GSTM1 and GSTT1 double null genotypes determining cell fate and proliferation as potential risk factors of relapse in children with hematological malignancies after hematopoietic stem cell transplantation

Abstract

Purpose: This study aimed to retrospectively evaluate the genetic association of null variants of glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with hematological malignancies (HMs) undergoing busulfan (BU)- containing allogeneic hematopoietic stem cell transplantation (HSCT) and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized lymphoblastoid cell lines (LCLs) and tumor THP1 GST gene-edited cell models.

Methods: GSTM1- and GSTT1-null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1-null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell death studies were conducted in GSTs- null and non-null LCLs and CRISPR-Cas9 gene-edited THP1 leukemia cell lines.

Results: Carrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76-15.42; p = 1.9 × 10-5]). BU-induced cell death preferentially in THP1GSTM1(non-null) and LCLsGSTM1(non-null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation.

Conclusion: The clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation.

Trial registration: ClinicalTrials.gov NCT01257854.

Keywords: Acute leukemia; Busulfan resistance; Hematological malignancies; Hematopoietic stem cell transplantation; Null genotypes of glutathione S-transferases; Post-transplant relapse.

Conflict of interest statement

The authors declare that they have no competing interests related to the submitted work.

© 2021. The Author(s).

Figures

Fig. 1

Cumulative incidence plots of relapse in univariable competing risk analyses stratified by GST-null variants. Results are plotted for (A) GSTT1(−/−) group versus groups GSTT1( ±) and GSTT1(+ / +) together presented as GSTT1( +); (B) GSTM1(−/−) group versus groups GSTM1( ±) and GSTM1(+ / +) together presented as GSTM1( +); (C) combined GSTM1(−/−)/GSTT1(−/−) versus other genotype combinations [GSTM1(−/−)/GSTT1( +), GSTM1( +)/GSTT1(−/−) and GSTM1( +)/GSTT1( +)]; (D) combined GSTM1(−/−)/GSTT1(−/−) versus grouped other genotype combinations [GSTM1( +)/GSTT1( +)]. p values for the difference in cumulative incidence of relapse were calculated with Gray’s test with death as a competing event. HR, hazard ratio; GSTM1-null and GSTT1-null are presented as GSTM1(−/−) and GSTT1(−/−), respectively. GSTM1 non-null and GSTT1 non-null genotypes are presented as GSTM1( +) and GSTT1( +), respectively

Fig. 2

IC50-values for BU in GSTM1-null and GSTT1-null LCLs, THP1GSTM1(−/−) and THP1GSTT1(−/−) cells. IC50 values for BU were stratified according to GSTM1- (A) and GSTT1-null variants (C) in LCLs and CRISPR–Cas9 gene-edited THP1GSTM1(−/−) (B) and THP1GSTT1(−/−) (D) cell models. Concentration–response titration points were fitted to a Hill equation for BU. The 50% inhibitory concentrations of BU (BU-IC50) were determined by dose–response curve fitting using Prism 5.02 software (GraphPad SoftwareInc., CA. USA). The coefficient of determination (R2) of each plate was used to assess experimental reproducibility and was set to be above 0.95. Independent experiments were repeated at least three times. Non-parametric unpaired t test was used in LCLs (A, C). Pairwise comparisons by t test between GST(−/−) variants in THP1-CRISPR–Cas9 models (B., D.) were used. In THP1-CRISPR–Cas9 cell models (B., D.), dots represented are specific clones with identified GST(−/−) variants (+ / + vs. −/−) based on Sanger DNA-sequencing. p values below 0.05 were considered statistically significant. ns, not significant. GSTM1-null and GSTT1-null are presented as GSTM1(−/−) and GSTT1(−/−), respectively. GSTM1 non-null and GSTT1 non-null genotypes are presented as GSTM1( +) and GSTT1( +), respectively

Fig. 3

Baseline cell growth against GSTM1-null and GSTT1-null genotypes in LCLs and THP1GSTT1(−/−) cells. Baseline cell growth was assessed against GSTM1-null (A) and GSTT1-null (B) genotypes in LCLs at 48 h end-point analysis and the (C) RealTime Cell Viability assay–Kinetics plot was performed for 72 h stratified by GSTT1-null genotype at baseline (proliferation profile) in THP1GSTT1(−/−) cell models. (A, B) On the y-axis, the basal cell growth rate (r) was calculated using the following formula, appropriate for the usual exponential kinetics of cell growth (N; the number of cells) after the defined time (t; h): Nt = No.2tr. (C) Measurement of baseline reducing the potential of viable cells according to GSTT1(−/−) variant in CRISPR–Cas9 gene-edited cell models was performed. The unpaired t test between GST genotypes in LCLs (A., B.) and Pairwise comparisons by t test between GSTT1 genotypes in THP1-CRISPR–Cas9 models were used. p values below 0.05 were considered statistically significant. GSTM1-null and GSTT1-null are presented as GSTM1(−/−) and GSTT1(−/−), respectively. GSTM1 non-null and GSTT1 non-null genotypes are presented as GSTM1( +) and GSTT1( +), respectively

Fig. 4

Number of necrotic and apoptotic cells in LCLs and caspase 3/7 activity in LCLs, THP1GSTM1(−/−) and THP1GSTT1(−/−) cells. Flow cytometric analysis (FACS) by using Annexin V/PI assay was used to assess primary necrosis (A), late apoptosis (B), live cells (C) and early apoptosis (D) in LCLs stratified according to GSTM1-null variants; and caspase 3/7 activity (E) in LCLs and THP-CRISPR–Cas9 models stratified according to GST-null variants at 250, 500 and 1000 μM BU 48 h post-treatment. Statistical analysis was performed by two-way ANOVA considering 250, 500 and 1000 μM BU concentrations (genotype and treatment factors); t tests between GST(−/−) variants in each condition separately were used; no statistically significant differences were observed between GSTM1 + and GSTM1(−/−) LCLs and THP-CRISPR–Cas9 models in either 1% DMSO or medium only; p values below 0.05 were considered statistically significant. GSTM1-null and GSTT1-null are presented as GSTM1(−/−) and GSTT1(−/−), respectively. GSTM1 non-null and GSTT1 non-null genotypes are presented as GSTM1( +) and GSTT1( +), respectively

Fig. 5

Glutathione levels in LCLs stratified according to GSTM1-null variant, THP1GSTM1(−/−) and THP1GSTT1(−/−) cells. [GSSG/GSHT] ratio (A) and GSHT (B) in LCLs were calculated stratified according to GSTM1-null variant; and [GSSG/GSHT] ratios in CRISPR–Cas9 edited THP1GSTM1(−/−) (C) and THP1GSTT1(−/−) (D) cell models after the treatment with 500 μM BU. Statistical analysis was performed by the two-way ANOVA considering 250 or 500 μM BU concentration (genotype factor); t tests between GST(−/−) variants in each condition separately were used; no statistically significant differences were observed between GST(−/−) variants in LCLs and THP-CRISPR–Cas9 models in either 1% DMSO or medium; p values below 0.05 were considered statistically significant. GSTM1-null and GSTT1-null are presented as GSTM1(−/−) and GSTT1(−/−), respectively. GSTM1 non-null and GSTT1 non-null genotypes are presented as GSTM1( +) and GSTT1( +), respectively