Accumulating evidence has confirmed that inherited genetic variations play multi-dimensional roles in childhood acute lymphoblastic leukemia (ALL), i.e., leukemia susceptibility, treatment response, chemotherapy tolerance, and relapse. Germline variants at the GATA3 locus increase the risk of developing Philadelphia chromosome-like ALL (Ph-like ALL) and inferior outcomes in childhood B-ALL among European and American cohorts. However, the role of inherited GATA3 variants in Han Chinese children with ALL still remains unclear.
To primarily identify the association of inherited GATA3 variants with treatment response, we retrospectively collected 273 childhood B-ALL blood samples after complete remission was achieved according to the Chinese Childhood Cancer Group ALL 2015. We then genotyped rs3824662 and rs3781093 in the GATA3 locus. The risk allele frequencies of rs3824662 and rs3781093 were 35.7% and 36.3%, respectively, consistent with the 1000 Genomes Project. Using a logistic regression model, we correlated the GATA3 genotype with minimal residual disease (MRD) level. In our single center, we found that GATA3 rs3824662 A allele and rs3781093 C allele statistically associated with positive MRD (the cut-off was >=0.01%, P=0.046 and 0.038, respectively). The A allele in rs3824662 and C allele in rs3781093 linked to 2-fold increase in the risk of MRD compared with their wildtype allele.
To explore the biological functions of these two germline SNP variants, we first utilized luciferase reporter assay to determine the impact of GATA3 variants on its transcription activity. Interestingly, the rs3824662 risk A allele significantly increased enhancer activity, while the rs3781093 did not show any effect. We next genetically modified rs3824662 from wild-type C allele to A allele in the lymphoblastoid cell GM12878 using clustered regularly interspaced short palindromic repeats/associated 9 (CRISPR/Cas9) gene editing system. Compared with wildtype GM12878 cells, ~3-folds higher GATA3 transcription level was found in GM12878 cells with A/A or A/C genotype.
Integrating the high risk of MRD and upregulated GATA3 expression, we proposed that GATA3 rs3824662 A allele might contribute to poor treatment response by promoting GATA3 transcription. To clarify the association of GATA3 expression with the sensitivity of ALL chemotherapeutic drugs, we retrieved GSE653 and GSE654 expression data for analysis and found that high GATA3 expression significantly correlated with L-asparaginase (L-Asp) and daunorubicin (DNR) resistance. To further confirm the correlation, we ectopically overexpressed GATA3 in B-ALL cell line (Nalm6) and only L-asp resistance was validated. L-asp resistance induced by GATA3 over-expression was rescued by GATA3 interference, consolidating the association between GATA3 and L-asp resistance in B-ALL cells.
Next, we probed the mechanism of GATA3-mediated L-asp resistance in B-ALL. We analyzed the association between inherited GATA3 variants and L-asp allergy, and did not identify statistical significance. Meanwhile, we couldn't find the correlation between GATA3 and ASNS, suggesting that ASNS might not be the cause either. Intriguingly, we found GATA3 over-expression induced the activation of autophagy-related genes, BECN1 and ATG5, which was reported to be associated with L-Asp resistance. Tests on primary B-ALL samples further confirmed this findings. To further confirm the effect of GATA3 on autophagy, we cloned highly conserved sequence BECN1 or ATG5 promoter region into luciferase reporter constructs. The Over-expression of GATA3 dramatically increased luciferase activity compared with the corresponding empty vector (P = 0.0098 and 0.0114, respectively), indicating that GATA3 expression were functionally activate their transcription. Taken together, all these data indicated that higher GATA3 expression might induce L-Asp resistance in B-ALL cells via autophagy activation.
In conclusion, we first identified that GATA3 rs3824662 associated with the risk of MRD in the childhood ALL cohorts of Han ethnicity. Mechanistic study showed that inherited GATA3 variants possibly contributed to L-Asp resistance via autophagy activation induced by promoting GATA3 enhancer activity, providing new insights into the rationale for the future development of combinational treatment of L-Asp and anti-autophagy regimen in ALL patients.
No relevant conflicts of interest to declare.
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