7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.

A Dynamic rRNA Ribomethylome Drives Stemness in Acute Myeloid Leukemia.

The development and regulation of malignant self-renewal remain unresolved issues. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2'-O-methylation regulate leukemia stem cell (LSC) activity in vivo. A comprehensive analysis of the rRNA 2'-O-methylation landscape of 94 patients with acute myeloid leukemia (AML) revealed dynamic 2'-O-methylation specifically at exterior sites of ribosomes. The rRNA 2'-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of the 2'-O-methyltransferase fibrillarin (FBL) induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2'-O-methylation redirected the ribosome translation program toward amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2'-O-methylation at specific sites on rRNAs shifts translational preferences and controls AML LSC self-renewal. SIGNIFICANCE: We establish the complete rRNA 2'-O-methylation landscape in human AML. Plasticity of rRNA 2'-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function. This article is highlighted in the In This Issue feature, p. 247.

Adrenomedullin-CALCRL axis controls relapse-initiating drug tolerant acute myeloid leukemia cells.

Drug tolerant/resistant leukemic stem cell (LSC) subpopulations may explain frequent relapses in acute myeloid leukemia (AML), suggesting that these relapse-initiating cells (RICs) persistent after chemotherapy represent bona fide targets to prevent drug resistance and relapse. We uncover that calcitonin receptor-like receptor (CALCRL) is expressed in RICs, and that the overexpression of CALCRL and/or of its ligand adrenomedullin (ADM), and not CGRP, correlates to adverse outcome in AML. CALCRL knockdown impairs leukemic growth, decreases LSC frequency, and sensitizes to cytarabine in patient-derived xenograft models. Mechanistically, the ADM-CALCRL axis drives cell cycle, DNA repair, and mitochondrial OxPHOS function of AML blasts dependent on E2F1 and BCL2. Finally, CALCRL depletion reduces LSC frequency of RICs post-chemotherapy in vivo. In summary, our data highlight a critical role of ADM-CALCRL in post-chemotherapy persistence of these cells, and disclose a promising therapeutic target to prevent relapse in AML.

Mitochondrial inhibitors circumvent adaptive resistance to venetoclax and cytarabine combination therapy in acute myeloid leukemia.

Therapy resistance represents a major clinical challenge in acute myeloid leukemia (AML). Here we define a 'MitoScore' signature, which identifies high mitochondrial oxidative phosphorylation in vivo and in patients with AML. Primary AML cells with cytarabine (AraC) resistance and a high MitoScore relied on mitochondrial Bcl2 and were highly sensitive to venetoclax (VEN) + AraC (but not to VEN + azacytidine). Single-cell transcriptomics of VEN + AraC-residual cell populations revealed adaptive resistance associated with changes in oxidative phosphorylation, electron transport chain complex and the TP53 pathway. Accordingly, treatment of VEN + AraC-resistant AML cells with electron transport chain complex inhibitors, pyruvate dehydrogenase inhibitors or mitochondrial ClpP protease agonists substantially delayed relapse following VEN + AraC. These findings highlight the central role of mitochondrial adaptation during AML therapy and provide a scientific rationale for alternating VEN + azacytidine with VEN + AraC in patients with a high MitoScore and to target mitochondrial metabolism to enhance the sensitivity of AML cells to currently approved therapies.

Extracellular ATP and CD39 Activate cAMP-Mediated Mitochondrial Stress Response to Promote Cytarabine Resistance in Acute Myeloid Leukemia.

Relapses driven by chemoresistant leukemic cell populations are the main cause of mortality for patients with acute myeloid leukemia (AML). Here, we show that the ectonucleotidase CD39 (ENTPD1) is upregulated in cytarabine-resistant leukemic cells from both AML cell lines and patient samples in vivo and in vitro. CD39 cell-surface expression and activity is increased in patients with AML upon chemotherapy compared with diagnosis, and enrichment in CD39-expressing blasts is a marker of adverse prognosis in the clinics. High CD39 activity promotes cytarabine resistance by enhancing mitochondrial activity and biogenesis through activation of a cAMP-mediated adaptive mitochondrial stress response. Finally, genetic and pharmacologic inhibition of CD39 ecto-ATPase activity blocks the mitochondrial reprogramming triggered by cytarabine treatment and markedly enhances its cytotoxicity in AML cells in vitro and in vivo. Together, these results reveal CD39 as a new residual disease marker and a promising therapeutic target to improve chemotherapy response in AML. SIGNIFICANCE: Extracellular ATP and CD39-P2RY13-cAMP-OxPHOS axis are key regulators of cytarabine resistance, offering a new promising therapeutic strategy in AML.This article is highlighted in the In This Issue feature, p. 1426.

