Screening Of Substrate-specific Shedding Inhibitor Of Notch1 And Its Application In T-ALL

Objective Abnormal activation of Notch1 signaling pathway plays pivotal roles in the pathogenesis ofT-cell acute lymphoblastic leukemia(T-ALL). The present study aims to screen the NOTCH1 gene mutation profile of the T-ALL patients at Shanghai Children’s Medical Center, in order to investigate the pathogenesis of T-ALL, as well as to clarify the characteristics of NOTCH1 mutations and the clinical significance in pediatric T-ALL patients. In addition, another purpose of the present study is to screen substrate-specific shedding inhibitor of Notch1 and detect its specificity against Notch1 shedding and its effects in T-ALL cells, in order to develop a new adjuvant therapy targeting Notch1 with high specificity and low toxicity.Methods 78 pediatric T-ALL patients diagnosed and treated at the Department of Hematology and Oncology of Shanghai Children’s Medical Center during January 2009 to December 2014 were randomly selected and included in the present study. Sanger sequencing after PCR was performed to screen the mutations within the heterodimerization(HD) domain and the proline-glutamicacid-serine-threonine(PEST) domain(encoded by exons 26, 27, 28 and 34) of the NOTCH1 gene, in order to explore the frequency, positions and types of the NOTCH1 mutations, as well as their correlation with disease prognosis in Chinese T-ALL children. The Notch1 activities in the patients were detected by Western blot, and their clinical correlation was also analyzed. Rabbit polyclonal antibody against the S2 cleavage site of Notch1 or mouse monoclonal antibody against the same epitope were prepared, and the effects of these antibodies in T-ALL cells were detected.Results 43 NOTCH1 mutations have been identified in the 78 T-ALL children, with an incidence of 55.13%(43/78), 12 of which were novel mutations not reported previously. 30 out of the 43 mutations were located in the HD domain only, 3 were in the PEST domain only, while 10 were in both the HD and PEST domains. The 1-year remission rate of all the followed-up patients was 66.67%(46/69). No significant difference(P = 0.9678) on the 1-year remission rate was observed between the Notch1-mutant group(85.37%, 35/41) and the non-mutant group(85.71%, 24/28). Results from western blot showed that the rabbit polyclonal antibody targeting the S2 cleavage site could bind to Notch1 specifically. Results from flow cytometry showed that the polyclonal antibody could bind T-ALL cell lines that have been pretreated with DLL4, a Notch1 ligand, and promote apoptosis of the T-ALL cell, suggesting that the rabbit polyclonal antibody could inhibit the activation of Notch1 signaling pathway by specifically and effectively inhibiting Notch1 shedding, leading to the inhibition of T-ALL cell proliferation. Similarly, the mouse monoclonal antibody against the same epitope also showed specifity and efficacy against Notch1 shedding and T-ALL cell proliferation, as evident by the results from western blot and flow cytometry.Conclusion The pediatric T-ALL patients had a high incidence of NOTCH1 mutations at various sites, which were correlated with the clinical characteristics and prognosis of the patients. The patients with NOTCH1 mutations had higher disease severity at diagnosis, but a similar short-term prognosis. Although the short-term recurrence rate of the Notch1-mutan group has a lower tendency compared to the non-mutant group, patients in the mutant group had a poor outcome of salvage therapy. The rabbit polyclonal antibody and mouse monoclonal antibody targeting the S2 cleavage site could specifically bind to Notch1 and effectively inhibit T-ALL cell proliferation.