JNK activation and Fas up-regulation precede proteasomal degradation of topoisomerase I in SN38-mediated cytotoxicity against multiple myeloma.

Abstract Topoisomerase I is an essential constitutive nuclear enzyme which is expressed in multiple myeloma (MM) cells as well as bone marrow stromal cells (BMSCs). SN38 is the highly potent active metabolite of topoisomerase I inhibitor irinotecan. Our investigations demonstrated significant differential toxicity of SN38 against MM cell lines and patient cells versus BMSCs. SN38 directly inhibited constitutive and inducible IL-6 and VEGF secretion by BMSCs, despite minimal impact upon BMSC viability, consistent with the S-phase specificity of camptothecin-induced apoptosis. In contrast to conventional therapy, adhesion of MM cells to BMSCs failed to confer tumor cell resistance to SN38. Proteasomal degradation of topoisomerase I in response to formation of topoisomerase I-cleavable complexes has been proposed as a resistance mechanism to camptothecins in solid malignancies. In our studies, proteasomal degradation of nuclear topoisomerase I was observed in MM.1S cells, but failed to protect from apoptosis. Moreover, RPMI-derived doxorubicin resistant Dox40 cells and dexamethasone-sensitive MM.1S cells were equally sensitive to SN38; in contrast to MM.1S, Dox40 displayed very little evidence of topoisomerase I degradation even at high concentrations of SN38. Furthermore, SUDHL4 lymphoma cells displayed significant resistance to SN38 despite lack of proteasomal degradation of topoisomerase I, indicating that proteasomal degradation is not a consistent mechanism of resistance to topoisomerase I inhibition in hematological malignancies. By elucidating the mechanisms of apoptosis downstream to SN38-induced DNA damage, approaches to augment the anti-tumor activity of this class of compounds may be revealed. We identified several intracellular signals translating topoisomerase I inhibition into apoptosis in MM. In MM.1S cells, SN38 at physiological concentrations induced JNK activation within 2 hours and cell surface Fas up-regulation that was maximal at 6–8 h, sustained for at least 24 h, and closely followed by cleavage of pro-caspase 8 and pro-caspase 3 independently from caspase 9. These pro-apoptotic signaling events were sustained despite proteasomal degradation of topoisomerase I, which occurred after JNK activation. Importantly, exogenous Fas activation by Fas-activator CH11 in combination with SN38 resulted in synergistic toxicity to MM cells. Downstream from caspase 8, PARP is an important DNA repair enzyme. PARP inhibitors have already shown significant activity in animal models, may augment chemotherapy, and may be potentially useful in combination with topoisomerase I inhibitors. In our experiments, PARP inhibition by NU1025 augmented SN38-induced apoptosis in a synergistic fashion, providing the rationale to target PARP in order to enhance the anti-tumor activity of DNA damaging agents in general, and irinotecan in particular. These findings have clinical significance because identification of downstream apoptotic signaling following topoisomerase I inhibition will both elucidate mechanisms of resistance and optimize future combination chemotherapy against MM. Furthermore, enhanced activity in combination with novel chemotherapeutic agents will provide the framework for future clinical trials of topoisomerase I inhibitors in MM.