Cellular ATP demand and mitochondrial ATP synthesis are tightly linked. ATP synthesis, in turn, depends on
(1) NADH generation in the TCA cycle and (2) recycling of the adenine nucleotide pool. When ATP is
depleted and AMP rises, the cell increases energy generation and curtails energy expenditure until
homeostasis is restored. In the context of mitochondrial pathology, the system becomes unhinged…
Reticular Dysgenesis (RD) is a rare hematologic disease, caused by biallelic loss-of-function mutations in the
mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the phosphorylation of AMP to ADP in the
inter-membrane space to generate substrate for ATP synthesis1. RD patients suffer from severe congenital
neutropenia, lymphopenia, and die early in life unless cured by hematopoietic stem cell transplantation5.
We have developed a novel biallelic CRISPR-knockout model of AK2 in primary human hematopoietic stem
and progenitor cells to precisely mimic the failure of human myelopoiesis in culture and after transplantation
into mice. Using broad metabolomic profiling, our preliminary studies revealed that AK2-deficient myeloid
progenitors exhibit a high NADH/NAD+ ratio and NAD+ depletion, consistent with reductive stress. In
addition, AK2-deficient myeloid progenitors displayed a decrease in mitochondrial metabolites, including TCA
cycle intermediaries and aspartate, while lipid carnitines were increased, and lipid droplets were found in the
cytoplasm. We also detected highly elevated levels of the purine intermediate inosine monophosphate
(IMP) and a decrease in rRNA and ribosome subunits. Interestingly, our studies suggest the high IMP stems
from deamination of AMP, rather than a block in purine de novo synthesis. Taken together, these observations
raise the possibility that AK2 deficiency causes mitochondrial reductive stress, curtailing TCA cycle activity and
diverting carbon and electron pools into lipid synthesis while counteracting the accumulation of AMP.
These findings led us to hypothesize that AK2 deficiency causes two interconnected but distinct pathologies:
I. Reductive stress redirecting energy metabolism into lipid storage rather than OXPHOS;
II. Accumulation of AMP and IMP, leading to defects in nucleotide metabolism. Our proposed studies will
test if failure of myelopoiesis is primarily a result of reductive stress and impaired energy utilization, versus
impaired purine metabolism, or both. We will determine if myelopoiesis can be rescued by correcting the
NADH/NAD+ ratio or nucleotide pools. Lastly, we will validate our findings in an in vivo model of RD and
investigate if different compensatory mechanisms in different blood lineages result in the RD phenotype.
We use RD as a model to dissect escape mechanisms at the juncture of energy metabolism, redox stress,
and nucleotide homeostasis. These insights will advance therapies for mitigating reductive stress and using
cell type-specific manipulation of purine metabolism as a strategy for immunosuppression and cancer therapy.