Absence of Spred, a Negative Regulator of Tyrosine Kinase Activity, in Acute Myeloid Leukemia Patients

The Sprouty family proteins, including Spred, were originally identified in Drosophila melanogaster as an antagonist of Breathless, the ortholog of Fibroblast Growth Factor Receptor (FGFR) in mammals. These proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. Their main function is to deregulate the RAS/MAPK and RAS/RAF/ERK signal pathways by physically interacting. The role of Spread proteins in haematological malignancies is still not been clarified.

The aim of this study was to investigate a possible involvement of Spred in maintaining the aberrant TK signalling in patients affected by acute myeloid leukaemia (AML).

Bone marrow (BM) cells were collected from 82 AML patients at diagnosis and 10 BM samples from healthy donors as control. In addition 20 patients were analyzed during follow-up at the time haematological remission. All the patients included had been characterized at the cytogenetic level by conventional karyotyping, and screened by reverse transcriptase-PCR for the presence of the most frequent fusion transcripts or mutations. We analysed Spread mRNA expression by RQ-PCR and the protein by Western blot and immunofluorescence assay. In addition gain of function experiments were performed by transfecting with Spred coding sequence different leukemic cells lines lacking Spred activity.

We found that Spread mRNA levels were significantly decreased in AML compared to healthy subjects (2^−DDct = 0,1 in AML compared to 0,6 in controls) (p<0,001). Western blot and immunofluorescence confirmed the Spred downregulation at the protein level. Spread expression did not correlate to FAB subtypes or cytogenetic risk. Interestingly, when patients obtained the complete hematological remission (HR) the levels of Spred mRNA and protein were significantly upregulated to a level similar to healthy subjects (p=0.1 between HR and normal controls). Transfection experiments of Kasumi cell line with Spred coding sequence resulted in a decrease of proliferation of 53%, a colony growth reduction of 50% and a significant increase of apoptosis (p<0.001). Finally, in order to establish the molecular basis for Spred1 deregulation we analyzed the expression levels of miRNA-21 which is a known regulator of Spred and we found a correlation between the expression of miRNA-21 and Spred.

A decreased expression and activity of Spred, a negative regulator of TK activity through Ras pathway, is implicated in sustaining the oncogenetic signalling and abnormal proliferation induced by tyrosine kinase proteins in acute myeloid leukemia patients.

No relevant conflicts of interest to declare.