Disruption of Intracellular Metal Homeostasis As a Vulnerability in SF3B1 Mutated AML

Acute Myeloid Leukemia (AML) is a heterogeneous disease in which outcome is highly dependent on cytogenetics and mutational profile. Adverse risk AML, recently defined and updated in the 2022 European LeukemiaNet (ELN) genetic risk classification, is associated with a poor overall survival.

To identify novel therapeutic approaches for AML, we screened a library of 10,000 compounds across 56 genetically diverse primary AML specimens. We then looked for active compounds with increased activity towards adverse risk AML samples and with low cytotoxicity to normal hematopoietic cells. Among the hits identified, we were able to validate that S767 molecule is preferentially active on adverse risk AML samples. Additionally, we found a strong association between SF3B1 mutations and sensitivity to S767.

To gain insights into S767 mechanism of action, we performed a CRISPR/Cas9 genome-wide functional screen, and identified multiples genes involved in mitochondrial processes both in synthetic rescue and synthetic lethal genes. In particular, knock-down of enzymes involved in heme biogenesis and iron-sulfur cluster biosynthesis appear to interfere with S767 cytotoxic activity. Given the central role of iron in these two synthetic pathways, and the structure of S767, we hypothesized that our molecule binds and disrupts intracellular iron homeostasis. Using UV-spectrometry analysis, we determined that S767 has metal affinity and binds to Fe²⁺, but also Cu²⁺ and Zn²⁺. We then quantified intracellular metal concentrations by ICP-MS (inductively coupled plasma mass spectrometry) and found that S767 exposure significantly decreases intracellular iron concentrations while increasing that of copper. Since metals are critical cellular cofactors involved in multiple enzymatic reactions, disruption of intracellular metal homeostasis can affect many processes. Mitochondrial respiratory chain is particularly sensitive to these perturbations as copper and iron-sulfur clusters are present in complexes I-IV of the electron transport chain and are essential for mitochondrial complexes function. By measuring oxygen consumption rate in leukemic cells exposed to S767, we found that the molecule indeed disrupts mitochondrial respiration.

SF3B1 is a splicing factor recurrently mutated in myeloid malignancies including myelodysplastic syndromes (MDS) and AML. Heterozygous mutations typically affect SF3B1 splicing functions and induces consequently mis-splicing of target mRNAs, including the ABCB7 iron-sulfur cluster transporter. Analysis of the Leucegene transcriptomic data of primary AML specimens revealed mis-splicing and downregulation of ABCB7 in SF3B1 mutated patients. Strikingly, ABCB7 was one of the top synthetic lethal gene identified by chemogenomic CRISPR/Cas9 screen following exposure to S767 molecule. Given that ABCB7 exports iron-sulfur clusters out of the mitochondria to be assembled by cytosolic proteins and plays a role in iron homeostasis, we hypothesized that its aberrant expression in SF3B1 mutated AML participates to the high sensitivity of these leukemias to S767.

We thus identified a novel molecule with selective activity in AML. Our data suggest that S767 is not a protein-targeting molecule but rather acts through the disruption of intracellular metal homeostasis, and ultimately affects mitochondrial respiration. Its mechanism of action highlighted a vulnerability in SF3B1 mutated AML which present a defect in ABCB7 expression.

Importantly, SF3B1 mutations are now recognized in the 2022 ELN risk classification as an adverse prognostic marker if they are associated with intermediate or adverse risk AML, giving a unique opportunity to develop novel therapeutic approaches for this subgroup of patients with poor clinical outcome. In addition, SF3B1 mutations are found in 25% of MDS cases, which are associated with ring sideroblasts and accumulation of mitochondrial iron, and that could also benefit from such therapeutic approach.

Hebert:BMS: Research Funding. Marinier:BMS: Research Funding. Sauvageau:ExCellThera: Consultancy, Current Employment, Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BMS: Research Funding.