A decreased Level of Shp1 provides an additive survival advantage to the Ph+ Cells of CML Patients and may account for Resistance to Imatinib Treatment
Authors: Esposito Nicola , Colavita Irene , Quarantelli Fabrizio , Izzo Barbara , Luciano Luigia , Vecchio Luigi Del , Hughes Timothy , Radich Jerald , Russo Domenico , Soverini Simona , Iacobucci Ilaria , Martinelli Giovanni , Saglio Giuseppe , Melo Junia , Ruoppolo Margherita , Kalebic Thea , Pane Fabrizio 
Published: April 2020

Abstract
Although rare in chronic phase myeloid leukemia (CML), primary or acquired resistance to the treatment with tyrosine kinase inhibitors (TKI) may be observed in the advanced phases of disease. Bcr/Abl related resistance has been well described, while the other mechanisms of resistance are poorly understood. In this study, we investigated the role of two SH2-containing, non-receptor protein tyrosine phosphatases (Shp1 and Shp2) in the resistance to Imatinib (Ima). To this aim, we have first used, as model system, a couple of Ima-sensitive (KCL22s) and Ima-resistant (KCL22r) KCL22 cell lines. In these cells, Ima resistance is independent by the oncogenic Bcr/Abl activity. We have found a very low level of Shp1 (both mRNA and protein), a protein with a tumour suppressor activity, in the KCL22r resistant cells, when compared to KCL22s sensitive cells. We have al shown the down-regulation of this gene to be related to the methylation level of SHP1 promoter. Indeed, 5-Azacytidine (5-AC) treatment, along with demethylation of the promoter region, re-induced expression of Shp1 in KCL22r. That treatment also re-established the Ima sensitivity, i.e. Ima growth inhibition, in these cells. At molecular level, the restored Ima sensitivity was associated to a significant reduction of phosphorylation of both STAT3 and ERK1/2. To better understand the functional role of Shp1, we carried out mass spectrometry to search for Shp1-binding proteins, and found that Shp1 interacts in these cells with Shp2, a protein phosphatase well known as positive regulator of oncogenic pathways, including the Ras/MAPK pathway. Gain-of-function mutations have been described in various hemopoietic neoplasias including Juvenile Chronic Myelomonocytic Leukemia. In Ph+ cells, oncogenic Bcr/Abl protein activates Shp2 through Gab2, an adaptor protein that, once phosphorylated is able to bind SH2 domain of Shp2. Through complex interactions that may involve the two carboxy-terminal tyrosine residues (542 and 580) Shp2 is also a signal transducer of growth factor receptor. We hypothesized that, Shp1, through dephosphorylation, might modulate the activity of Shp2 and constitute an important mechanism of Ima resistance. Knock-down of Shp1 in KCL22s cell line resulted in complete phosphorylation of Shp2 both 542 and 580 tyrosine residues and in its reduced sensitivity to the drug, thus supporting the role of this protein in Ima sensitivity. On the other hand, knock-down of Shp2 in KCL22r, that shows low Shp1 level, resulted in growth inhibition, restored Ima sensitivity and is associated to a significant reduction of phosphorylation of both STAT3 (60%) and ERK1/2 (70%). The data on primary cells support the role of Shp1 in Ima resistance in patients. Indeed, we analyzed 60 CML patients classified, according to the ENL definitions, as optimal (n =35), suboptimal (n=17) Ima responder, and primary (n=5) or secondary resistant (n=3) to Ima. The levels of Shp1 mRNA were significantly reduced in resistant patients [ratio of SHP1/ABL 3.2 ± 1.04, (mean±SD), *p&lt;0.05] when compared to the suboptimal (3.8±1.54) and optimal responders (5.8±1.77). Moreover, the Shp1 decrease was observed in CD34+ cells isolated from 6 resistant patients in comparison to 6 optimal responders. In conclusion, our study suggests that an aberrant balance between the Shp1 and 2 levels play a role in the Bcr-Abl independent resistance to Ima through activation of Ras/MAPK pathway and that lower levels of Shp1 are associated with non responsive patients.