CITED2 Affects Leukemic Cell Survival By Interfering with p53 Activation
Authors: Mattes Katharina , Berger Gerbrig , Geugien Marjan , Martens Joost H.A. , Vellenga Edo , Schepers Hein 
Published: October 2019

Abstract
Recently it was demonstrated that CITED2 (CBP/p300-interacting-transactivator-with-an ED-rich-tail 2) is upregulated in 70% of AML cases and that CITED2 levels are critical for the survival of leukemic stem cells. To address the role of CITED2 in leukemia maintenance, NSG mice were transplanted with human AML CD34+ cells transduced with a lentiviral construct for RNAi-mediated knockdown of CITED2. The results demonstrated that these mice had a significantly longer survival compared to mice transplanted with control AML cells (p<0.02).
In order to identify the molecular pathways affected in hematopoietic stem and progenitor cells upon loss of CITED2, differential gene expression after CITED2 knockdown was analyzed using microarray. Gene ontology analysis revealed an enrichment of p53 targets among those genes. We observed increased levels of acetylated p53 and its downstream target p21 upon down-regulation of CITED2 in both primary CD34+ cord blood cells and in the leukemic cells lines NB4 and MOLM13, whereas levels of the anti-apoptotic protein BCL2 were strongly reduced. In line with these observations, we found increased levels of apoptosis in CITED2 knockdown cells, which could be rescued by simultaneous knockdown of p53.
Further analysis of p53 targets by qPCR in CITED2 knockdown cells revealed an increased expression of PHLDA3. PHLDA3 is known as a repressor of the AKT signaling pathway, which promotes cell growth and survival. In line with this finding, we observed decreased AKT phosphorylation in CITED2 knockdown cells. Notably, we could rescue the CITED2 knockdown- mediated repression of AKT signaling by simultaneous knockdown of PHLDA3. Additionally, simultaneous knockdown of PHLDA3 led to a partial rescue of the apoptotic phenotype induced by CITED2 RNAi.
Similar, knockdown of CITED2 in patient AML CD34+ cells (n=17) cultured on MS5 stromal layer led to impaired growth of AMLs cells, whereas a simultaneous knockdown of CITED2 and p53 could rescue this phenotype. These data suggest that de novo AML with p53 mutations will be resistant to CITED2 knockdown-mediated cell death and we currently investigate this hypothesis. Additionally, we aim to get insight into the mechanism of increased p53 acetylation and activation upon knockdown of CITED2. By performing immunoprecipitation studies, we found a mutually exclusive binding of CITED2 and p53 to p300, suggesting that knockdown of CITED2 potentially alters p300 protein stability and acetylation activity towards p53. Furthermore, ChIP-Seq data revealed that knockdown of CITED2 in AML cells causes a global increase of H3K27 acetylation, thereby providing further evidence for altered p300 activity.
Our results demonstrate that knockdown of CITED2 triggers apoptosis in both normal hematopoietic- and leukemic cells in a p53-dependent manner. Therefore, CITED2 represents a highly interesting target for the treatment of p53 wild type AML patients.

Disclosures
No relevant conflicts of interest to declare.