Abstract
ATM (Ataxia telangiectasia mutated) kinase plays important roles in hematopoiesis. Two co-factors participate in ATM activation in a signal-dependent manner: NBS1 during ionising radiation induced DNA damage and ATMIN under hypotonic stress or chloroquine treatment, presumably due to changes in chromatin structure. Such artificial treatments might not have a genuine biological significance. However, we have recently shown a potential role for ATMIN in B cells. CD19-Cre mediated deletion of Atmin led to impaired class switch recombination, greatly increased genomic instability and consequently the development of B cell lymphomas. Considerable (but not total) overlap in phenotypes was found in ATM and ATMIN deficient B cells suggesting that ablation of ATMIN alters ATM function. As Atm-/- HSCs have impaired quiescence, maintenance and reconstitution capacity due to elevated reactive oxygen species, we sought to investigate if the absence of ATMIN would affect the primitive hematopoietic compartment. We used a Vav-Cre approach to delete Atmin to test ATMIN’s functions in the hematopoietic system (simply referred as AtminΔ/Δ).
At steady state, AtminΔ/Δ mice have ∼75% reduction in total splenocytes as compared to control mice. This is mainly due to reduced B cell, but also T cell, numbers. However, AtminΔ/Δ mice do not develop B cell lymphomas but they present differentiation block at the pre-B cell and at the immature/mature re-circulating B (IgM+/hi) cell stages with more than a 2-fold increase in apoptosis in these cell fractions compared to controls. The lack of lymphoma development in these mice suggests that the lack of ATMIN would also affect the more primitive compartment (e.g. CLP), not allowing tumorigenic clones to accumulate. Indeed, in the bone marrow (BM), a ∼40% reduction in total cellularity was observed in ATMIN deficient mice compared to littermate controls at 8-12 wks of age. This reduction in total cellularity is reflected across the board, from LT-HSCs (LSK/CD34-/lo/Flt3-) to the more mature cells and, more evidently, B cells. Apart from a similar B cell maturation block seen in the spleen, the frequency of CMPs (LS-K/IL7R-/CD34+/FcγR−) and GMPs (LS-K/IL7R-/CD34+/FcγR+) were reduced by 3- and 2-fold, respectively, in ATMIN deficient mice. The frequencies of other primitive compartments were not different in ATMIN deficient mice vs controls, including the CLP (LS-Klo/IL7R+) fraction. This reduction in CMP and GMP fractions translated into a ∼50% reduction in CFU capacity. Interestingly, only LT-HSCs, ST-HSCs and MPPs were found to be more cycling in the absence of ATMIN, suggesting a compensatory mechanism due to the reduction in some of the compartments downstream of LSK. Unlike in B cells, no increase in apoptosis was found in any other ATMIN deficient BM cell types, implying that the function of ATMIN is dependent on the context of the cell.
When transplanting similar numbers of ATMIN or control LT-HSCs, the regenerative capacity of AtminΔ/Δ-Vav-Cre cells was reduced by ∼16-fold in peripheral blood at 16 wks post-transplantation, with an early pronounced ∼10-fold reduction in B220+ cell production that was maintained over time. We found a steady decline in the production of myeloid cells over time, reaching a ∼13-fold difference between AtminΔ/Δ-Vav-Cre and control transplanted cells. However, in secondary recipients transplanted with a similar number of LSK cells, the regenerative capacity of AtminΔ/Δ-Vav-Cre cells was only further reduced to ∼24-fold as compared to control cells at 16 wks post-transplant. Interestingly, when transplanting ST-HSCs, the regenerative capacity of this fraction was even more drastically reduced, reaching ∼51-fold difference between AtminΔ/Δ-Vav-Cre and control cells.
Contrary to our predictions, oxidative stress does not seem to play any evident role in reducing CMP and GMP fractions. Interestingly, however, we observed a mild but significant phosphorylation of H2AX and a specific ATM substrate, KAP1, in only these myeloid progenitors suggesting activation of ATM signaling. Further studies are on going to test whether this to due to replicative stress.
Overall, our studies highlight that ATMIN appears to have different roles in different cells and potential novel ATM-independent functions. Certainly, in the absence of ATMIN, hematopoiesis is severely altered, directly and/or indirectly affecting the function of LT-HSCs.
Disclosures:
No relevant conflicts of interest to declare.
NIAM-Deficient Mice Are Predisposed to the Development of Proliferative Lesions including B-Cell Lymphomas