NIPA As a Novel Regulator of Aging and Stress Response of the Primitive HSC Pool

Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated.

NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p<0.001). All LSK-subgroups, LT-HSCs (p<0.001), ST-HSCs (CD34+Flt3-LSK; p<0.01) and MPPs (CD34+Flt3+LSK; p<0.05) of NIPA deficient animals are affected and failed to age-related increase, whereas the lineage differentiation of Nipa^ko/ko progenitor cells showed no gross differences. Myeloid depression by 5-FU treatment led to severely reduced HSC self renewal in Nipa^ko/ko mice independent of age (p<0.001). Moreover, weekly 5-FU activation showed reduced survival of Nipa^ko/ko vs. wt animals (11 vs. 14.5 days).

To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulation experiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipa^ko/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p<0.0001) in Nipa^ko/ko animals. Serial LSK transplantation assays revealed loss of Nipa-deficient LSKs shortly after TX, whereas long-term repopulation capacity seemed to be maintained, suggesting a role of NIPA in critical stress response.

To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population.

Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool.