methionine in the cerebrospinal fluids of such children following chemotherapy. Gene expression analysis reveals differential expression of certain folate pathway genes in subtypes of acute lymphoblastic leukaemia, based on lineage (T-cell or B-cell), hyperploidy or non-hyperploidy and presence or absence of gene fusion, suggesting that methotrexate dynamics might vary, depending on subtypes of acute lymphoblastic leukemia. To conclude, folate pathway gene expression and genetic analysis might help in predicting patients at risk of developing all types of toxicity, including hematological toxicity in leukemia.

Abstract Purpose: Defective S-methylation of 6-mercarptopurine (6-MP) either due to reduced activity of thiopurine s-methyltransferase (TPMT) or due to reduced synthesis of S-adenosyl methionine (SAM) induces hematological toxicity in children undergoing 6-MP treatment for acute lymphoblastic leukemia (ALL). Recently, we have demonstrated the genetic variants of TMPT results in hematological toxicity when on 6-MP. In the current study, we have aimed to explore the role of seven functional polymorphisms in folate metabolism as pharmacogenomic determinants of 6-MP mediated toxicity. Experimental Procedures: In 126 children with ALL being treated with 6-MP, Glutamate carboxypeptidase II (GCPII) C1561T, Reduced folate carrier 1 (RFC1) G80A, cytosolic serine hydroxymethyl transferase (cSHMT) C1420T, Thymidylate synthase (TYMS) 3′-UTR ins6/del6, Methylene tetrahydrofolate reductase (MTHFR) C677T, Methionine synthase (MTR) A2756G polymorphisms were analysed by PCR-RFLP method while TYMS 5′-UTR 28 bp tandem repeat analysis was done by PCR-AFLP method. Summary: Univariate analysis showed positive association of GCPII C1561T (r= 0.23) and cSHMT C1420T (r=0.15) variants with hematological toxicity. Multi dimensionality reduction analysis confirmed synergetic gene-gene interaction between these two loci (×2=5.9, p=0.02) apart from showing an interesting tri-variate interaction i.e, RFC1 G80A/cSHMT C1420T/TYMS 5′-2R3R. RFC1 G80A showed additive interaction with cSHMT while TYMS 2R alleles showed counteracting interaction. Toxicity grade prediction model was developed using multiple linear regression approach, which showed moderate predictability of aberrations in folate metabolism and TPMT*3C (Area under the receiver operating characteristic curve=0.69) thus explaining 36% variation in toxicity. Conclusion: Aberrations in folate metabolism and TPMT*3C contribute to 36% 6-MP mediated toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1874. doi:1538-7445.AM2012-1874

AIM:The rationale of this study was to explore the contribution of genetic variants of the folate pathway to toxicity of 6-mercaptopurine (6-MP)-mediated hematological toxicity in children with acute lymphoblastic leukemia (ALL) and to explore the interaction of these variants with TPMT and ITPA haplotypes using multifactor dimensionality reduction analysis.MATERIALS & METHODS:Children with ALL (n = 96) were screened for GCPII C1561T, RFC1 G80A, cSHMT C1420T, TYMS 5´-UTR 2R3R, TYMS 3´-UTR ins6/del6, MTHFR C677T, MTR A2756G polymorphisms using PCR-RFLP and PCR-amplified fragment length polymorphism techniques.RESULTS:GCPII C1561T showed independent association with toxicity. The following synergetic interactions appeared to increase the toxicity of 6-mercaptopurine: TPMT*12 × RFC1 G80A; TPMT CTTAT haplotype × RFC1 G80A; TPMT CTTAT haplotype × RFC1 G80A × TYMS 2R3R. The genetic variants of thiopurine and folate pathway cumulatively appeared to increase the predictability of toxicity (r(2) = 0.41) in a multiple linear regression model. For the observed toxicity grades of 1, 2, 3 and 4, the respective predicted toxicity grades were 1.65 ± 0.29, 1.68 ± 0.24, 2.56 ± 0.58 and 2.99 ± 1.03, p(trend) < 0.0001.CONCLUSION:Gene-gene interaction between thiopurine and folate pathways inflate the 6-MP-mediated toxicity in Indian children with ALL illustrating the importance of ethnicity in the toxicity of 6-MP.

