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Substantial racial differences exist in both the incidence and treatment outcome of childhood acute lymphoblastic leukemia among major racial/ethnic groups in the US1-4. However, biological basis of such racial disparities are largely unknown. Because different race/ethnic groups are often well distinguished by populationâ€specific genetic polymorphisms5, we hypothesize that ancestryâ€related genomic variations may contribute to racial disparities in the incidence and outcome of ALL.
In a recent publication, genomic variations characteristic of Native American (NA) ancestry are at least partly responsible for the higher risk of ALL relapse in Hispanic children4, and one of such genetic variations is in the ARID5B locus on chromosome 10. Interestingly, a few groups have previously identified ARID5B single nucleotide polymorphisms (SNPs) as the strongest genetic predisposition factor for childhood ALL6,7, and the risk allele frequencies by race correlate with racial differences in ALL incidence8,9. These observations collectively point to ARID5B as a novel and critical determinant in both pathogenesis and drug response of ALL, likely contributing to racial disparities in this disease. Herein, we propose 4 specific aims:
characterize reported genetic variation at ARID5B (10q21.2) in diverse Asian populations (Chinese, Malay and Indian) and to evaluate their associations with ALL susceptibility and ALL relapse in the Malaysia-Singapore (Ma-Spore) ALL cohort
explore the molecular mechanisms by which ARID5B regulates response to anti-leukemic agents
ALL is the most common type of cancer in children10. Despite impressive improvement in cure rates of childhood ALL, substantial racial disparities have persisted11. Differences exist in both the incidence12,13 and treatment outcome of ALL by race1-4. For example, Hispanic children not only exhibit the highest incidence of ALL but also the lowest survival among major race/ethnic groups in the U.S. In contrast, although African American children are least likely to develop ALL, they fare worse with ALL therapy than Caucasian Americans and Asians. Without a clear understanding of the biological basis of racial differences in ALL and development of treatment approaches to overcome such racial disparities, this catastrophic disease is likely to continue to disproportionately affect children from certain race/ethnic background.
Although racial differences in cancer susceptibility and treatment outcome have long be indicated, it is often difficult to ascertain the genetic or biological basis for the racial differences, partly due to the inaccuracy in selfâ€reported categorical race classification and the complex pattern of racial mixture. Recent genomeâ€wide study of population worldwide provides compelling evidence for genetic underpinning of racial/ethnic differences and similarities5,14. Capitalizing on these sophisticated genomic tools, Yang et al. developed strategies to identify germline genetic variations defining ancestral components in children with ALL, and to determine relative contribution of these ancestry related SNPs to racial disparities in ALL4.
Genomeâ€wide association studies (GWAS) identify ARID5B SNPs predispose children to ALL
Recent GWAS interrogating ~500,000 germline SNPs across the genome identified 3 genomic loci (7p12.2, 10q21.2, and 14q11.2) with genomeâ€wide significant association with ALL susceptibility6,7. SNPs in ARID5B exhibited the strongest association with ALL (e.g. rs10821936, P = 1.4Ã-10â€15, odds ratio = 1.91)6. Multiple validation studies subsequently replicated the association of ARID5B with ALL incidence8,15-17. In contrast to prior studies focusing on genes in the xenobiotic and folate pathway that yielded conflicting results18-20, the association of ARID5B SNPs with ALL represents the most robust and reproducible evidence thus far that inherited genetic variations contribute to ALL susceptibility. The link between ARID5B and leukemogenesis is further supported by the observations that ARID5B deficiency leads to impaired hematopoiesis and lymphocyte development in mice21.
It should be noted that previous GWAS were performed with ALL subjects of European descent and have also been replicated in African-American and Hispanic ALL patients8,9. In the only published study on an Asian population, a detailed analysis of the aforementioned loci in a Korean cohort identified weak association for ARID5B17. However, the Korean study consisted of only 50 ALL cases, an exceedingly small sample size to detect meaningful associations.
ALL susceptibilityâ€related ARID5B SNPs are also associated with ALL relapse, plausibly through modulation of response to methotrexate (MTX)
Previous GWAS have also reported significant association of ARID5B SNPs with ALL subtypes that have distinct prognosis6,7, question arises as to whether ARID5B are related to treatment outcome of ALL. In a recent study, 49 ARID5B SNPs (within 30Kb upâ€ and downstream of ARID5B) were tested for association with ALL relapse in 1,605 children treated on the Children's Oncology Group (COG) P9904/9905 clinical trials9. Three SNPs at this locus were significantly associated with ALL relapse, after adjusting for known treatment-related and prognostic treatment factors. For example, patients carrying the T allele at rs6479778 were at a much higher risk of ALL relapse (P = 8.1Ã-10â€5). More interestingly, the same allele was also associated with higher incidence of ALL as observed in the original GWAS of ALL susceptibility (whites: P = 0.0029, Hispanics: P = 0.0296)9. This SNP was associated with minimal residual disease (MRD) at the end of induction therapy (P = 1.3Ã-10â€4), but remained prognostic even after adjusting MRD (P = 0.035).
