R01HL165489

Adenylate Kinase 2 Deficiency and the Failure of Myelopoiesis - Project Summary/Abstract

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 synthesis. RD patients suffer from severe congenital neutropenia, lymphopenia, and die early in life unless cured by hematopoietic stem cell transplantation.

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.

The Leland Stanford Junior University

THE DIVISION OF BLOOD DISEASES AND RESOURCES SUPPORTS RESEARCH AND RESEARCH TRAINING ON THE PATHOPHYSIOLOGY, DIAGNOSIS, TREATMENT, AND PREVENTION OF NON-MALIGNANT BLOOD DISEASES, INCLUDING ANEMIAS, SICKLE CELL DISEASE, THALASSEMIA, LEUKOCYTE BIOLOGY, PRE-MALIGNANT PROCESSES SUCH AS MYELODYSPLASIA AND MYELOPROLIFERATIVE DISORDERS, HEMOPHILIA AND OTHER ABNORMALITIES OF HEMOSTASIS AND THROMBOSIS, AND IMMUNE DYSFUNCTION. FUNDING ENCOMPASSES A BROAD SPECTRUM OF HEMATOLOGIC INQUIRY, RANGING FROM STEM CELL BIOLOGY TO MEDICAL MANAGEMENT OF BLOOD DISEASES AND TO ASSURING THE ADEQUACY AND SAFETY OF THE NATION'S BLOOD SUPPLY. PROGRAMS ALSO SUPPORT THE DEVELOPMENT OF NOVEL CELL-BASED THERAPIES TO BRING THE EXPERTISE OF TRANSFUSION MEDICINE AND STEM CELL TECHNOLOGY TO THE REPAIR AND REGENERATION OF HUMAN TISSUES AND ORGANS. SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM: TO STIMULATE TECHNOLOGICAL INNOVATION, USE SMALL BUSINESS TO MEET FEDERAL RESEARCH AND DEVELOPMENT NEEDS, FOSTER AND ENCOURAGE PARTICIPATION IN INNOVATION AND ENTREPRENEURSHIP BY SOCIALLY AND ECONOMICALLY DISADVANTAGED PERSONS, AND INCREASE PRIVATE-SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT FUNDING. SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM: TO STIMULATE TECHNOLOGICAL INNOVATION, FOSTER TECHNOLOGY TRANSFER THROUGH COOPERATIVE R&D BETWEEN SMALL BUSINESSES AND RESEARCH INSTITUTIONS, AND INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL R&D.

93.837 - Cardiovascular Diseases Research

National Heart Lung and Blood Institute (NHLBI) [HHS - NIH]

Palo Alto, California 943041049 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 402% from $599,120 to $3,005,346.