Mechanisms of Drug Resistance in Leukemia

This doctoral study explores and comprehensively evaluates various candidate drug resistance genes and transporters as well as cytogenetic and molecular markers to explain the efficacy of cytarabine in primary AML cells. We analyzed and validated the role of expression of the major genes involved in cytarabine transport and metabolism on ex-vivo Ara-C response as well as on the clinical outcome and compared it with cytogenetic and molecular markers in AML. 1. Genetic variants and RNA expression of the major Ara-C metabolizing genes associated with ex-vivo cytarabine in primary AML cells: RNA expression of candidate Ara-C metabolizing genes was identified as major predictors of Ara-C resistance ex-vivo. DCK expression was identified to have a significant association with ex-vivo cytotoxicity, where patients with high DCK levels have better ex-vivo sensitivity towards Ara-C. Similarly ENT1 the major drug influx transporter and RRM1 the regulator subunit of RR were also found associated with increase ex-vivo sensitivity when its expression is high. CDA expression was found to be significantly lower in patients who were sensitive to Ara-C when compared to those who were resistant. Based on the expression data on ex-vivo Ara-C sensitivity, Ara-C resistance index (RI) was proposed. RI incorporates candidate Ara-C metabolizing genes whose RNA expression significantly associated with ex-vivo cytotoxicity towards Ara-C. RI was found to be significantly associated with ex-vivo Ara-C sensitivity where patients with higher RI values were found likely to have high IC-50 as well as high AUC values. Patients who presented with relapse, also showed higher RI values similar to that of ex-vivo resistant samples at diagnosis which again affirm the genuineness of this index. Further, the role of genetic variants on candidate genes’ expression was evaluated. CDA which showed the maximum variation in gene expression was found to be most polymorphic in terms of genetic variants. DCK and ENT1 which were not having much variation in gene expression were less polymorphic. These genetic variants identified from each gene were evaluated for its influence on mRNA expression and Ara-C cytotoxicity. Genetic variants were identified from CDA and NT5C2 to have role in influencing the mRNA expression of these genes. Also CDA and RRM1 genetic variants were identified associated with Ara-C cytotoxicity. 2. Clinical validation of genetic variants and RNA expression of Ara-C metabolizing genes: The validation of the gene expression and genetic variants in candidate Ara-C metabolizing genes with clinical outcomes like CR, induction death, EFS and DFS was done. Higher expression of DCK and RRM1 were associated with better DFS, actually substantiate the data obtained through ex-vivo experiments. There are no reports from AML where the regulatory subunit of RR is shown associated with survival, though a recent report has shown association of RRM1 SNPs and treatment outcome.213 It is speculated that RRM1 might not be a direct molecular target for Ara-C as that in case of gemcitabine, even though both the drugs have similar metabolic activation. When RI was evaluated in the clinical scenario, in contrary to higher Ara-C sensitivity in ex-vivo, low RI patients were found to have shorter EFS. Further evaluation of these low RI patients recognizes higher incidence of induction death in this group which probably resulted in shorter EFS. Higher Ara-C sensitivity leading to higher drug related toxicity could be of great relevance as induction death is still a major challenge in AML treatment particularly in developing countries. Further validation of this index in an independent cohort of AML patients is warranted. The DCK/CDA expression data also supports the idea of higher Ara-C sensitivity leading to drug associated complications as well as TRM. Two of the CDA SNPs rs2072671 (79A>C) and rs602950 (-92 A>G) when in the mutant form had a higher risk to have a shorter EFS as well as DFS in our study. RRM1 synonymous variant rs9937 in heterozygous state (AG) was found to be associated with shorter EFS/DFS when compared to its homozygous wild type (AA) as well as mutant forms (GG). 3. Other salient outcomes of the doctoral work: This study evaluated the association of prognostically relevant cytogenetic and molecular markers with candidate Ara-C metabolizing enzymes and transporter association. When the effect of these markers in AML on expression of individual Ara-C metabolizing genes and RI was evaluated, no significant association was found between any of these markers except NPM1. Our data have shown for the first time that the good prognosis associated with the NPM1 mutation might be due to the over expression of the enzyme DCK in NPM1 mutated patients. Both NPM1 mutations and DCK expression were associated with better DFS in the clinical scenario, even though not reaching statistical significance. The role of ABC transporters on Ara-C resistance was evaluated. Of the 14 ABC transporter screened ABCC4 expression was shown associated better ex-vivo Ara-C sensitivity, though the biological explanation for this association still remains to be elucidated. Lower ABCB6 expression in FLT3-ITD patients is also an interesting finding as ABCB6 is considered to play a major role in free-radical detoxification. Preliminary in-vitro observations from our laboratory suggests that patients with FLT3-ITD, owing to inherently low ABCB6 expression and high basal ROS levels may get benefited from therapeutic agents which brings cell kill by inducing ROS.258 We undertook a genome-wide gene expression profiling to address other possible mechanisms of Ara-C resistance apart from the candidate gene approach. Of the differentially expressed gene families, genes involved under apoptosis and metabolism were studied with special interest. It was noted that in Ara-C sensitive samples pro-apoptotic genes like BAX, PMAIP1 (Noxa), NDUFA13, DAPK3, CASP 3, CASP6 were upregulated, and at the same time anti-apoptotic genes like TEGT (BAXI 1), MCL1, SERPINB2, NOD2, PAK1 were down-regulated. Interestingly, several genes involved in cellular metabolism were found to be down regulated in Ara-C sensitive group. Major down-regulated metabolic genes include SLC2A3, SLC2A8, SLC11A1, HK3, GK, ACSL1, PFKFB4, PFKFB3, PDK4, ME1 and PC. This study has come up with previously unrecognized aspects in Ara-C resistance in AML, and is suggestive of Ara-C sensitive samples to have decreased expression of ‘Warburg genes’ and the ‘anti-apoptotic’ genes