Abstract
Significant progress has been made in treating childhood leukaemia's in recent decades, with a 5-year survival rate of 65%-95%. However, KMT2A-rearranged infant leukaemia remains a significant challenge. Classic chemotherapies can induce long-term side effects through toxicity, including neurocognitive deficits, cardiomyopathy, impaired growth and fertility. Despite recent success of CD19 targeted immunotherapies, some infants show evidence of CD19 negative escape or lineage switch, even after achieving complete remission, and additional conventional chemotherapy or haematopoietic transplantation is still required. Therefore, a pressing need remains to develop more effective, less toxic treatments for infant leukaemia. Earlier studies in solid tumours have identified dietary removal of the non-essential amino-acids serine and glycine (-S/G) as a potential therapeutic approach, but their use in leukaemia remains unexplored.
To explore this novel therapeutic approach in leukaemia, we subjected KMT2A-rearranged (KMT2Ar) leukaemia cell lines to S/G starvation in vitro and observed a reduction in proliferation. Supplementation of -S/G cells with exogenous glycine and formate restored proliferation, indicating that -S/G affects one-carbon metabolism in the folate cycle. This was further supported by metabolomic data that revealed an impact of -S/G on downstream metabolic pathways of the folate cycle, including glutathione (GSH) recycling to eliminate reactive oxygen species (ROS). RNASeq results validated our findings, showing an increased gene expression of all serine synthesis pathway (SSP) enzymes and key enzymes involved in the folate cycle (including MTHFD2). Additionally, it showed an increase in expression of the cystine importer SLC7A11. Cystine is relevant for GSH synthesis; therefore, increased cystine import further indicates increased dependency on antioxidant defence under S/G starvation.
Interestingly, LC-MS showed an intracellular accumulation of asparagine under S/G-starved conditions despite the lack of asparagine in the media. As ALL cells are known for their incapacity to synthesise asparagine via asparagine synthetase (ASNS), making asparaginase an effective clinical treatment, RNA expression levels of ASNS were observed and found to be increased.
The increased levels of SSP enzymes, SLC7A11 and GSH synthesis indicated increased oxidative stress under S/G-starvation, and the increased ASNS expression led to investigating the integrated stress response (ISR) as MTHFD2, SLC7A11, ASNS and the SSP enzymes are targets of ATF4. Indeed, ATF4 target genes DIIT3, TRIB3, AARS1, SESN2 and DDIT4 also showed significantly increased expression under S/G-starved conditions in RNASeq. Next, Western Blotting showed increased ATF4 expression with only 10µM serine and glycine present in the media, indicating cellular stress of KMT2Ar infant leukaemia at not only complete S/G depletion from the media, but also low levels of S/G, thus potentially sensitising the cells to any drug targeting ATF4 regulated stress responses or ATF4 itself. An FDA-approved drug screen comparing S/G starved and non-starved cells revealed selective sensitivity to drugs targeting upstream pathways of ATF4 activation, such as Trazodone, Metformin and Rapamycin. Additionally, ATF4-mediated compensatory pathways consume ATP and NAD+ at high rates, requiring OxPhos for ATP production and NADH recycling. This was supported by significant sensitivity of the cells to OxPhos inhibitors Metformin, Rotenone and Phenformin.
In conclusion, S/G starved KMT2Ar cells rely on the ATF4-mediated ISR for survival. Therefore, repurposing drugs active upstream or downstream of ATF4 could open potential new, more effective, less toxic therapeutic windows in the treatment of infant leukaemia.
