Abstract 727

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al. Blood. 2010;116:193. ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. In the current study we have analyzed the role of p53 in RPS19-deficient hematopoiesis by crossing the transgenic mice into Trp53 null background. To isolate the hematopoietic phenotype we transplanted bone marrow cells from these mice into lethally irradiated wild-type recipients. We have previously shown that the severity of the hematopoietic phenotype in transplanted recipients is highly dependent on the level of RPS19 downregulation, and the recipients with low RPS19 expression die 2–3 weeks after induction because of bone marrow failure. Remarkably, the inactivation of Trp53 rescued the early mortality in these recipients. However, although the inactivation of Trp53 completely reversed the erythrocyte and leukocyte numbers in the recipients with intermediate RPS19 downregulation, the recipients with low RPS19 expression still developed a mild anemia and macrocytosis. p53 activation is known to inhibit the AKT/mTOR pathway, a central regulator of cell growth and survival. Although the role of this pathway in DBA pathogenesis remains poorly defined, some patients positively respond to treatment with amino acid L-leucine, a nutrient signal that stimulates mTOR activity. Currently we are studying the role of L-leucine in RPS19-deficient hematopoiesis both in vitro and in vivo. Our preliminary data confirm that L-leucine modestly enhances the proliferation of RPS19-deficient c-Kit -enriched hematopoietic progenitors in vitro (1.2 fold in 8 days), while there is no effect on wild-type cultures. Interestingly, the proliferative response in RPS19-deficient cultures appears more pronounced when cells are cultured in low cytokine concentration (1.6 fold in 8 days). Since primary cells from DBA patients are highly responsive to stem cell factor (SCF), which also mediates its effect partly via PI3K/AKT/mTOR pathway, we are studying whether L-leucine has a synergistic role with SCF enhancing the proliferation of hematopoietic progenitors. Finally, a 15% L-leucine supplement in drinking water partly rescues the erythrocyte and leukocyte number in RPS19-deficient mice. In summary, our results demonstrate a key role of p53 activation in RPS19-deficient DBA, although they also suggest that p53-independent pathways may contribute towards phenotype upon severe RPS19 deficiency. Furthermore, our preliminary data supports the role of L-leucine as a therapeutic agent in the treatment of DBA.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.

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