In addition, previous observation that ARID5B SNP genotype was associated with MTXâ€polyglutamates (MTXâ€PG) level in leukemic blasts6, suggest modulation of response to anti-metabolite might be one of the mechanisms by which ARID5B is linked to ALL relapse.
Taken together, the existing knowledge builds a compelling case that genetic variations of the ARID5B gene are strongly related to ALL susceptibility and treatment outcome, and may significantly influence racial disparities of this disease. Therefore, we pose 2 questions in this proposal:
are the previously reported ARID5B ALL susceptibility and relapse-associated SNPs important in a diverse Asian cohort
what are the molecular mechanisms by which ARID5B modulates leukemia drug response.
Aim 1: Characterize reported genetic variation at ARID5B (10q21.2) in the Malaysia-Singapore (Ma-Spore) ALL cohort and to evaluate their associations with ALL susceptibility and ALL relapse
Studies of inherited genetic factors influencing the susceptibility and relapse of ALL in Asian children are sparse and Singapore's multi-ethnic population provides a unique environment to assess these genetic risk factors. The Ma-Spore study group's DNA bank of over 500 ALL patients samples (120 ALL patients per ethnicity) and 1000 non-ALL cord blood samples offer an ideal opportunity to comprehensively evaluate the ALL susceptibility and relapse genetic risk factors in an Asian cohort.
We plan to first genotype the ARID5B rs10821936 SNP, that has been well replicated in various ethnic population (whites, Blacks, NA) using a well-established allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) procedure in our laboratory. We will genotype ALL cases and controls from 3 ethnic groups: Chinese, Malays and Indians, with 120 individuals from each population. Subsequently, we will genotype the ARID5B polymorphisms associated with risk of ALL relapse reported previously9. To determine the relationships between ARID5B SNP genotypes and ALL susceptibility/relapse in each race group, we will compare allele frequency distribution between ALL cases and ancestryâ€matched controls using the logistic regression test6.
Anticipated results and analyses plans:
We anticipate that only some of the previously reported ARID5B SNPs will be associated with ALL susceptibility/relapse. With an odds ratio of 1.8 and 40% risk allele frequency in the general population, to detect such an association with a sample size of 120 cases and 360 controls for each ethnicity at the P = 0.05 significance level, our statistical power is 76%.
Pitfalls and alternatives:
Although ARID5B genetic variation associated with ALL susceptibility has been replicated in different ethnic population, ALL relapsed associated polymorphisms have yet to be replicated and the risk variants are in relatively low frequency in the general population (based on the Singapore Genome Variation Project data14, see table below). These genotype-phenotype association analyses are not designed to substitute experimental characterization of ARID5B genetic variants, thus we have already started to characterize functions of ARID5B sequence variation (e.g. effects on ARID5B transcription).
Aim 2: Explore molecular mechanisms by which ARID5B regulates antiâ€leukemic drug response
To explore the mechanisms by which ARID5B is related to ALL treatment outcome, we plan to examine the effect of ARID5B expression on anti-leukemic drug response in ALL cell lines. We are have currently established shRNA mediated stable knockdown of ARID5B in 3 Bâ€precursor ALL lines (NALM6, SEM, and UOCâ€B1) and have consistently observed significant increase in resistance to both MTX and 6MP, whereas sensitivity to were not affected. We've also observed significantly reduction in intracellular accumulation of MTXâ€polyglutamates (MTXâ€PG), active metabolites of MTX, in ALL cells with ARID5B knockdown compared to controls. This is in line with previous observations that ARID5B SNP genotype was associated with MTXâ€PG level in leukemic blasts6, suggesting modulating response to anti-metabolite might be one of the mechanisms by which ARID5B is linked to ALL relapse.
