Attempts to Induce t(11;14) during Redifferentiation of Normal B Cell-Derived Ips Cells to Study Myeloma-Initiating Cells

The cellular origin of multiple myeloma (MM) has not yet been identified. Based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomies and chromosomal translocation (cTr) play a critical role in the early tumorigenesis of MM. We hypothesized that the abnormal cells from which myeloma cells originate might be mature B cells with chromosomal or genetic changes in the reprogrammed state that enable them to acquire the potential to become malignant tumors in the process of redifferentiation into B cells. We established induced pluripotent stem cells (iPSs) from normal B cells (BiPSCs: BiPSC13 and MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B cells (Sci Rep, 2017). We then established a method to induce reciprocal cTr t(11;14), which is a reciprocal cTr between IgH and CCND1 and the most frequent cTr in MM, using the CRISPR/Cas9 system; cTr was induced by infection of IgH-CCND1lentiCRISPRv2 lentivirus, which targets the human IgH Eµ region and 13 kb upstream of the CCND1 coding sequence, in BiPSCs (Oncol Lett, 2019). Subsequently, we established cell lines carrying reciprocal cTr t(11;14) between CCND1and either an allele in which VDJ rearrangement of IgH had been completed or an allele in which VDJ rearrangement had not been completed (stopped at DJ joining) in BiPSC13 t(11;14) （AZ and AX）and MIB2-6 t(11;14) (BC and BG), respectively. These BiPSCs differentiated into CD34⁺/CD38^-/CD45^+/-/CD43^+/- hematopoietic progenitor cells in stem cell differentiation medium; this was subsequently confirmed by the differentiation into granulocytes, macrophages, and erythroblasts in a colony-formation assay (Sci Rep, 2021).

We are now trying to produce BiPSCs in which cTr t(11;14) is induced when they differentiate into mature B cells expressing CD19. First, we tried the Cre-loxP recombination system to induce cTr t(11;14) in MIB2-6. MIB2-6 were transfected with an IgH loxP-Neo-loxP knock-in vector and IgH lentiCRISPRv2 vector. Subsequently, G418-resistantMIB2-6 were transfected with iCre-EGFP. After removing the loxP-Neo site from EGFP-positive cells, MIB2-6 carrying IgH-loxP were transfected with CCND1 loxP-FRT3-Neo-FRT3 knock-in vector and CCND1 lentiCRISPRv2 vector. Subsequently, G418-resistant MIB2-6 were transfected with Flpo-EGFP. After removing the FRT3-Neo site from EGFP-positive cells, MIB2-6 carrying IgH-loxP in IgH gene and CCND1-loxP-FRT3 in CCND1 gene (MIB2-6 with the Cre-loxP system) were transfected with iCre-HygR. We then confirmed that the Cre-loxP recombination system works by detecting the reciprocal cTr t(11;14) in hygromycin B-resistant cells using PCR.

Next, we developed a promoter trap for the CD19 gene system in which Cre is expressed with CD19 in MIB2-6 cells by transfecting a CD19 exon 1-Cre FRT3-Puro-FRT3 knock-in vector or a CD19 exon 1-EGFP FRT3-Puro-FRT3knock-in vector with CD19 exon 1-targeting lentiCRISPR v2 vectors. We then established MIB2-6 carrying the Cre-loxP system and CD19-promoter trap with Cre or EGFP gene after removing the FRT3-Puro site from puromycin-resistant cells. We also confirmed that CD19-promoter trap with EGFP gene works in Raji cells. Therefore, if this modified MIB2-6 can be differentiated into B cells, the reciprocal cTr t(11;14) could be induced and be useful for studying myeloma-initiating cells. The final challenge will be to determine how to induce them to differentiate into CD19⁺ cells.

Ikezoe:AsahiKasei Pharma: Research Funding; Nippon Shinyaku Co., Ltd: Research Funding.