Our clinical association study performed in 86 children with HMs undergoing HSCT following BU-based conditioning regimens demonstrated that patients harboring homozygous deletions in both GSTM1 and GSTT1 genes presented a high risk of relapse (HR 7.2 [95% Cl, 2.2 - 23.9; p = 0.002]). After adjustment for known risk factors (diagnosis, disease status, the intensity of conditioning regimen and BU exposure), the association remained significant demonstrating that the deletion of both GST genes is an independent risk factor for relapse (adjusted HR 6.52 [95% CI, 2.8 – 15.4; p = 1.9 x 10-5]). Although it is a small cohort, this is the first report on the risk of post-HSCT relapse in relation to the germ-line GSTM1- and GSTT1-null variants in children with HMs. Until now, only one study conducted in BU/CY-based HSCT settings although in adults showed increased relapse rates in patients carrying GSTM1-null genotype, while no association was identified with GSTT1-null genotype (27). Concerning non-transplant-based studies in pediatric or adult patients, a similar association between GSTM1/GSTT1 double null carriers and increased risk of relapse (19, 28-30), lower complete remission rate (31) and lower event-free survival was demonstrated (18, 19, 32-38). There are nevertheless a few studies showing no such an association (20, 39), in which the small number of patients or the different treatment regimens may have mainly precluded defining a relationship between GST variants.
Based on the known detoxifying role of GSTs, our results from the clinical association are contradictory. Although GSTA1 is the main enzyme involved in BU detoxification, GSTM1 is also highly expressed in the liver and recognized as involved in BU conjugation (9, 11, 22, 40), precluding the BU to cross-link with the DNA strands. Functional variants of the genes coding for GSTs may then interfere in HSCT by affecting BU metabolism. It is known that low BU exposure (CumAUC < 59 mg×h/L) is associated with graft failure and relapse (5, 22, 41) whereas high BU exposure (CumAUC > 98.6 mg×h/L) could reduce post-HSCT relapse in leukemia at the cost of an increase in organ toxicities, and therefore transplantation-related mortality (5, 22, 41-43). However, at the level of HCs, less is known about the direct effect of BU.
We compared BU-related cell death mechanisms in LCLs and THP1 with and without GSTM1 and/or GSTT1 genes after exposure to BU. We demonstrated that only GSTM1-null (but not GSTT1-null) is associated with higher resistance to BU as determined by higher BU-IC50 values of GSTM1-null LCLs and THP1(GSTM1-/-) in comparison to GSTM1-non-null cells. This could be due to a change in the redox equilibrium as demonstrated by lower levels of oxidized GSH, lower primary necrosis and higher early apoptosis. An increase of GSTM1-null LCL`s viability was confirmed either by continuous follow-up of redox potential within 72h. Apoptosis/necrosis kinetic results demonstrate that BU-induced apoptotic processes are more pronounced in GSTM1-null LCLs. In contrast, primary necrotic cell death was more pronounced in GSTM1-non-null cells when comparing with the GSTM1 -null cells. In addition, primary necrosis was significantly induced at an earlier stage in GSTM1-non-null cells. These results show that GSTM1-null variants can modulate BU-induced cell death, which were supplemented further by increased activation of known apoptotic markers Caspase -3 or -7 in GSTM1-null LCLs and THP1 in comparison to GSTM1-non-null cells. Importantly, observed reduced rates of GSTM1-dependent cell death cannot be attributed to the increased baseline cell proliferation.
The findings of higher primary necrosis, lower early apoptosis and lower cell viability in GSTM1-non-null hematopoietic cells compared to GSTM1-null cells treated with BU were unexpected. Contrary to our observations, many studies showed associations between increased expression or activity of GSTs and resistance mechanisms against a range of cytotoxic drugs (44, 45). These results could potentially be explained by not only direct detoxification with GSH but also through negative regulation of pro-apoptotic protein kinases, such as apoptosis signal-regulating kinase 1 (ASK1) (13, 14). For instance, stress conditions cause the release of ASK1 from GSTM1, thereby leading to induction of apoptosis, which was shown in our experiments after induction with BU. In addition, GSTM1-null cells carrying more free ASK1 for phosphorylation activation are expected to have more apoptosis upon BU-induced stress in comparison to GSTM1-null cells which is in accordance with our in vitro results.
