Assistant Professor Cleveland Clinic Lerner Research Institute, United States
Introduction: There exists, as of yet, no genetically engineered mouse model (GEMM) with which to simultaneously investigate multiple genetic perturbations in vivo. This is due to (i) lack of a non-leaky germinal center B cell specific Cre mouse, and (ii) lack of an inducible Cre mouse that can simultaneously induce dysregulation of multiple genes in the adult animal. Many genes of interest in MM are either embryonically lethal or cause other neoplasia development in AID-Cre, KRASG12D mice and in Cγ1-Cre, NRASQ61R mice leading, in the latter, to death prior to overt MM development. For these reasons, studies of MM tumorigenesis in the conditional transgenic mouse have been severely restricted by the “leakiness” of AID-Cre mice and Cγ1-Cre mice currently in use. There is therefore an urgent need for a newer system with which to activate genetic mutations or their expression simultaneously in the germinal-center B-cells only in adult mice.
Methods: We recently created the brand new AIDCreERT2 +/-, EYFP LSL/-, P53 L/L, KrasG12D LSL/-, cMYCLSL/- (AEY-PKM) mouse model, with germinal center B cell restricted overexpression of KrasG12D and cMYC, along with homozygous deletion of P53. Unlike AID-Cre+/-mice, AIDCreERT2 +/- mice express Cre recombinase under the control of an AID promoter and tamoxifen.
Results: Mice carrying a combination of these traits developed clonal expansion of clonal CD138+ MM cells in the bone marrow and experienced MGUS and MM as they aged (100% of mice after 6 months of age). These studies are the first demonstration of the driving impact of this combination of genes in MM development. AEY-P(K/Q/V)M mice required only ~5 months to develop MGUS and then MM. By comparing the WGS data from these murine MM cell lines, we found most of them to consist of missense mutations and C to T mutations indicative of the germinal center AID process in these cell lines. Fewer secondary genetic mutations are needed for transformation in AEY-PKM and AEY-PM cells to MM cells in comparation with VK*MYC MM cell lines. We also identified similar common mutations in all four mouse myeloma cells lines, including for example Sp140, which was also found in recently published MM patients’ data. In our 10X scRNA seq studies using bone marrow samples from 3 AEY-PM, 3 AEY-PKM, 3 AEY-PQM and 3 AEY-PVM bone marrow cells to identify distinct gene signatures from different immune cell populations. We stratified bone marrow immune cells from MM patients into 19 cell clusters. By means of scRNA seq analysis, we then identified the topmost upregulated and downregulated genes in each immune cell cluster. In combination with 5’BCR and 5’TCR seq, we also verified that all the MM tumor developed from AEY-PM, AEY-PKM, AEY-PQM, and AEY-PVM were IgG1 type.
Conclusions: Our newly generated third generation AEY-PK/V/QM mice with short latency and high penetrance are feasible for preclinical studies without the need of transplantation avoided potential Graft vs. Host Disease (GVHD) from different mice.
