Targeting Metabolism to Restore Hematopoiesis in Inherited Bone Marrow Failure Syndromes: Preclinical Insights from Zebrafish Models

Inherited bone marrow failure syndromes (IBMFS) are a clinically diverse group of rare genetic disorders characterized by cytopenia of one or more hematopoietic lineages. IBMFS account for 10-15% of marrow aplasia and >50% of chronic pediatric bone marrow failure disorders. Definitive treatment for all IBMFS currently requires allogeneic hematopoietic stem cell transplantation. However, post-transplant complications such as organ toxicity and engraftment failure are common in this population. A thorough understanding of the underlying disease biology would enable the development of targeted therapeutic interventions that could rescue marrow failure, and potentially prevent progression to myeloid malignancies. We hypothesized that in addition to the transcriptional dysregulation, known to underpin IBMFS, perturbations in metabolism are essential for the transition of hematopoietic cells from a state of hypo-proliferation in IBMFS to one of hyperproliferation in leukemia. Given the rarity of IBMFS, large numbers of primary human samples are not readily available for mechanistic studies, warranting the use of animal models. Zebrafish (Danio rerio) are ideal given their highly conserved and rapid hematopoiesis and access to early blood progenitors.

Here, we used loss-of-function zebrafish mutants to study two IBMFS subtypes with increased propensity for malignancies, namely, DNAJC21-mutant Shwachman-Diamond syndrome (SDS) and PARN-mutant dyskeratosis congenita (DC). We recently showed that in dnajc21^-/- embryos, poor DNA damage responses caused by nucleotide deficiency impedes cell cycle progression, contributing to neutropenia. Treatment of dnajc21^-/- embryos with 100 mM uridine or thymidine nucleoside relieved the cell cycle block and restored neutrophil counts (Ketharnathan et al. Leukemia, in press). We extended these findings to our parn^-/- zebrafish that also present with neutropenia and anemia at 48 hours post-fertilization (hpf). Reduced telomerase activity and shortened telomeres are inherent features of DC. Zebrafish telomere lengths are strikingly similar to that of humans (5-15 kb in zebrafish versus 20-150 kb in mice). Preliminary analysis revealed reduced telomerase activity in parn^-/- whole kidney marrows (WKMs, human bone marrow equivalent) by 12 months of age. Thymidine treatment has been shown to support telomere elongation in human cells. Hence, we treated parn^-/- embryos with thymidine (100 mM from 3 to 48 hpf). We found that thymidine treatment rescued neutropenia, but only partially improved erythrocyte counts, suggesting differences in underlying mechanisms.

In addition to nucleotide imbalance, metabolomic analyses of dnajc21^-/- embryos and WKMs identified deficiencies in vitamin B6 (pyridoxine) and its active form, pyridoxal 5-phosphate. We are currently evaluating the effectiveness of exogenous pyridoxal 5-phosphate supplementation for rescuing cytopenia in the dnajc21^-/- mutants. In parn^-/- WKMs, we identified several metabolic processes that are dysregulated at the transcriptional level: linoleic acid metabolism, fatty acid biosynthesis and glycine, serine and threonine metabolism were downregulated whereas, cholesterol biosynthesis, arachidonic acid metabolism, cysteine and methionine metabolism were upregulated. Importantly, squalene epoxidase (zebrafish sqlea), the second rate-limiting enzyme in the cholesterol biosynthesis pathway and a marker that is upregulated in various cancers, was elevated in the parn^-/- mutants. We are currently investigating the effects of altered cholesterol metabolism on hematopoietic differentiation, and the potential for SQLE inhibitors such as terbinafine for rescuing cytopenia in parn-mutant DC.

In summary, our zebrafish models of SDS and DC serve as promising in vivo platforms for revealing disease mechanisms and preclinical screening of targeted therapies.

Mannherz:The Children's Medical Center Corporation: Patents & Royalties: Inventor on a patent application on use of nucleosides for telomere elongation. Agarwal:The Children's Medical Center Corporation: Patents & Royalties: Inventor on a patent application on use of nucleosides for telomere elongation; Cimeio Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Orbimed: Consultancy. Berman:Oxford Immune Algorithimics: Membership on an entity's Board of Directors or advisory committees.