In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: LinSca-1c-kitCD48CD150) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.

ULK1 inhibition increases the sensitivity of leukemia stem cells to TKI therapy by modulating energy and redox balance.

Despite the development of selective BCR-ABL-targeting tyrosine kinase inhibitors (TKIs) transforming the management of chronic myeloid leukaemia (CML), therapy-resistant leukaemic stem cells (LSCs) persist after TKI treatment and present an obstacle to a CML cure. Recently, we and others have made significant contributions to the field by unravelling survival dependencies in LSCs to work towards the goal of eradicating LSCs in CML patients. In this review, we describe these findings focusing on autophagy and mitochondrial metabolism, which have recently been uncovered as two essential processes for LSCs quiescence and survival respectively. In addition, we discuss the therapeutic potential of autophagy and mitochondrial metabolism inhibition as a strategy to eliminate CML cells in patients where the resistance to TKI is driven by BCR-ABL-independent mechanism(s).

Supporting cell proliferation through nucleotide biosynthesis is an essential requirement for cancer cells. Hence, inhibition of folate-mediated one carbon (1C) metabolism, which is required for nucleotide synthesis, has been successfully exploited in anti-cancer therapy. Here, we reveal that mitochondrial folate metabolism is upregulated in patient-derived leukaemic stem cells (LSCs). We demonstrate that inhibition of mitochondrial 1C metabolism through impairment of de novo purine synthesis has a cytostatic effect on chronic myeloid leukaemia (CML) cells. Consequently, changes in purine nucleotide levels lead to activation of AMPK signalling and suppression of mTORC1 activity. Notably, suppression of mitochondrial 1C metabolism increases expression of erythroid differentiation markers. Moreover, we find that increased differentiation occurs independently of AMPK signalling and can be reversed through reconstitution of purine levels and reactivation of mTORC1. Of clinical relevance, we identify that combination of 1C metabolism inhibition with imatinib, a frontline treatment for CML patients, decreases the number of therapy-resistant CML LSCs in a patient-derived xenograft model. Our results highlight a novel role for folate metabolism and purine sensing in stem cell fate decisions and leukaemogenesis.

Metabolic rewiring is an important hallmark of cancer. The folate metabolism pathway, also known as one-carbon (1C) metabolism, allows for transfer of 1C units through folate intermediates for biosynthetic processes, including precursors for DNA synthesis. Recent studies have shown that enzymes involved in the mitochondrial arm of 1C metabolism are overexpressed in a subset of aggressive cancers and that their expression affects responses to anti-metabolite drug treatments. However, the role of 1C metabolism in therapy resistant leukemic stem cells (LSCs) is currently unknown. Therefore, we aimed to investigate the activity and the impact of genetic and pharmacological inhibition of folate enzymes in primitive chronic myeloid leukaemia (CML) cells. We initially performed transcriptomic analysis of CD34+38- cells, from individuals with chronic phase CML (E-MTAB-2581). This revealed a significant upregulation of folate metabolism genes in CML LSCs, including serine hydroxymethyltransferase (SHMT2; p≤0.05), a key mitochondrial enzyme. To assess the activity of 1C metabolism in primitive cells we performed gas chromatography-mass spectrometry-mediated secretomic analysis using patient-derived, c-KIT enriched CML cells, which revealed a significant increase in the exchange rate of formate (folate intermediate necessary for purine synthesis) in CML cells, when compared to the secretome of normal counterparts (p<0.05). This reinforced the idea that 1C metabolism may be a metabolic dependency in CML. Following CRISPR-Cas9-mediated SHMT2 knockout (KO) in CML cell line, we observed a significant decrease in growth rate, together with a decrease in glycolytic capacity and oxygen consumption rate (p<0.01), suggesting impairment in proliferation and central carbon metabolism. Further metabolic characterisation of CML SHMT2 KO cells using liquid chromatography-mass spectrometry demonstrated a significant increase in AICAR, a purine biosynthesis intermediate and an AMP activated kinase (AMPK) activator. This prompted us to investigate the effect of 1C metabolism inhibition on AMPK. We found that AMPK phosphorylation on the conserved Thr 172 (a site that is phosphorylated under energy stress) was increased in SHMT2 KO cells, with similar effect seen following pharmacological inhibition of both SHMT2 and its cytosolic counterpart SHMT1 using SHIN1,which also promoted AMPK-dependent phosphorylation of the autophagy-inducing kinase ULK1 and downstream ULK1 target ATG13. Moreover, analysis of mitochondrial fraction revealed accumulation of mitochondrial fission related protein DRP1 and the mitophagy receptor NIX on mitochondria, hinting towards cellular interplay between 1C metabolism and mitochondrial homeostasis. Phenotypically, both pharmacological and genetic inhibition of SHMT1/2 induced the expression of erythropoiesis markers CD71 and Glycophorin A, which was reversed following formate supplementation. CRISPR-Cas9-mediated double AMPKα1/α2 KO revealed that the increased expression of these erythropoiesis markers following SHMT1/2 inhibition was independent of AMPK activity. Conversely, while NIX KO had no effect, pharmacological inhibition of ULK1 kinase activity, or genetic inhibition of ULK1 and ATG7 (protein important for autophagosome formation), prevented increased expression of CD71/Glycophorin A following SHMT1/2 inhibition. We next investigated the effect of 1C metabolism inhibition on differentiation and survival of primary CML cells. Of clinical relevance, pharmacological inhibition of SHMT1/2 promoted erythroid maturation of CD34+ CML cells (measured by expression of CD71, CD44, CD36 and Glycophorin A) when challenged with erythropoietin, which sensitises primitive cells to erythroid lineage commitment. Lastly, pharmacological inhibition of 1C metabolism decreased the colony formation capacity of CD34+ CML by 50%, with minimum effect on normal CD34+ cells. Moreover, combination treatment of SHIN1 with imatinib, a frontline treatment for CML patients, further increased the sensitivity of primary CML cells to imatinib by 40%. Overall, our novel findings indicate that disruption of the folate metabolism pathway inhibits central carbon metabolism in CML cells, promotes autophagy dependent, but AMPK independent maturation phenotype and has detrimental effect on the survival of primitive CML cells. Disclosures No relevant conflicts of interest to declare.