Targeting Myeloperoxidase Disrupts Mitochondrial Redox Balance and Overcomes Cytarabine Resistance in Human Acute Myeloid Leukemia.

Chemotherapies alter cellular redox balance and reactive oxygen species (ROS) content. Recent studies have reported that chemoresistant cells have an increased oxidative state in hematologic malignancies. In this study, we demonstrated that chemoresistant acute myeloid leukemia (AML) cells had a lower level of mitochondrial and cytosolic ROS in response to cytarabine (AraC) and overexpressed myeloperoxidase (MPO), a heme protein that converts hydrogen peroxide to hypochlorous acid (HOCl), compared with sensitive AML cells. High MPO-expressing AML cells were less sensitive to AraC in vitro and in vivo. They also produced higher levels of HOCl and exhibited an increased rate of mitochondrial oxygen consumption when compared with low MPO-expressing AML cells. Targeting MPO expression or enzyme activity sensitized AML cells to AraC treatment by triggering oxidative damage and sustaining oxidative stress, particularly in high MPO-expressing AML cells. This sensitization stemmed from mitochondrial superoxide accumulation, which impaired oxidative phosphorylation and cellular energetic balance, driving apoptotic death and selective eradication of chemoresistant AML cells in vitro and in vivo. Altogether, this study uncovers a noncanonical function of MPO enzyme in maintaining redox balance and mitochondrial energetic metabolism, therefore affecting downstream pathways involved in AML chemoresistance. SIGNIFICANCE: These findings demonstrate the role of myeloperoxidase in the regulation of ROS levels and sensitivity of AML cells to cytarabine, an essential chemotherapeutic backbone in the therapy of AML.

Chemotherapy-Resistant Human Acute Myeloid Leukemia Cells Are Not Enriched for Leukemic Stem Cells but Require Oxidative Metabolism.

Chemotherapy-resistant human acute myeloid leukemia (AML) cells are thought to be enriched in quiescent immature leukemic stem cells (LSC). To validate this hypothesis in vivo, we developed a clinically relevant chemotherapeutic approach treating patient-derived xenografts (PDX) with cytarabine (AraC). AraC residual AML cells are enriched in neither immature, quiescent cells nor LSCs. Strikingly, AraC-resistant preexisting and persisting cells displayed high levels of reactive oxygen species, showed increased mitochondrial mass, and retained active polarized mitochondria, consistent with a high oxidative phosphorylation (OXPHOS) status. AraC residual cells exhibited increased fatty-acid oxidation, upregulated CD36 expression, and a high OXPHOS gene signature predictive for treatment response in PDX and patients with AML. High OXPHOS but not low OXPHOS human AML cell lines were chemoresistant in vivo. Targeting mitochondrial protein synthesis, electron transfer, or fatty-acid oxidation induced an energetic shift toward low OXPHOS and markedly enhanced antileukemic effects of AraC. Together, this study demonstrates that essential mitochondrial functions contribute to AraC resistance in AML and are a robust hallmark of AraC sensitivity and a promising therapeutic avenue to treat AML residual disease.Significance: AraC-resistant AML cells exhibit metabolic features and gene signatures consistent with a high OXPHOS status. In these cells, targeting mitochondrial metabolism through the CD36-FAO-OXPHOS axis induces an energetic shift toward low OXPHOS and strongly enhanced antileukemic effects of AraC, offering a promising avenue to design new therapeutic strategies and fight AraC resistance in AML. Cancer Discov; 7(7); 716-35. ©2017 AACR.See related commentary by Schimmer, p. 670This article is highlighted in the In This Issue feature, p. 653.