Paper Details

ABSTRACT

Iron is an elemental nutrient utilized in DNA replication and repair, protein translation, and oxidative metabolism and is essential for anabolic metabolism. Blasts from B-cell acute lymphoblastic leukemia (B-ALL) retain elements of their developmental trajectory, albeit in a dysregulated manner, and the developmental stage of blasts correlates with their degree of anabolic drive. However, the role of iron in driving anabolic metabolism during physiologic B-cell development and in B-cell malignancies are not understood. Using single-cell RNA sequencing data from healthy B-cell development_{[Iacobucci et.al. PMID: 40579588]}, we identified coordinated upregulation of genes involved in iron-dependent processes during the transition from the Pro-B to Pre-B state. This transition is characterized by rapid expansion driven by mTORC1 signaling downstream of the newly recombined IGH gene. We next developed an iron-utilization gene signature based on the 11 most differentially expressed iron-related genes along this transition and applied it to publicly available transcriptomic data from primary patients_{[Mullighan et.al. PMID: 19129520, Chang et.al. PMID: 39121442]} to stratify patients with B-ALL into “high-utilization” and “low-utilization” groups. Patients with tumors possessing an iron score above the median (IS^high) have significantly worse event-free survival (EFS) and overall survival (OS) [EFS_NCI-TARGET HR=1.85, P=0.00033; OS_NCI-TARGET HR=1.96, P=0.00109; OS_MP2PRT HR=1.64, P=0.00833].

In vitro, we found that B-ALL cell lines were uniquely sensitive to the iron chelator deferoxamine (DFO) and underwent apoptotic cell death with low-dose exposure (10µM). In contrast, equivalent doses of DFO in T-ALL, AML, and HeLa cells resulted in minimal to no cell death, underpinning iron chelation as a B-ALL specific vulnerability.  We next asked whether mTORC1 directly regulated iron acquisition, functioning as an upstream factor involved in establishing the IShigh state. We found that sensitivity to DFO was correlated with strength of mTORC1 signaling, as measured by level of pS6. Furthermore, pS6high cells had increased expression of CD71 (TFRC; transferrin receptor), the principal receptor for iron uptake, and increased endocytosis of iron-bound transferrin protein. Treatment of B-ALL cells with rapamycin, a potent mTORC1 inhibitor, reduced CD71 expression and transferrin uptake, and abrogated DFO-mediated cell-death. We next performed an unbiased, genome-wide CRISPR screen in a pS6high B-ALL cell line to identify additional genes and pathways which modulate sensitivity to iron chelation. We found that deletion of genes involved in mitochondrial biogenesis and mitochondrial iron-utilizing processes protected against iron chelation induced cell-death. Furthermore, expression of key enzymes in the heme-biosynthetic pathway, a mitochondrial-dependent process, are upregulated in IShigh B-ALL tumors and heme supplementation rescues chelation-sensitive B-ALL cells from apoptosis in vitro. To corroborate our findings, we analyzed the publicly available Cancer Dependency Map[https://depmap.org/portal] CRISPR screen data for B-ALL-unique vulnerabilities compared to all other tumor types. We found that B-ALL-specific vulnerabilities were enriched for genes involved in iron-dependent processes, including cellular iron acquisition, mitochondrial function, iron-sulfur cluster biogenesis, and heme-biosynthesis. Together, these data suggest a fundamental role for increased mitochondrial iron utilization and de-novo heme-biosynthesis in a prognostically poor subset of B-ALL tumors which can be targeted using pharmacologic iron chelators.