Establishment and Characterization of a Novel Human Myeloma Cell Line, AMU-MM1, From a Multiple Myeloma Patient Involving Central Nerve System After Treatment with Bortezomib

Abstract 4991

Bortezomib improved the survival in patients with multiple myeloma (MM), however, cannot cure this disease even when it is combined with autologous stem cell transplant(s) and other new drugs. Myeloma cells cannot be excluded completely from a patient due to drug refractoriness and/or inefficiency of drug delivery in extramedullary sites such as cerebrospinal fluid (CSF). Therefore, it is important to clarify the mechanisms of bortezomib resistance and invasion to central nerve system (CNS) of myeloma for the progress of myeloma therapy.

In this study, we established a novel human myeloma cell line from a myeloma patient involved with CNS and analyzed its characters including sensitivity to bortezomib. The patient had been treated with bortezomib for 1.5 years when her CNS was involved with myeloma. The cells from patient's CSF were cultured in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS) and rhIL-6. After 3 months of culture, cell proliferation became continuous with 10% FBS and without rhIL-6. The cell line, named AMU-MM1 was established and negative for EBV. A doubling time of AMU-MM1 cells was about 48 hours. AMU-MM1 cells were positive for CD38, CD54, CD138 and cytoplasmic kappa chain and negative for CD19, CD20, CD33 and CD56 by flow cytometry analysis while those in patient's bone marrow were positive for CD56. AMU-MM1 showed hypo- and pseudodiploid karyotypes with t(4;14), t(8;13), t(1;19), del13q, amp1q21 and others but without del17p by cytogenetic analyses including FISH. The G322A mutation in the proteosome beta 5 subunit (PSMB5) gene, which is reported as a mutation found in bortezomib-resistant cell lines induced via repeated drug selection, was not detected in AMU-MM1 by direct sequencing. Apoptosis analysis using Annexin V/PI assay indicated that AMU-MM1 was sensitive to bortezomib. Our data suggest that AMU-MM1 was derived from the cells that invaded to CSF in which bortezomib concentration was very low and not resistant to bortezomib, and the downregulation of CD56 might play a role in the pathogenesis of CNS involvement as reported before. In addition, we are now focusing on new chimera or dysregulated genes in t(8;13), t(1;19) and other sites as well.

In conclusion AMU-MM1 is a useful cell line for analysis of mechanisms of CNS involvement and also possibly the acquired resistance to bortezomib.