In the mouse plasmacytoma system, we have documented that at least 4-5 genes are involved in determining the susceptibility patterns seen in BALB/cAn mice. Three of these genes, Pctr1-3, reside in the mid and distal portions of mouse Chr4. The Pctr1 region shares linkage homology with human 9p21 and 1p31. The Pctr2 and 3 regions in the mouse also share linkage homology with human Chr 1p, abnormalities of which have been documented in a variety of human cancers, including breast, neuroblastoma, melanoma, Burkitt's lymphoma and multiple myeloma.The cyclin dependent kinase inhibitor Cdkn2a (p16, p19) was mapped within the Pctr1 critical region. Two sequence variants were identified between susceptible and resistant mice. Site-directed mutagenesis experiments of Cdkn2a were performed to construct GST-fusion proteins containing the allelic variants associated with the susceptible strain. These proteins did not inhibit phosphorylation of RB in kinase assays and did not bind to CDK4-CyclinD2 complexes. Furthermore, when the DBA/2 p16 variant was transfected into plasmacytoma cell lines, many cells were blocked at G1. In contrast, cells transfected with the BALB/c variants continue to cycle. Plasmacytomagenesis is accelerated in knock-out mice. Furthermore, BALB/c p16 fails to suppress ras-transformation of NIH3T3 cells. DBA p16, as well as, both BALB and DBA p16 and p19 are capable of suppressing ras transformation. These data strongly implicate the Cdkn2a locus as the Pctr1 tumor susceptibility gene. Genetic variation was also found in the p16 promoter and differences in promoter activity have been associated with one of the variants. The ras related element binding protein, RREB was found to bind preferentially to the BALB promoter and acts to repress p16 expression. We have developed siRNA vectors to knock-down RREB expression. We are evaluating RREB inhibition as a potential molecular target for reactivation of the p16 tumor suppressor gene as a potential treatment for these B cell tumors. Progress has been made in generating relevant knock-in alleles of p16 for phenotyping and validation;mice are currently being evaluated in tumorigenicity assays. Several plasma cell tumor cell lines were treated with zebularine and gene expression profiles were generated and some of the genes differentially regulated have been validated. In the Pctr2 region, a series of tumor induction studies coupled with allelotypic analyses of a series of recombinant congenic strains have narrowed the genetic interval harboring the susceptibility/resistance gene to 1-2 cM. The Pctr2 locus was found to act in a codominant fashion in studies of F1 hybrids between resistant and susceptible congenic strains of mice. Phenotypic effects of the Pctr2 locus were evident in delaying the onset of tumorigenesis and reducing tumor incidences. Sample sequencing and EST matches identified at least 11 new genes in the Pctr2 interval. These genes were evaluated as candidates for the Pctr2 locus. Only one of the genes (Frap, mTOR) differs in amino acid sequence between BALB/c and DBA. Functional assays have demonstrated a defect in kinase activity associated with the susceptible allele. Both p53 and Phas1 are downstream substrates of FRAP kinase activity and for both substrates, BALB/c mTOR did not phosphorylate the proteins as well as DBA/2 mTOR. Additional studies have shown that BALB/c mTOR does not suppress ras transformation as effectively as DBA/2 FRAP. Subsequent studies with the mTOR inhibitor, rapamycin, have demonstrated that plasmacytoma cells are non-responsive to rapamycin. Because mTOR knock-outs are embryonic lethal, we generated a viable hypomorphic mouse by neo-insertion that partially disrupts mTOR transcription and creates a potential physiologic model of mTORC1/TORC2 inhibition. Homozygous knock-in mice exhibited reductions in body, organ and cell size. Although reductions in most organ sizes were proportional to decreased body weight, spleenswere disproportionately smaller. Decreases in the total number of T cells, particularly memory cells, and reduced responses to chemokines suggested alterations in T cell homing/homeostasis. TCR-stimulated T cells proliferated less, produced lower cytokine levels and expressed FoxP3. Decreased neutrophil numbers were also observed in the spleen, despite normal development and migration in the bone marrow. However, B cell effects were most pronounced, with a partial block in B cell development in the bone marrow, altered splenic populations, and decreases in proliferation, antibody production and migration to chemokines. Moreover, increased AKTSer473 phosphorylation was observed in activated B cells, reminiscent of cancers treated with rapamycin, and was reduced by DNA-pk inhibitors. Thus, mTOR is required for the maturation and differentiation of multiple immune cell lineages. These mice provide a novel platform for studying consequences of constitutively reduced mTORC1/TORC2 activity.
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