Fanconi Anemia (FA), results from mutations in genes necessary for DNA damage repair and often leads to progressive bone marrow failure. Although the exhaustion of the bone marrow leads to cytopenias in FA patients as they age, evidence from human FA and mouse model fetal livers suggests hematopoietic defects originate in utero which may lead to deficient seeding of the bone marrow. To address this possibility, we examined the consequences of loss of Fancd2, a central component of the FA pathway. Examination of E14.5 Fancd2 knockout (KO) fetal livers showed a decrease in total cellularity and specific declines in long-term and short-term hematopoietic stem cell (LT- and ST-HSC) numbers. Fancd2 KO fetal liver cells display similar functional defects to Fancd2 adult bone marrow cells including reduced colony forming units, increased mitomycin C sensitivity, increased LT-HSC apoptosis and heavily impaired competitive repopulation, implying these defects are intrinsic to the fetal liver and not dependent on the accumulation of DNA damage during aging. Telomere shortening, an aging-related mechanism proposed to contribute to HSC apoptosis and bone marrow failure in FA, was not observed in Fancd2 KO fetal livers. In summary, loss of Fancd2 yields significant defects to fetal liver hematopoiesis, particularly the HSC population, which mimic key phenotypes from adult Fancd2 KO bone marrow independent of aging-accrued DNA damage.

The purpose of this study was to investigate how CD44 impaired Akt phosphorylation, EGR-1 expression and cell proliferation. E6.1 Jurkat cells, which lack endogenous CD44 expression, were engineered to express CD44. Previously we showed that Akt is hypophosphorylated, EGR-1 expression is reduced and proliferation is impaired in CD44 expressing E6.1 Jurkat cells. The cell cycle was studied using flow cytometry and the role of calcium (Ca2+) in Akt phosphorylation and EGR-1 expression was investigated using Western blotting. Phosphatase activity was assessed using a commercially available kit. CD44 expressing cells showed disruption at the G1 to S transition. Chelation of Ca2+ from the culture media impaired Akt phosphorylation and EGR-1 expression in both CD44 expressing cells and the open vector control. Moreover, Ni2+ disrupted cell proliferation in both cell types suggesting Ca2+ import through calcium release activated calcium channels (CRAC). Staining of cells with fura-2 AM showed significantly higher Ca2+ in CD44 expressing cells as compared with the vehicle control. Finally, non-calcium mediated phosphatase activity was significantly greater in CD44 expressing cells. We propose that the enhanced phosphatase activity in the CD44 cells increased the dephosphorylation rate of Akt; at the same time, the increased intracellular concentration of Ca2+ in the CD44 cells ensured that the phosphorylation of Akt remains intact albeit at lower concentrations as compared with the vector control. Reduced Akt phosphorylation resulted in lowered expression of EGR-1 and hence, reduced the cell proliferation rate.

Ott1(Rbm15) is essential for engraftment and maintaining hematopoietic stem cell (HSC) quiescence during proliferative stress; therefore we sought to establish whether Ott1 has a regulatory role within the cell cycle. Ott1 knockout (KO) E14.5 murine embryonic fibroblasts (MEFs) were analyzed using BrdU labelling and demonstrated a higher basal proliferative rate. However, when subjected to oncogenic stress induced by infection with a constitutively active N-Ras expressing retrovirus, Ott1-deleted MEFs undergo immortalization and morphologic transformation in contrast to wild type (WT) MEFs which undergo senescence. Oncogene-induced senescence is a p53-facilitated process. P53 protein levels were shown by western blot to decrease in Ras-infected Ott1 KO MEFs rather than increase as observed in WT Ras-infected MEFs. Consistent with this finding, p16Ink4a, which is a transcriptional target of p53, is not upregulated in Ras-infected Ott1 KO MEFs. Gamma irradiation was still able to induce p53 in Ott1 KO MEFS, demonstrating Ott1 regulation of p53 is specific to the oncogenic stress pathway, but not the DNA damage pathway. Measurement of p53 mRNA levels in Ras-infected Ott1 KO MEFs showed a modest increase compared to WT, indicating the p53 protein decrease must occur at a post-transcriptional level. Classical p53 induction by oncogenic stress occurs through inhibition of ubiquitin-mediated degradation of p53 by ligases such as Mdm2 and Mdm4. To determine why Ras induction of p53 is defective in Ott1 KO MEFS, Ras-infected cells were incubated with the proteasome inhibitor, MG132, which was able to rescue p53 induction, implicating a ubiquitination-dependent mechanism. Furthermore, incubation with Nutlin3, an Mdm2-specific inhibitor, also showed significant rescue of p53 induction, signifying Ott1 is required for Mdm2-mediated degradation of p53 during oncogenic stress. P53 has an essential, non-apoptotic role in HSC function and has also been shown to help maintain HSC quiescence and self-renewal. We previously identified an Ott1-dependent mechanism for down-regulating Thrombopoietin response via its receptor Mpl in Ott1 KO HSCs through expression of a dominant negative alternatively spliced isoform, Mpl-TR. Although Mpl-TR expression is sufficient to reduce Mpl signaling and competitive repopulation in Ott1 KO HSCs, full length Mpl alone is unable to rescue engraftment of Ott1 -deleted HSCs suggesting Ott1 has other critical targets. Based on the Ott1-dependence of p53 function in MEFs, we hypothesized a similar dysfunction of the p53 pathway exists in Ott1 KO HSCs undergoing proliferative stress. Ott1 KO and WT HSCs were analyzed before and after incubation in a cytokine-rich medium to stimulate proliferation. At baseline, Ott1 KO HSCs have similar p53 protein levels as WT HSCs. However, after cytokine stimulation, Ott1 KO HSCs shift into active cell cycle more readily and now demonstrate a significant decrease in p53 protein levels as measured by intracellular flow cytometry. In summary, Ott1 is required for p53 response during oncogenic stress via inhibition of Mdm2. Ott1 is similarly required to maintain p53 levels during proliferative stress in HSCs and may thereby promote quiescence and self-renewal. Moreover, OTT1 is the 5' fusion partner in the chimeric OTT1-MAL (RBM15-MKL1) product in t(1;22)-associated acute megakaryocytic leukemia, raising the possibility that dysregulation of p53 pathways may contribute to the pathogenesis of t(1;22)-derived leukemias. Disclosures No relevant conflicts of interest to declare.