ABSTRACT

The studies included in this PhD dissertations were carried out at the Dept. of Hematology and Clinical Biochemistry, H:S Rigshospitalet, Copenhagen.

Despite high dose chemotherapy and autologous transplantation multiple myeloma is incurable. Supportive treatment with bisphosphonates has ameliorated the invalidating myeloma bone disease. Apart from the palliative effect, recent experimental in vitro data indicated an additional antineoplastic effect of amino-group containing BP-derivates (N-BP). However, clinical data are more inconsistent.

The study was designed to clarify the discrepancy between the observed in vitro efficiency and the lack of proven in vivo anti-tumor effects in order to determine the antineoplastic potential of N-BP in the treatment of multiple myeloma. First, the in vitro effects of N-BP on myeloma cells were verified and characterized. N-BP induced apoptosis and inhibited proliferation of the multiple myeloma cell lines in a time and dose dependent manner. N-BP treated myeloma cells showed typical signs of apoptosis, i.e. cleavage of the caspase-3 substrate PARP, externalization of phosphatidylserine, fragmentation of DNA, and typical changes of cellular morphology. However, upon continuous exposure to N-BP myeloma cell lines developed resistance to the antineoplastic effects. This de novo N-BP resistance resulted at least in part from upregulation of the enzyme activity of the N-BP target enzyme farnesyl pyrophosphate synthase (FPPS), which was not due to increased gene transcription as measured by quantitative PCR. Intracellular drug accumulation was unchanged and both extrinsic and intrinsic apoptotic signaling pathways were functional. Of note, N-BP resistance did not induce cross-resistance to conventional myeloma chemotherapy. While investigating the resistance mechanism a high-resolution gel electrophoresis with albumin calibrators was developed, that allows quantifying free light chains (FLC) in cell culture supernatant and urine and has been implemented in routine procedures.

In conclusion, the presented data demonstrate that N-BP do have impressive anti-tumor effects in vitro, but that the clinical antineoplastic application is limited by the development of resistance. Fortunately, the observed de novo N-BP resistance derives from an alteration of the molecular drug target and thus does not impair conventional therapy through cross resistance.