Screening Key Target Genes Of MLL And Studying On The Underlying Mechanisms

[Background and objective]:The competition and imbalance growing state between leukemia stem cells and normal hematopoietic stem cells is the crux of therapeutical approaches and outcome of leukemia as accompanying the whole process of generation and development of leukemia. The latest research reveals that PI3K/PTEN/AKT/mTOR signaling pathway may be a new target for correcting the imbalance between leukemia stem cells and normal hematopoietic stem cells. Two generations of mTOR complex inhibitor have been developed, however, the practical clincal applications have received unfavorable outputs. In present study, we use cytoblot high throughput assay to identify cell-specific inhibitor of mTOR complex 2. Then to investigate the degradation effect of protein Rictor in parts of MLL cell lines induced by lead compound Z1, and to supply some theories for gene targeted therapy to MLL. This study was designed to investigate the role of Rictor in the colony forming, proliferation and apoptosis-regulation of MLL leukemic cells in vitro. To study the effect of Rictor in the pathogenesis and maintain of leukemia in vivo.[Methods]:1. Searching for specific inhibitor of mTOR complex 2 by developing a high-throughput cytoblot assay:specific inhibitor of mTOR complex 2 was screened from small molecule monomeric compound library of NCPC new drug research center by using cytoblot high throughput assay. Western blot was then used to determine the inhibiting effect of compound against the key component protein in mTOR complex.2. An approach to the mechanism of lead compound Zl in degradation protein Rictor: THP-1 cell line was taked as the cell model, Western blot was employed to detect the expressions of protein rictor after the treatment of CHX and Z1-MG-132-combined treatment, respectively; Luciferase-Proteasome-Glo Cell-Based Assay was used to measure the poteasome activity changes of THP-1 after treated by Zl; ubiquitylation in vitro model was constructed, and the situation of ubiquitylation in protein rictor was also detected.3. Study on the role of Rictor to MLL Ieukemic cells in vitro:Rictor-RNA interference (RNAi) by transcription of short hairpin RNAs (shRNAs) from a lentiviral system was applied to silence Rictor gene expression in MLL leukemic cells, non-MLL leukemic cells and normal cells; and then GFP-Rictor-RNAi cells were isolated by Flow cytometry using the targeting-GFP in this lentivirus RNAi system; after established by flow cytometry, cellular immunofluorescence and Western blot, methyl cellulose colony forming assay was used to detect the differences among these Rictor-RNAi groups. Meanwhile, the proliferation dynamics of DDAO-SE labeled cells was detected by flow cytometry and analyzed with ModFit software, and flow cytometry was also used to observe apoptosis of each cell group.4. Preliminary Study on the influence of Rictor in the pathogenesis and maintain of MLL leukemia in vivo:Mouse model of hukemia was established in NOD/SCID mice. To evaluate the effect of xenograft by dynamic monitoring the percentages of hCD45 positive cells and hCD45/GFP double positive cells in mice peripheral blood, meanwhile, it could be used as a means for evaluating transplant rates of group Rictor-RNAi distinguishing from the other two control groups. At the end point of observation post transplantation, the aspects, the sizes of livers and spleens, also with the number of tumor nodes in liver and spleen were compared among each group. Flow cytometry was used to analyze the percentages of hCD45/GFP double positive cells in bone marrow, liver and spleen. The expression of the hCD45 in the livers, spleens, kidneys and lungs of the mice post transplantation was detected by immunocytochemistry. The rearrangements of MLL gene in mice peripheral blood was detected by RT-PCR. The survival curve of THP-1 transplantation mice was draw, and used to evaluate the differences of survival time among each group by Kaplan-Meier method.[Results]:1. The screen results indicated that compound Z1 had selectively block activation on Rictor protein which was a conserved component of mTOR complex 2, this phenomenon appeared only in parts of MLL leukemia cell lines. Meanwhile, only decreased level of phosphorylated mTOR complex 2 downstream molecules (such as p-AKT ser473) was appeared in other leukemia cell lines treated by compound Z1.2. No degradation effect of rictor in THP-1 was detected after the treatment of 10μmol/L cycloheximide in different time points by western blot; MG-132 could reverse the degradation effect of rictor in THP-1 cell line by Z1; the activity of poteasome in THP-1 was inhibted by Z1; GST targeting Rictor fragment could be ubiquitylated by THP-1 S-100 fraction which was treated by Z1.3. After certified by flow cytometry, the positive rate of each sorted cell group was over 90%, meanwhile, both of cellular immunofluorescence and Western blot confirmed that protein Rictor was blocked obviously; colony forming ability of group Rictor-RNAi in MLL leukemic cells was found to significantly lower than group NC-RNAi (P< 0.001) There was no significant difference in colony forming ability between Rictor-RNAi and NC-RNAi groups in non-MLL leukemic cells and normal cells (P>0.05). Furthermore, proliferation of group Rictor-RNAi in MLL leukemic cells was also found to significantly lower than group NC-RNAi (P< 0.01).The effects of Z1 in combination with proteasome inhibitor MG-132 reversing the induced apoptosis by Z1 disappeared in group Rictor-RNAi of MLL leukemic cells, not in non-MLL leukemic cells or normal cells.4. The results of dynamic monitoring showed that the percentages of hCD45 positive cells in THP-1 xenograft mice peripheral blood increased with the increase of storage time, and it illustrated the success of this transplantation model. The percentages of hCD45 positive cells in THP-1 infected Rictor-RNAi group was not evidently different from those in the THP-1 blank group or THP-1 infected NC-GFP group during the nine-week dynamic monitoring period (P>0.05). However, the percentages of hCD45 positive cells in THP-1 infected Rictor-RNAi group had became significantly lower than those in the THP-1 blank or NC-GFP group from the seventh week (P<0.01). At the end point of observation post transplantation, the aspect of Rictor-RNAi group mice was better than that of THP-1 blank or NC-GFP group. Meanwhile, the sizes of the livers in Rictor-RNAi group were smaller than those in other two groups, and the numbers of liver tumor nodes in Rictor-RNAi group were less than those in other two groups. The percentages of hCD45/GFP double positive cells in mice liver nodes, spleens and bone marrow of Rictor-RNAi group were significantly lower than those of NC-GFP group(P<0.01).The analysis by Kaplan-Meier method showed that survival time of Rictor-RNAi group was longer than those of control groups (P=0.033).[Conclusion]:1. Taking cytoblot high throughput assay, a specific small molecule monomeric compound was found out which could degrade a conserved component of mTOR complex 2—Rictor selectively, and it will provide new target molecules for targeted gene therapy of MLL leukemia.2. A highly conserved protein Rictor is stable in normal situation, and the protein rictor could be degradated by ubiquitin-proteasome pathway(UPP), the degradation is likely to be achieved through the activation of E3 ubiquitin ligase by Z1, and has no relationship with the activation of proteasome.3. Rictor could play a critical role in the colony forming, proliferation and apoptosis regulation of MLL leukemia. It revealed that Rictor might be a potential key target gene in target therapy for MLL leukemia.4. Rictor was necessary for the pathogenesis and maintain of MLL leukemia in vivo.