CREB Controls C/Ebpd to Impair Myeloid Differentiation Process in Acute Myeloid Leukemia

Purpose. The cAMP response element binding protein (CREB) is a transcription factor documented to be crucial for normal and neoplastic hematopoiesis. Its overexpression has already been demonstrated to impair myelopoiesis and to aberrantly control cell proliferation, apoptosis and cell cycle progression, both in vitro and in vivo. Its protein overexpression has been found in patients with acute myeloid leukemia (AML) contributing to decrease survival. CREB guides the expression of more than 5000 different targets in a tissue/cell-type specific fashion, and to date its main network towards leukemogenesis remains unknown. Here we aim to identify through which genes CREB triggers AML by using a CREB overexpressing transgenic zebrafish which developed AML in adulthood.

Patients and methods. We performed gene expression profile of RNA from the kidney marrow of 14-months old CREB-zebrafish (n=5) and control zebrafish (n=5). Principal component analysis was performed using Partek Genomic Suite software to integrate zebrafish AML signature with human signature found in pediatric AML to find common CREB target genes. AML cell lines were used to validate CREB targets in vitro.

Results. By GEP analysis and integration data we found 20 differentially expressed genes in both the zebrafish and human leukemia. Several of them were involved in the myeloid differentiation process, such as JUN, FOS and C/EBPδ. We confirmed that C/EBPδ protein levels highy correlated with phosphoCREB levels in zebrafish tumor as well as in a cohort of 66 pediatric patients with AML (r=0.79). We silenced and overexpressed CREB in AML or healthy bone marrow primary cultures respectively, and found that C/EBPδ was controlled by CREB transcriptional activity. We took advantages of a previously created HL60^(CREB-) cell line, where CREB protein levels was absent compared to the control cell line called HL60^(CREB+), where CREB was overexpressed. HL60^(CREB-) cell line expressed lowered C/EBPδ levels compared to HL60^(CREB+). To investigate CREB's role on differentiation program, cell lines were treated with all-trans-retinoic acid (ATRA). The evaluation of CD11b expression revealed that HL60^(CREB-) differentiated to a higher extent compared to HL60^(CREB+)_.Enhanced myeloid differentiation was also confirmed by cell morphology examination. Then we silenced C/EBPδ gene in HL60^(CREB+) cell line and treated with ATRA, confirming that C/EBPδ lowered expression increased CD11b expression, phenocopying the HL60^(CREB-) behavior.

Conclusion. These results indicated that C/EBPδ is a CREB target crucial for myeloid differentiation. The new role discovered for CREB proto-oncogene in blocking myeloid differentiation process opens for further opportunities with differentiation agents to cure AML.