However, the observed paradox in increased cell death of GSTM1 well expressed cells upon BU treatment could additionally be explained by findings of the study of DeLeve et al. (46) demonstrating that in murine hepatocytes BU is cytotoxic also through oxidative stress caused by BU metabolites (BU glutathione S- conjugate thiophenium ion, GS+THT) and by the depletion of GSH in addition to DNA alkylation. The toxic metabolites of BU/GSH metabolism are mainly oxidized by Flavin-containing monooxygenases (FMOs, e.g. FMO3) and cytochromes (CYPs, e.g. CYP3A4) (47) to water-soluble non-toxic metabolites (e.g. sulfolane, (48)). However, CYP3A4 and FMO3 are mainly expressed in the liver (accounting for 54% of overall tetrahydrothiophene [THT] disappearance, the metabolite of BU), and less in LCLs, as observed in our laboratory (data not shown) and by others (https://www.proteinatlas.org). After RNA sequencing in LCLs, very low or no gene expressions of CYP 2D6, 2C19, 2C9, 2B6, 2C8, 4A11, 3A4, FMO1 and FMO3 were identified. In this context, the oxidative burst caused by electrophilic molecules from BU-GSH conjugation (49, 50) in addition to the absence of CYP3A4 and FMO3 could be a reason for the lower sensitivity of GSTM1-null HCs to BU, as observed in LCLs and THP1. In contrast, higher total expressions of CYPs and FMOs in hepatocytes (47) could explain why GSTA1-slow BU metabolizing individuals in addition to the absence of GSTM1 activity show potentially more treatment-related toxicities (e.g. SOS (51) and aGvHD (52)) than carriers with normal GST`s enzyme activities. A hypothetical comparative model of the difference in BU fate between hepatocytes and lymphocytes is suggested in Supplementary Figure 4 and warrants further investigation.
The genetically-determined different cell fate after BU exposure might explain the apparently discordant results between the relapse incidence in patients carrying GSTM1-null genotype (in combination with GSTT1-null) and the cellular resistance to BU in GSTM1 null-LCLs and THP1GSTM1(-/-). The higher rates of necrosis in GSTM1-non null cells might predict a pro-inflammatory cell death of the malignant cells, resulting in enhanced immunogenicity (53). Unlike the other chemotherapeutic regimens including autologous transplantation, the efficacy of the allogeneic transplantation relies on the graft-versus-leukemia effect, especially in HMs (54, 55), but that theory should be further explored.
Another relevant observation is the significantly increased post-HSCT relapse in GSTT1-null when combined with GSTM1-null genotype in children with HMs. The link between GSTT1 and post-HSCT relapse is not clear yet. Our in vitro observations cannot be attributed to the BU-related differences in IC50 values or [GSSG/GSHT] ratios. Other pharmacogenomics studies also demonstrated that genetic variations in GSTT1 are not associated with BU clearance or liver toxicity (11, 51, 56, 57). Nevertheless, we observed faster baseline proliferation in GSTT1-non-null LCLs/THP1 and a slightly higher baseline increase of Caspase 3/7 activation compared to those with GSTT1-null genotype, indicating GSTT1 potential involvement of BU- independent mechanisms in the relapse development.
The results of the present clinical study are limited by the retrospective study design and relatively small pediatric sample size with no clinical validation cohort. However, the sample size of 86 patients has at least 80% power with 10% of observed combined GSTM1/GSTT1 double null variants frequency and relapse incidence with the estimated observed effect size of ≥ 7.0 and alpha value of 0.05. The primary diagnosis of HMs was made at the referring institution and was not centrally reviewed. Well-known risk factors such as somatic genetic/cytogenetics abnormalities, the donor DNA and the initial response to the treatment (e.g. MRD) were not available. However, as described in Supplementary Table 2 similar characteristics were present between the GST genetic subgroups (p- values > 0.05). The GST-null variants were not associated with the status of the disease before HSCT and we are assuming that the germline genotype impact on protein expression was present in malignant cells as shown by Weiss et al. (16). The majority of cases in our study underwent a BU-CY conditioning regimen, however, it is not known if this association is specific to a BU-CY conditioning regimen only or unspecific to other chemotherapeutics used in HSCT setting (e.g.Thio or Mel) (7). For instance, active metabolites of CY (e.g. acrolein) are also eliminated by GSH conjugation catalyzed by GSTs (48). This needs to be evaluated in the future with a focus on whether GSTs play a major role in determining clinical outcomes. This aspect is currently being evaluated by our group using a cohort from multiple centers with the usage of multiple conditioning regimens. Furthermore, the transplant-related mortality or combined toxicities were not associated with the GSTM1- and GSTT1-null variants (data are not shown), suggesting compensation of BU conjugation by other GSTs, especially GSTA1, which is mainly expressed in hepatocytes and other somatic cells.