Upregulated heme biosynthesis, an exploitable vulnerability in MYCN-driven leukemogenesis
Yu Fukuda, Yao Wang, Shangli Lian, John Lynch, Shinjiro Nagai, Bruce Fanshawe, Ayten Kandilci, Laura J. Janke, Geoffrey Neale, Yiping Fan, Brian P. Sorrentino, Martine F. Roussel, Gerard Grosveld, John D. Schuetz
Yu Fukuda, Yao Wang, Shangli Lian, John Lynch, Shinjiro Nagai, Bruce Fanshawe, Ayten Kandilci, Laura J. Janke, Geoffrey Neale, Yiping Fan, Brian P. Sorrentino, Martine F. Roussel, Gerard Grosveld, John D. Schuetz
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Research Article Metabolism

Upregulated heme biosynthesis, an exploitable vulnerability in MYCN-driven leukemogenesis

Abstract

The increased heme biosynthesis long observed in leukemia was previously of unknown significance. Heme, synthesized from porphyrin precursors, plays a central role in oxygen metabolism and mitochondrial function, yet little is known about its role in leukemogenesis. Here, we show increased expression of heme biosynthetic genes, including UROD, only in pediatric AML samples that have high MYCN expression. High expression of both UROD and MYCN predicts poor overall survival and unfavorable outcomes in adult AML. Murine leukemic progenitors derived from hematopoietic progenitor cells (HPCs) overexpressing a MYCN cDNA (MYCN-HPCs) require heme/porphyrin biosynthesis, accompanied by increased oxygen consumption, to fully engage in self-renewal and oncogenic transformation. Blocking heme biosynthesis reduced mitochondrial oxygen consumption and markedly suppressed self-renewal. Leukemic progenitors rely on balanced production of heme and heme intermediates, the porphyrins. Porphyrin homeostasis is required because absence of the porphyrin exporter, ABCG2, increased death of leukemic progenitors in vitro and prolonged the survival of mice transplanted with Abcg2-KO MYCN-HPCs. Pediatric AML patients with elevated MYCN mRNA display strong activation of TP53 target genes. Abcg2-KO MYCN-HPCs were rescued from porphyrin toxicity by p53 loss. This vulnerability was exploited to show that treatment with a porphyrin precursor, coupled with the absence of ABCG2, blocked MYCN-driven leukemogenesis in vivo, thereby demonstrating that porphyrin homeostasis is a pathway crucial to MYCN leukemogenesis.

Authors

Yu Fukuda, Yao Wang, Shangli Lian, John Lynch, Shinjiro Nagai, Bruce Fanshawe, Ayten Kandilci, Laura J. Janke, Geoffrey Neale, Yiping Fan, Brian P. Sorrentino, Martine F. Roussel, Gerard Grosveld, John D. Schuetz

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Figure 2

MYCN upregulates heme biosynthesis, which is required for self-renewal and maximal mitochondrial respiration.

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MYCN upregulates heme biosynthesis, which is required for self-renewal a...
(A) Intracellular heme levels were measured by spectrophotometry in murine hematopoietic progenitors (HPCs) transduced with either a retrovirus encoding MYCN cDNA or empty vector (n = 3, mean ± SD). (B) Mitochondrial number was measured by Mitotracker Red (n = 3, mean ± SD) and by (C) citrate synthase (CS) activity in the murine HPCs overexpressing MYCN or the empty vector (n = 3, mean ± SD). (D) Oxygen consumption rates (OCRs) and extracellular acidification rate (ECARs) in vector- or MYCN-transduced HPCs were measured using the Seahorse analyzer (n = 5, mean ± SEM). (E) Gene set enrichment analysis (GSEA) shows strong upregulation in MYCN-transduced HPCs. Genes involved in succinyl CoA ligase are denoted with asterisks. (F) Methylcellulose replating assay shows that heme biosynthesis is critical for sustaining MYCN-mediated self-renewal (n = 3, mean ± SEM). The methylcellulose-replating steps are labeled MC, where cells are harvested from MC1 (or MC2) after 7 days in culture and replated for step MC2 (or MC3). (G) Microarray analysis shows heatmap of genes contributing to complex IV assembly and mitochondrial membrane (inner and outer) assembly. See also Supplemental Figure 1C for gene names. (H and I) Oxygen consumption (OCR) was measured in MYCN cells that were cultured in media containing vehicle, succinylacetone (SA), or N-methyl protoporphyrin (N-MPP) (representative graph with triplicate data points of 3 independent experiments). Maximal respiration was estimated as the value after FCCP (uncoupler) addition, and spare respiratory capacity was calculated as the difference between basal and maximal respiration (n = 3, mean ± SEM). (J) Inhibition of heme biosynthesis produced a small increase in glycolytic activity (ECAR) in MYCN-hematopoietic progenitors (n = 3, mean ± SEM). (*P < 0.05, ****P < 0.0001). Student’s t test was used for A, D, and J, and 2-way ANOVA was used for I and F.