Selenocysteine dependency renders central nervous system leukaemia therapeutically sensitive to ferroptosis

Abstract

Abstract Background and significance: Central nervous system (CNS) directed therapy in acute lymphoblastic leukemia (ALL) remains a formidable clinical challenge. The leptomeningeal niche restricts drug penetration and immune surveillance, limiting the efficacy of immunotherapies, and maintaining the current reliance on neurotoxic intrathecal methotrexate chemotherapy. Despite this, there have been no new CNS treatments licensed since the 1960's. The CNS microenvironment presents unique metabolic constraints, with cerebrospinal fluid (CSF) providing limited nutrient and oxygen availability. Here we describe how low CSF selenium and cystine bioavailability drives a unique niche dependent vulnerability to ferroptosis, a distinct form of non-apoptotic cell death characterised by lipid peroxidation. We show that targeting ferroptosis protective mechanisms by genetic and pharmacological inhibition, including use of the repurposed FDA approved agent Auranofin, eradicates CNS-ALL in vivo. Results: Public transcriptomic datasets (GSE60926, GSE18518) of CNS and bone marrow (BM) resident ALL cells were analysed, revealing a distinct ferroptosis-protective signature in CNS-ALL centred around selenium- and glutathione- metabolism. Metabolomic profiling of CSF from ALL patients and healthy controls revealed significant changes to cystine, glutamate and glycine, precursors required for glutathione (GSH) synthesis. Intracellular GSH levels were markedly reduced in CNS-resident ALL (CNS-ALL) cells relative to BM counterparts. GSH supports the uptake of selenium, a micronutrient required to fuel the activity and expression of the selenocysteine-containing glutathione peroxidase 4 (GPX4). GPX4 is the only enzyme capable of quenching reactive lipid species in cell membrane phospholipids, that if unchecked can result in ferroptosis. Indeed, CNS-ALL cells demonstrated elevated lipid peroxidation indicative of reduced GPX4 function. To model this biochemically restrictive environment in vitro we developed a custom physiological media that closely mimics human CSF. Exposure of both B- and T-ALL cells to this media resulted in significantly increased lipid peroxidation, triggered the GSH stress marker CHAC1, and increased sensitivity to the ferroptosis-inducing compounds RSL3 (inhibitor of GPX4) and Erastin (inhibitor of cystine antiporter System XC-). We hypothesised that leukemic cells survive in the CNS by relying on LRP8-mediated uptake of SELENOP-derived organic selenium, bypassing GSH-dependent selenium processing. Consistent with our hypothesis, we found that CNS-ALL cells retrieved from a murine xenograft model showed increased LRP8expression compared to BM-resident cells, mirroring mRNA data obtained from patient-derived CNS-ALL cells. Together, this confirmed that the CNS imposes a GSH-restricted microenvironment on ALL cells, leading to enhanced lipid peroxidation, and induction of LRP8 to maintain GPX4 expression. We therefore generated LRP8 and SEPHS2 (another key enzyme involved in handling selenium) CRISPR-Cas9 knock out (KO) ALL cell lines. GSHdisruption (by cystine deprivation in CSF media, or Erastin) in LRP8KO and SEPHS2KOcells resulted in increased lipid peroxidation, selective loss of GPX4 expression and enhanced ferroptosis. In vivo, LRP8KO and SEPHS2KO cells demonstrated significantly impaired survival within the CNS of xenografted NOD scid gamma (NSG) mice, with increased lipid peroxidation in recovered cells. We then identified the FDA-approved agent Auranofin, which disrupts cellular selenium utilisation, as a suitable therapeutic for potential repurposing against CNS-ALL. In vivo experiments using the CNS tropic BCP-ALL cell line 018Z and high CNS-risk patient-derived xenografts (PDXs) treated with Auranofin, following systemic engraftment, resulted in significant reduction in disease burden specifically in the CNS compartment (p<0.05). Conclusions: By comprehensive in vitro and in vivo characterisation we have identified a pivotal mechanism underpinning leukaemic survival in the CNS-niche. We show that the unique biochemical composition of CSF drives a dependence on selenocysteine biosynthesis which presents a targetable metabolic vulnerability. Our demonstration that Auranofin, a repurposed FDA-approved oral compound with excellent tolerability, leads to selective eradication of CNS-leukaemia in PDX models paves the way for clinical advances in treatment of CNS-ALL.