The associations between ARID5B genetic polymorphisms and ALL relapse highlight its potential value as a novel prognostic marker, but also beg the question as to what is the mechanism by which ARID5B is linked to treatment response. In our preliminary studies, we first tested which of the 6 chemotherapeutic agents (prednisolone, methotrexate, 6-mercaptopurine, asparaginase, vincristine, and daunorubicin) commonly used in ALL therapy are modulated by ARID5B. Thus, we established isogenic cell line models (stable knockâ€down by shRNA) to examine the effects of ARID5B expression on anti-leukemic drug responses. In 3 preâ€B ALL cell lines (NALM6, SEM, and UOCâ€B1), we consistently observed dramatically increased resistance to MTX and 6MP, but not for prednisolone, asparaginase, vincristine, or daunorubicin. We further demonstrated that resistance to MTX in ARID5B knockdown cell might be attributed to reduced accumulation of MTXâ€PGs. Aim 2 of the proposal poses three questions:
are ARID5B genetic variations associated with clinical response to MTX in children with ALL?;
how does ARID5B regulate the metabolism of MTX and 6MP; and
because MTX and 6MP belong to the same class of anticancer drug (i.e. anti-metabolite) and their mechanisms of action converge on DNA synthesis/cell cycle, how is ARID5B linked to genes involved in these processes?
In collaboration with Dr. William E. Evans and Dr Jun J. Yang from from St. Jude Children's Research Hospital (SJCRH), we aim to determine the relationships between ARID5B and MTX response in patients. We will capitalize on a wellâ€annotated St. Jude patient cohort treated on St. Jude Total XV and XIIIB protocols for whom MTX response was measured as change in white cell count before and after a single drug "MTX window therapy" and for whom genomeâ€wide genotyping was performed by Affymetrix 500K (germline DNA) and genomeâ€wide gene expression was profiled by Affymetrix Gene Expression array (diagnostic ALL blasts)22. Thus, we will perform association analyses of germline SNP genotype and ARID5B expression in ALL blast with MTX response phenotype, following well-established statistical procedures at SJCRH22,23.
Although some reports suggest that ARID5B is a transcription factor with possible histone demethylase activity24 and possible interaction with PDGF25, physiological functions of ARID5B are largely unknown particularly in the hematopoietic system. Therefore, we plan to take an agnostic approach to comprehensively characterize the genes regulated by ARID5B in ALL. We will compare global gene expression pattern in ARID5B knockâ€down vs. control cells (in all 3 ALL cell lines), using Affymetrix gene expression array. In parallel, we will also obtain 2 publically available gene expression datasets of childhood ALL (GSE12995, N=221 and GSE11877, N=202)26,27 to identify genes for which expression is correlated with ARID5B. A final "ARID5B signature" will be defined as genes whose expression was both significantly affected by ARID5B knockdown in isogenic cell line and was associated with ARID5B expression in primary ALL. This signature will then be used for gene set enrichment analysis to identify pathways regulated by ARID5B. In addition to this genomeâ€wide approach, we also plan to focus on a number of specific pathways. Both MTX and 6MP undergo anabolism to achieve (or improve) cytotoxicity. Because downâ€regulation of ARID5B expression led to reduced level of MTXâ€PG in ALL cell line, we suspect that certainly key enzymes responsible for MTXâ€PG conversion might be targeted by ARID5B. To this end, we will compare the expression (mRNA and protein levels) and enzymatic activity of folyploglutamate synthetase (FPGS) and gammaglutamyl hydrolase (GGH) in ARID5B knockâ€down vs. controls cells. For 6MP, we plan to measure thioguanine nucleotides (TGN, the active metabolite of 6MP) and activity of HPRT and TPMT (enzymes key to TGN conversion) in ARID5B knockdown vs. controls, following well-established HPLC procedures28,29. Finally, because cytotoxicity of both MTX and 6MP is highly correlated with the rate of cell cycle and DNA synthesis (particularly % cells in S phase), we hypothesize that ARID5B might modulate genes involved in cell proliferation/apoptosis. We will first compare distribution of cell cycle of ARID5B knockdown vs. control in all 3 ALL cell lines, by flow cytometryâ€based cell cycle assay. We stipulate that ARID5B knockdown cells might be somewhat arrested in the nonâ€proliferative G0/G1 phase. For examples, we will probe a panel of G1/S checkpoint genes (e.g. CDK2/4/6, CyclinD/E) and regulators of checkpoint genes (e.g. p21, p27, p53) by Western blot. The combination of genomeâ€wide and hypothesisâ€driven approaches is likely to shed lights on potential pharmacologic and molecular mechanisms for ARID5Bâ€related drug resistance in ALL.
The timing of this grant award is opportune for the conduct of this research study to make significant progress in the next 2 years as: 1) we have already generated compelling preliminary data to lay a solid basis of the project; 2) we are collaborating with SJRCH who has clinical samples and gene expression data from adequately powered clinical trials; 3) we have already established laboratory experimental models to be used for mechanistic/functional studies. Our longâ€term goal of is to translate our pharmacogenomic findings to individualized ALL therapy and further improve treatment rates.