Abstract
Background and Aims: Although recent therapeutic advances have led to an improvement in the outcome of Multiple Myeloma (MM), it still remains an incurable disease, and therefore, new drugs with novel mechanisms of action are needed for myeloma patients. Zalypsis is a new synthetic alkaloid derived from certain marine compounds which has demonstrated significant in vitro and in vivo antitumor activity in different malignancies. It is currently under late Phase I development in solid tumours, with preliminary evidence of activity. In this study, we have analysed the preclinical activity and mechanism of action of Zalypsis in MM.
Material and methods: Nine different MM cell lines and BM samples from MM patients and normal donors were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and gene expression profile analysis. The in vivo activity was explored in a human subcutaneous plasmocytoma model and immunohistochemistry was performed in selected tumours.
Results: Zalypsis turned out to be the most potent antimyeloma agent we have tested so far in our laboratory, with IC50s in picomolar or low nanomolar ranges depending on the cell lines studied. Interestingly, the sensitivity to Zalypsis was independent of the pattern of resistance of the cell lines to conventional antimyeloma agents such as Dexamethasone or Melphalan. It also showed remarkable ex vivo potency in freshly isolated plasma cells from six patients (including two with plasma cell leukemia) and synergized with many other antimyeloma compounds, being the combination of Zalypsis + Lenalidomide + Dexamethasone particularly attractive. Regarding toxicity, Zalypsis preserved the CD34+ hematopoietic progenitor cells from MM and normal donor BM samples. This remarkable activity prompted us to investigate the mechanism of action of the drug. Besides the induction of apoptosis and cell cycle arrest, Zalypsis provoked DNA double strand breaks, which were evidenced by an increase in phospho Histone H2AX and phospho CHK2, followed by a striking overexpression of p53 in MM cell lines bearing wild type forms of this protein. Of note, no other compound currently used in the MM clinic induced such an increase in p53 protein levels. In addition, in a subset of MM cell lines in which p53 was mutated, Zalypsis also provoked DNA double strand breaks and induced cell death, although higher concentrations were required. Changes in the gene expression profile of MM cells treated with Zalypsis were concordant with these results, since important genes involved in DNA damage response were deregulated. This include genes implicated in the ATM repair pathway, such as TLK2, ATR, CHEK2, RAD5 and BRIP1 and other mRNAs related to DNA repair, such as RAD23B, XPC, XRCC1, XRCC5 and GADD45A. These results were confirmed in vivo in a model of human subcutaneous plasmocytoma in SCID mice. Zalypsis (0.8 and 1 mg/Kg) decreased tumour growth and improved survival of mice implanted with MM1S (wild type p53) and OPM-1 (mutated p53) plasmocytomas. Immunohistochemical studies in tumours from treated animals also demonstrated DNA damage with H2AX phosphorylation and p53 overexpression.
Conclusion: The potent in vitro and in vivo antimyeloma activity and the singular mechanism of action of Zalypsis uncovers the high sensitivity of tumour plasma cells to double strand breaks, and strongly supports the potential use of this compound in multiple myeloma patients.
Association of Rad9 with Double-Strand Breaks through a Mec1-Dependent Mechanism