To explore the role of genetic variants of thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) in 6-mercaptopurine (6-MP)-induced toxicity in Indian children with acute lymphoblastic leukemia (ALL).

BACKGROUND & OBJECTIVES The aetiology of gastric ulcers is not completely understood and continuous use of anti-ulcer agents leads to many side effects. In this study we evaluated the anti-ulcer efficacy of a polyherbal formulation with potent antioxidant activity in aspirin and pyloric ligature induced gastric ulcers in rats. METHODS The efficacy of the polyherbal formulation NR-ANX-C (composed of the extracts from Withania somnifera, Camellia sinensis, Ocimum sanctum, shilajith and triphala) was evaluated in terms of antioxidant potential as assessed in terms of protection from lipid peroxidation and the antiulcer activity as seen by the area of gastric lesions, gastric juice volume, gastric pH, total acidity and total adherent gastric mucus content. RESULTS In our study, NR-ANX-C (25 and 50 mg/kg) was more efficacious than ranitidine in reducing ulcer index in both the models. At the highest dose tested (50 mg/kg), NR-ANX-C was comparable to omeprazole in preventing ulcer formation in the pyloric ligature model. NR-ANX-C showed a dose- dependent decrease in gastric juice volume and total acidity in both the models. A dose-dependent increase in gastric pH and total adherent gastric mucus was also seen in NR-ANX-C treated groups. The extent of lipid peroxidation was also reduced in the test drug treated groups. INTERPRETATION & CONCLUSION Based on our findings, we presume that the cytoprotective, anti-secretary and antioxidant properties of NR-ANX-C were responsible for its anti-ulcer activity. These findings suggest the potential for use of NR-ANX-C as an adjuvant in the treatment of gastric ulcer.

Neuroleptics that are commonly used in the treatment of schizophrenia and other affective disorders are often associated with distressing extrapyramidal side effects (catalepsy). Catalepsy induced by neuroleptics in animals has been used as a model for the extrapyramidal side effects associated with antipsychotic agents in human beings. In the present study, we have attempted to evaluate the protective effect of Triphala on haloperidol induced catalepsy in mice. Inbred albino mice were divided into five groups, each containing six animals. Both, the test drug, the aqueous extract of Triphala and the standard drug scopolamine were dissolved in 1% gum acacia solution. Catalepsy was induced with haloperidol (1mg/kg). The first group received the vehicle (10ml/kg), the second group received scopolamine (1mg/kg) and the remaining three groups of animals received the test compound, Triphala (2.5, 6.25 and 12.5 mg/kg respectively) orally. In the acute study, a single dose of vehicle and the test drug were administered, while in the chronic study, they were given once a day for seven days, 30 minutes prior to haloperidol administration. Catalepsy was determined by the standard bar test after 30 minutes of haloperidol administration and was scored as described by Ahtee and Benumbe. In the acute study, the aqueous extract of Triphala at all the doses tested, significantly (P<0.01) reduced the cataleptic score after the latency of 60 minutes. However, in the chronic study, the reduction in the cataleptic score was seen throughout the period of observations. These effects were comparable to that produced by the standard drug scopolamine. Pretreatment of Triphala decreased haloperidol induced catalepsy in mice, which is comparable to that produced by the standard drug scopolamine. Triphala seems to be more effective when it is repeatedly administered than with a single administration. It can be used as an alternative drug or with a combination of currently available drugs in treating drug induced extrapyramidal side effects.

