Down-Modulation of Shp1 and Hsp70 Provides a Survival Advantage to the Ph+ Cells of CML Patients Additive to That Related to the Oncogenic BCR/ABL Protein and May Account for Resistance to Ima Treatment.

Some chronic myeloid leukemia (CML) patients in chronic-phase and even more in advanced-phase, demonstrate primary resistance to imatinib or develop secondary resistance during treatment. In this study we investigate the mechanisms of resistance in a couple of Ima-sensitive and -resistant Kcl22 cell lines (Kcl22-s and Kcl22-r) combining a gene expression profile and proteomics approach. This couple of cells is interesting for a series of reasons: none of the known mechanisms of Ima resistance, i.e. Bcr-Abl point mutations or its overexpression or MDR up-regulation, has been described in these cells; in addition, differently respect to the various Ph+ cell lines and more similarly to a Ph+ stem cells, the original parental Kcl22-s show a high level of resistance to the Ima-induced apoptosis. Microarray indicated that 153 genes are up-regulated and 103 are down-regulated in the Kcl22-r respect to the Kcl22-s cells. Up-regulated genes comprise genes encoding for some membrane transporters, cell signalling proteins including MAPK kinase pathway molecules, cancer testis antigens, apoptosis negative regulators and DNA repair proteins. Among the down-regulated genes, that comprised also Annexin-1 and the HSP-70 and HSP27 proteins, we found a very low levels of the non-receptor, SH2 domain-containing protein tyrosin phosphatase-1 (Shp1). This protein is widely expressed in hematopoietic cells, and is directly involved in the negative regulation of cytokine/protein tyrosine kinase-mediated signalling. Methylation-specific PCR analysis of specific regions of SHP-1 and HSP70 promoters showed that the down-regulation of these genes is related to the hypermethylation of their promoters. Treatment of Kcl22-r with the 5-Azacytidine (5-AC), a demethylant agent, resulted in induction of Shp1 thus providing a further confirmation for the role of promoter methylation in the down regulation of this gene. Interestingly, the 5-AC-induced expression of Shp1 correlated, in Kcl22-r, with a significant reduction of phosphorylation of both pSTAT3 and pERK1/2, as well as of proliferation capacity of these cells. The phosphorylation of these latter signalling proteins is rather low in Kcl22-s cells independently by the Bcr-Abl p210 activity and it is not changed by the 5-AC treatment. We also show that Shp1 interacts in these cells with the Shp2, another SH2-containing phosphatase with different biological properties. Indeed, it has been shown that Shp2 is able to positively regulate signalling pathways, including the Ras/MAPK pathway. Moreover, the Shp2-dependent activation of the Ras/MAPK pathway in Kcl22-r cells may be also due to Hsp70 protein down-regulation. Indeed, this chaperone protein has been recently shown to stabilize the inactive form of the oncogenic Shp2 phosphatase. Taken together, our results indicate that a complex pathway of proliferation and survival in Ph+ cells based on the balance between the levels of Shp1 and 2 phosphatases and Hsp70 may account for the Bcr-Abl independent activation of Ras/MAPK pathway and for the resistance to Ima.