Search

The Wilms’ tumor gene is overexpressed in many types of haematological malignancies including acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML) and Ph negative myeloproliferative disorders. WT1 holds great promise for immunotherapy of leukaemia since it is expressed at high levels in blast cells but not in normal tissues, it is involved in the maintenance of malignant phenotype and it is spontaneously immunogenic. Many clinical trials using MHC class I-restricted WT1 peptide, have been recently performed in patients affected by AML, MDS, lung and breast cancer with satisfactory clinical results and without relevant toxicity. The first aim of the study was to analyze the natural immunity against WT1 in different types of haematological malignancies and the second was to set up a vaccination approach in the mouse model using the WT1 full length protein and to test the safety and efficacy of this vaccine. Using a dot blot technique, we analyzed for the presence of WT1 specific antibodies 107 patients including: 18 AML, 20 MDS, 22 Multiple Myeloma, 14 CML, 12 Idiopatic Myelofibrosis (IM), 6 CMML, and 15 ALL and 30 healthy subjects as control. For the second aim, we purified the WT1 full length protein. Complete coding sequence was cloned in a pGEX bacterial expression vector for the production of the fusion protein GST-WT1 that was subsequently purified by glutathione conjugated beads. Twenty C57BL7/6 mice were immunized with 50 μg of purified WT1 protein and 50 μL of Freund adjuvant every 15 days, for a total of 3 immunizations, 20 mice were injected with GST and adjuvant and 20 with PBS alone as control. 2 weeks after the last administration, 10 vaccinated mice and 10 controls have been injected with 200.000 TRAMP-C2 cells. Ten mice for each group were sacrificed and lymphocytes, cultured in presence of IL-2 to perform a cytotoxicity assay. Antibodies against WT1 have been evaluated. Toxicity on normal tissues has been rouled out using the histochemical analysis of different tissues. Results: Dot blot analysis demonstrated the presence of significant levels of WT1 IgG antibodies in 81% and IgM in 76% of the patients but not in normal subjects so demonstrating that WT1 is highly immunogenic. Particularly 88% of AML, 85% of MDS, 63% of MM, 100% of CML, 83% of IF and 66% of CMML presented high levels of antibodies. After mice vaccination, no organ toxicity has been observed. Importantly, immunized mice showed high levels of both, IgM and IgG antibodies with a mean value of luminescence of 690000 in WT1 injected mice vs 7500 in control mice and high levels of CTLs against WT1 as compared to mice injected with PBS or GST alone. Importantly, in control mice the mean size of the tumor was 1,6±0,4 cm, by contrast in vaccinated mice the tumor was unable to develop. Data obtained from this study clearly demonstrated that WT1 is immunogenic. The full length protein vaccination approach is able to elicit both, umoral and cytotoxic response and it results in a significant antitumor effect in vivo. Although further studies are required to compare the efficacy of this approach to the peptide based vaccine, WT1 full length protein could represent a valid vaccination strategy allowing to overcome the HLA restriction limit of the peptide approach.

Spred proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. They are implicated in negative feedback interactions that regulate intracellular pathways. The repressive function of Spred proteins targets several TK receptors so resulting in a variety of biological effects. Spred proteins, after growth factor stimulation, translocate to the plasma membrane, become tyrosine phosphorilated and interact with components of the Ras/MAPK and Ras/Raf/Erk pathways. Abnormal activation of Ras/MAPK pathway has been demonstrated in many haematological disorders including AML. In order to assess whether the activation of this pathway, leading to the disruption of many biological process, could be ascribed not only to the constitutive activation of TKs but also to defective signalling inhibition, we studied the function of Spred1 in AML. Using a Real Time PCR we studied the expression level of Spred1 in 80 AML samples (15 PB and 65 BM), 26 normal controls (16 PB and 10 BM) and 12 CD34+ samples sorted from AML patients and 12 from healthy subjects. We found that Spred1 transcript amount is significant reduced in AML samples when compare to normal controls (2−DeltaDeltaCt =0,084 vs 2,4 p=0,0000001) (range in AML 0,3–0,003). This difference is even more sound in AML CD34+ cells where the Spred1 transcript is 3 logs lower compared to normal CD34+ (2−DeltaDeltaCt =0,001 vs 2,27 p=0,0000001). Western blot using an antibody against Spred1 demonstrated the reduction or the absence of Spred1 protein in AML cells. Sequence analysis allowed to exclude the presence of mutations in the coding and promoter regions. In order to better understand the mechanism leading to the abrogation of Spred1 we analyzed the factors responsible for Spred1 transcription. We demonstrated that the transcription factor WT1 binds to and activates the promoter region of Spred1. WT1 KTS- is the isoform with transcriptional activity, while the isoform KTS+ is mainly localized at the cytoplasmatic level as confirmed by immunofluorescence analysis. Immunofluorescence assay and western blot carried out in NIH3T3 cells transfected with plasmids containing KTS+ and KTS- isoforms demonstrated that Spred1 protein is present at high levels in WT1 KTS- cells but not in KTS+ transfected cells. Finally we demonstrated, using capillary electrophoresis, that in AML patients the WT1 KTS- isoform was reduced or completely absent (ratio KTS+/KTS- ranging from 2 to 9,6 compared to a normal ratio ranging from 1, to 1,4 in cells from healthy subjects. Finally, in patients who achieved a complete remission after chemotherapy, WT1 KTS+/KTS- ratio returned within the normal range and Spred1 transcript and protein were significantly upregulated. This study clearly demonstrates that WT1 transcription activity is altered in AML cells in which WT1 may probably play a role of oncogene rather than of tumor suppressor gene. This leads to the absence of Spred1 transcript and protein which is a physiological inhibitor of RTK mediated signalling.