Neuroleptic drugs used in the treatment of schizophrenia and other affective disorders are known to produce extrapyramidal side effects. Catalepsy was induced by these drugs in animals and these have been used as models for the extrapyramidal side effects associated with antipsychotic agents in human beings. In the present study, we have attempted to evaluate the protective effect of the aqueous extract of the fruits of Emblica officinalis (EO) on haloperidol (1.0mg/kg intraperitoneal administration) induced catalepsy in mice by employing the standard bar test. Mice were allocated to seven groups, each group containing six animals. The effects of the test drug EO (0.8, 2.0 and 4.0mg/kg doses) and the standard drugs scopolamine (1.0mg/kg) and ondansetron (0.5 and 1.0mg/kg doses) were assessed after single and repeated dose administration for seven days, 30 minutes prior to the haloperidol. Mice were sacrificed on the seventh day and super oxide dismutase (SOD) activity in the brain tissue was estimated by using the Beauchamp and Fridovich method. A significant (P<0.001) reduction in the cataleptic scores was observed in all the test drug treated groups as compared to the control, with maximum reduction in the dose 4.0mg/kg group. Similarly, the maximum reduction in SOD activity (P<0.01) was observed in the dose 4.0mg/kg group. Our study suggests that EO has significantly reduced oxidative stress and the cataleptic score induced by haloperidol. It could be used to prevent drug- induced extrapyramidal side effects.

Background & objectives: Use of typical antipsychotics like haloperidol in treatment of schizophrenia is associated with a high incidence of extrapyramidal side effects. In rodents, administration of haloperidol leads to the development of a behavioural state called catalepsy, in which the animal is not able to correct an externally imposed posture. In the present study we evaluated the anticataleptic efficacy of NR-ANX-C, a polyherbal formulation containing bioactives of Withania somnifera, Ocimum sanctum, Camellia sinensis, triphala and shilajit in haloperidol induced catalepsy in mice.Methods: Five groups (n = 6) of male albino mice were used in the study. Catalepsy was induced by ip administration of haloperidol (1mg/kg). The degree of catalepsy (cataleptic score) was measured as the time the animal maintained an imposed posture. We compared the anticataleptic efficacy of NR-ANX-C (10, 25 and 50 mg/kg) with scopolamine (1mg/kg). The superoxide dismutase (SOD) level in brain tissue was also estimated to correlate the levels of oxidative stress and degree of catalepsy in the animal.Results: Significant (P<0.01) reduction in the cataleptic scores was observed in all NR-ANX-C treated groups and maximum reduction was observed in the NR-ANX-C (25 mg/kg) treated group. Significant (P<0.05) reduction in SOD activity was observed in NR-ANX-C (25 and 50 mg/kg) treated groups and maximum reduction was observed in NR-ANX-C (25mg/kg) treated group.Interpretation & conclusions: In our study, maximum reduction in cataleptic score was observed in NR-ANX-C (25 mg/kg) treated group. The maximum reduction in SOD activity was also observed in the same group. These findings suggest a possible involvement of the antioxidant potential of NR-ANX-C in alleviating haloperidol induced catalepsy.

Neuroleptic drugs used in the treatment of schizophrenia and other affective disorders are known to produce extrapyramidal side effects. Catalepsy induced by these drugs in animals has been used as a model for the extrapyramidal side effects associated with antipsychotic agents in human beings. In the present study, we have attempted to evaluate the protective effect of the ethanolic leaf extractof Ocimum sanctum (OS) on haloperidol (1.0 mg/kg, intraperitoneal administration)-induced catalepsy in mice by employing the standard bar test. Mice were allocated to seven groups, each group containing six animals. The effects of the test drug OS (at 1.75, 4.25 and 8.5 mg/kg doses) and the standard drugs, scopolamine (1.0 mg/kg) and ondansetron (0.5 and 1.0 mg/kg doses) were assessed after single and repeat dose administration for seven days, 30 minutes prior to the haloperidol. The results suggest that OS has a protective effect against haloperidol-induced catalepsy, which is comparable to the standard drugs used for the same purpose. Our study indicates that OS could be used to prevent drug-induced extrapyramidal side effects.