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1205 A Novel Conditional Knockout of the Diamond Blackfan Anemia Gene Rpl11 Shows Failure of Erythropoiesis, a Marked Increase in BFU-E Progenitors By Phenotype That Proliferate Poorly in Culture, and Activation of p53 Target Genes

Bone Marrow Failure
Program: Oral and Poster Abstracts
Session: 508. Bone Marrow Failure: Poster I
Saturday, December 5, 2015, 5:30 PM-7:30 PM
Hall A, Level 2 (Orange County Convention Center)

Cailin E Joyce, PhD1*, Meaghan McGuinness, BS2*, Rebekka K Schneider, MD3*, Mark D. Fleming, MD, DPhil4, Dean R Campagna, BS4*, Paul J Schmidt, PhD4, Benjamin L Ebert, MD, PhD5, Carl D. Novina, MD, PhD6 and Colin A. Sieff, MB.BCh2

1Dana Farber Cancer Institute, Boston, MA
2Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA
3Brigham and Women's Hospital, Boston, MA
4Department of Pathology, Boston Children's Hospital, Boston, MA
5Division of Hematology, Brigham and Women's Hospital, Boston, MA
6Dana-Farber Cancer Institute, Boston, MA

Diamond Blackman Anemia (DBA) is a severe erythroid hypoplasia that usually presents during the first year of life. Most cases are due to heterozygous mutations in ribosomal protein genes including RPS19, RPL5 and RPL11. Ribosomal stress-induced p53 activation is thought to play a major role in DBA, leading to apoptosis and/or cell cycle arrest in the erythroid lineage. This response is dependent on accumulation of RPL5 and RPL11 subunits that bind and sequester MDM2, the ubiquitin ligase that naturally degrades p53. Thus, it is difficult to understand how this pathway can operate in the presence RPL5 or RPL11 mutations. Here we show that Rpl11 haploinsufficiency in mice leads to severe anemia characteristic of DBA and, surprisingly, p53 activation in the bone marrow.

We created a conditional Rpl11 mouse with loxP sites that flank exon 2. These animals were crossed with MxCre heterozygotes, and treated with pIpC. After three weeks, Rpl11+/- animals became severely anemic, with increased MCV, decreased reticulocyte counts and normal leukocyte and platelet counts. Sacrifice of survivors after 4 months showed increased spleen weight due to the murine stress erythroid response. FACS analysis of bone marrow cells showed a marked increase in BFU-E and relative decrease in CFU-E in Rpl11+/- animals. However, methylcellulose colony forming assays revealed a decrease in large BFU-E colonies from Rpl11+/- animals, coupled with an increase in smaller, atypical BFU-E colonies, which fail to proliferate at a normal rate. To identify transcriptional signatures associated with severe anemia in Rpl11+/- animals, we sequenced cDNA libraries from total BM of induced Rpl11+/- mice and identified 508 differentially expressed genes relative to controls. Unexpectedly, p53 pathway genes were significantly upregulated in Rpl11+/- mice, which may suggest that Rpl5 accumulation alone or with haploinsufficient amounts of Rpl11 can sequester MDM2, or that p53 activation occurs through an alternative mechanism. In conclusion, we created a unique genetic heterozygous Rpl11 conditional knockout mouse that recapitulates the severe macrocytic anemia of DBA. We wish to use this model to identify novel molecular mechanisms of pathogenesis and test emerging therapies.

Disclosures: Ebert: Genoptix: Consultancy , Patents & Royalties ; Celgene: Consultancy ; H3 Biomedicine: Consultancy .

*signifies non-member of ASH