Riboflavin Drives Nucleotide Biosynthesis and Iron-Sulfur Metabolism to Promote Acute Myeloid Leukemia

Cofactors, many of which are vitamin-derived, are organic molecules with diverse biological functions. They are required for many metabolic pathways and enzymatic reactions, and thus can influence cell proliferation and differentiation. Though the role of vitamins in non-malignant contexts is relatively well characterized, their role in carcinogenesis is not well defined. Here, we sought to molecularly characterize how vitamins contribute to oncogenesis in acute myeloid leukemia (AML).

We performed vitamin depletion screens in AML cells using culture medium designed to mimic human plasma. These screens revealed deprivation of riboflavin (Vitamin B₂) as detrimental to AML proliferation. Analysis of the Cancer Dependency Map (DepMap) and in vivo CRISPR-Cas9 screens identified the first, rate-limiting enzyme of riboflavin metabolism, riboflavin kinase (RFK), as a hematological cancer dependency. Genetic knockout (KO) of RFK or exogenous depletion of riboflavin in genetically diverse AML cell lines and patient-derived xenograft (PDX) cells induced AML differentiation and apoptosis. Riboflavin depletion was rescued by addition of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), the cofactor products of riboflavin metabolism. We validated that the growth of non-hematological cancer cells was largely unperturbed by exogenous deprivation of riboflavin or RFK KO, highlighting the specificity of this vulnerability in AML.

To understand the mechanistic basis of riboflavin dependency in AML, we performed mass spectrometry-based metabolomics studies. Whilst RFK loss dramatically reduced FMN and FAD pools, it also selectively depleted pyrimidine nucleotide species. A subsequent loss-of-function screen using riboflavin restricted culture medium and a metabolism-focused CRISPR-Cas9 library showed that loss of multiple genes encoding purine nucleotide biosynthesis enzymes promoted cell growth in riboflavin-restricted conditions, thereby alleviating the nucleotide imbalance induced by RFK loss. Accordingly, uridine supplementation partially restored proliferation in riboflavin deficient media. Nucleotide imbalance was accompanied by strongly impaired assembly of mitochondrial complex I and II which led to concomitant collapse in oxidative phosphorylation; indeed, loss of respiratory complex I and II subunits enhanced the anti-leukemic effects of riboflavin depletion in our screen.

Our CRISPR-Cas9 screen also revealed, unexpectedly, that loss of iron-sulfur (Fe-S) cluster assembly factors enhanced the anti-leukemic effects of riboflavin depletion. Consistent with these findings, in a subsequent CRISPR-Cas9 screen in an AML cell line insensitive to RFK KO, NDOR1, a member of the cytosolic Fe-S cluster assembly machinery, scored as the top sensitizer to RFK loss. Fe-S clusters are ancient cofactors required for the function and stability of enzymes involved in electron transport and redox reactions. Indeed, global proteomics analysis in cells lacking RFK, or in riboflavin restricted media, showed a strong decrease in Fe-S cluster-dependent enzymes. We demonstrated that RFK KO induced a potent iron starvation response analogous to Fe-S cluster starvation, characterized by increased iron responsive element protein and transferrin receptor levels, with a concomitant decrease in the activity and stability of Fe-S cluster-containing enzymes such as aconitases.

To explore perturbation of riboflavin metabolism as an anti-leukemic strategy, we took advantage of previous reporting of a correlation between reduced mitochondrial respiratory activity and sensitivity to BCL-2 inhibition. Consistent with our findings that perturbing riboflavin metabolism ablates oxidative phosphorylation, RFK KO or depletion of exogenous riboflavin strongly sensitized AML cell lines and cultured PDX cells to the BCL-2 inhibitor venetoclax. These findings were phenocopied using roseoflavin, a riboflavin antimetabolite, which was highly synergistic in killing AML cells in combination with venetoclax.

Collectively, this work describes a mechanistic framework of riboflavin dependency in AML and defines a previously unappreciated interplay between riboflavin and pyrimidine biosynthesis and Fe-S metabolism. Dietary modulation of riboflavin in combination with venetoclax is being tested for anti-leukemic potential in vivo.

Bjelosevic:Ramsay Health Care Limited: Current equity holder in publicly-traded company. Root:Merck: Research Funding; Addgene, Inc: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Abbvie: Research Funding; BMS: Research Funding. Vander Heiden:iTeos Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sage Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Drioa Ventures: Membership on an entity's Board of Directors or advisory committees; Auron Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pretzel Therapeutics: Membership on an entity's Board of Directors or advisory committees; Faeth Therapeutics: Membership on an entity's Board of Directors or advisory committees; Lime Therapeutics: Membership on an entity's Board of Directors or advisory committees. Stegmaier:Novartis/DFCI Drug Discovery Program: Research Funding; Kronos Bio: Research Funding; Auron Therapeutics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.