Molecular Mechanisms Involved in the Development of Extramedullary Disease Following the Administration of All-Trans Retinoic Acid to Patients with Acute Promyelocytic Leukemia.

Abstract 1596

Poster Board I-622

The administration of all-trans retinoic acid (ATRA) has been associated with the occurrence of extramedullary disease (EMD), despite improvement in the prognosis of patients with acute promyelocytic leukemia (APL) treated with ATRA. EMD has been reported to occur in 3%–8% of patients with APL primarily in the central nervous system and skin. We postulate that, similar to the processes responsible for the development of metastasis, changes in the expression of proteins participating in adhesion, migration and homing may enable malignant hematopoietic cells to inhabit extramedullary sites. Our objective is to identify the molecular and cellular changes associated with exposure of the APL cell lines, NB4 and HL60 to ATRA and to establish the role of these changes in the development of EMD associated with ATRA in APL. We found that 30% of NB4 and HL60 cells, treated with ATRA adhere to fibronectin as opposed to untreated cells which show virtually no adhesion ability. A microarray screen revealed that many of the genes whose expression was altered following ATRA treatment participate in migration and adhesion processes. Among them was PYK2 whose expression was increased by 3-fold. PYK2 is an intracellular non-receptor tyrosine kinase which plays a role in intracellular signaling pathways that regulate processes such as cell adhesion and migration, which have been shown to correlate with tumor development and aggression. It is established that upon stimulation, PYK2 migrates to the membrane, where it is phosphorylated and activated leading to recruitment of additional proteins ultimately initiating cell adhesion. We found that pyk2 mRNA levels were upregulated in a time-dependent and ATRA-dependent manner. pyk2 mRNA levels were reduced to their basal level following ATRA withdrawal. The increase detected in PYK2 mRNA expression was due to enhanced transcription. In addition, PYK2 protein expression and phosphorylation levels were also upregulated in a time-dependent and ATRA-dependent manner. Interestingly, PYK2 protein expression level remained high even 5 days after ATRA withdrawal. Accordingly, NB4 cells maintained their adhesion ability for at least 5 days after ATRA depletion. Several additional PYK2-associated proteins: paxillin, vinculin, talin, GSK3a, integrin β7 and integrin β2 were upregulated at least 2-fold following treatment. Unexpectedly, we found 2 PYK2 isoforms in NB4 cells - the 116KDa known PYK2 isoform and a novel ∼80kDa isoform. Although both isoforms were upregulated and significantly phosphorylated following ATRA treatment, only the 80kDa isoform co-precipitated with paxillin following treatment. In conclusion, we show that following ATRA treatment, the expression of PYK2 and many PYK2-associated proteins is upregulated; PYK2 is extensively phosphorylated, initiating its activation and association with paxillin, possibly leading to the observed adherence of NB4 cells to fibronectin. These findings may contribute to our understanding of the molecular events associated with EMD in APL patients treated with ATRA. This in turn might help in the prevention and treatment of this phenomenon.