Studies of overcoming acquired resistance : molecular mechanisms and development of novel drugs

Abstract: Chemotherapeutic agents have become widely applied for treatment of various types of malignancies. Drug resistance unfortunately remains as a major obstacle for the effectiveness of chemotherapy. Cancer drug resistance includes two broad categories: intrinsic and acquired. In this thesis I have examined the problem of acquired drug resistance and have aimed to develop novel approaches to overcome acquired resistance. Clofarabine is a second-generation nucleoside analogue which has been employed primarily for the treatment of hematological malignancies. In paper I, we found that clofarabine inhibited [14C]-thymidine uptake, presumably by decreasing DNA synthesis. Clofarabine was also found to induce apoptosis of a solid tumor cell line, a finding which may open new fields of application of this drug. In paper II, 5’-nucleotidases, a family of enzymes known to confer resistance to nucleoside analogues, were found to be expressed at various levels in samples from CLL patients. Moreover, degradation of fludarabine monophosphate was found to be associated with CN2 activity, and degradation of cladribine monophosphate was associated with CN1 activity. This result helps to explain why some leukemic patients may show resistance to different nucleoside analogues. In paper III, we screened the RPMI 8226 myeloma cell line and its multidrug resistant subline 8226/Dox40 for the response to 3,000 chemically diverse compounds. We found one compound, designated VLX40, which showed strong cytotoxicity to the drug resistant cells. VLX40 was found to be cytotoxic to myeloid and lymphoid leukemia cells. Mechanistic studies showed that VLX40 is a novel microtubule inhibitor. The efficacy of the compound may potentially be improved leading to the development of novel tubulin active agents that are insensitive to common mechanisms of cancer drug resistance. b-AP15 is a novel small molecule inhibitor of the ubiquitin-proteasome system. b- AP15 inhibits the USP14/UCHL5 deubiquitinases of the 19S proteasome and shows anti-tumor activity in a number of tumor models. In paper IV, we examined the anti- multiple myeloma activity of b-AP15. We found that b-AP15 has significant efficacy in multiple myeloma (MM) disease models, including cells resistant to the proteasome inhibitor bortezomib. The finding provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in MM. In paper V, we show that despite the fact that b-AP15 is a reversible enzyme inhibitor, it induces rapid commitment to apoptosis/cell death. We show that the compound is rapidly taken up and enriched in cells, findings that explain the difference in potency of b-AP15 in biochemical and cellular assays. Based on the effective anti-cancer activity of b-AP15, we performed a lead optimization procedure aimed to identify efficient b-AP15 analogues with improved solubility. In paper VI, we identified the analogue VLX1570 which has similar biochemical activity as b-AP15. VLX1570 has strong antineoplastic activity in multiple myeloma cells and is capable of overcoming bortezomib resistance. We conclude that VLX1570 is a promising candidate for the clinical management of multiple myeloma.