All Trans Retinoic Acid (atRA) Differentiation Markers in Normal and Retinoid-Resistant Acute Promyelocytic Leukemia Cells Revealed Induction of atRA Metabolism as Relevant Prognostic of APL Sensitivity to Therapy.

It is now well established that all trans Retinoic Acid (atRA), administered at pharmacological doses to Acute Promyelocytic Leukemia (APL) patients, provides the first example of therapy by differentiation. Clinical remission is often transient as resistance develops. Because mechanisms of installation are still unclear, gene expression changes during APL cells differentiation were identified by Serial Analysis of Gene Expression (SAGE). Construction of proliferative and 48 hours atRA-treated NB4 cells libraries allowed us to identify a set of new transcriptional markers. Expression profiles of atRA response were performed on NB4, two atRA-resistant cell lines (NB4-LR2 and UF1) and APL blasts by real time PCR analyze on Microfluidic Card. We choose a hundred genes from multiple functional classes since there are several potential mechanisms for atRA resistance: target gene expression, transcription regulation, atRA metabolism, proteasome pathways≡ Reliability between SAGE and Microfluidic technologies is high since 95% of significantly modulated SAGE transcripts (p<0.01) show the same modulation by PCR. Analyzes provide valuable markers of the granulocytic phenotype (ICAM3, S100A9, CYP4F3, TMSB10), relevant and new atRA target genes (HIC1, ID2). Moreover, SAGE highlights chromatin remodeling factors, histone deacetylase (HDAC11) or coactivator (NCOA3) which may play a crucial role in the differentiation process. Finally, we insulate markers that correlate with resistance (CEBPA, CRABP2, NDRG1, CYP26). Transcriptome studies were conducted onto blast of patients with distinct long-term sensitivity, established by correlation to the in vitro differentiation rate (Cassinat, B. et al. Blood, 2001). As a result, all patients show transcriptional response to retinoid. However, once blast differentiation reached, induction of atRA-response element genes in high sensitive blasts is reduced. In opposition, transcripts expression of low sensitive patients is still high, revealing a delay in differentiation establishment. Conversely, expression of cytochrome p450 CYP26, involved in the atRA catabolism, is maintained in highly sensitive blast whereas no modulation is observed in low sensitive blast. Because promoter analysis reveals Homeobox response element, involvement of HOX factors found in SAGE librairies was investigated. In cell lines, HOX factors cooperate with retinoid receptors to increase CYP26 transcription. Furthermore, high-pressure chromatography shows a switch between atRA and its metabolites, 6 hours after atRA addition but only in sensitive cell line. To conclude, CYP26 is a relevant marker of resistance prognostic. Since metabolites show similar efficiency for cell growth inhibition and differentiation than atRA, their implication in resistance installation is investigated.