Targeting Autophagy Augments In Vitro and In Vivo Antimyeloma Activity of DNA-Damaging Chemotherapy

Abstract

Purpose: Although autophagy occurs in most tumor cells following DNA damage, it is still a mystery how this DNA-damaging event turns on the autophagy machinery in multiple myeloma (MM) and how the functional status of autophagy impacts on its susceptibility to death in response to DNA-damaging chemotherapy.Experimental Design: We investigate the effects of DNA damage on autophagy in MM cells and elucidate its underlying molecular mechanism. Then, we examined the impacts of pharmacologic or genetic inhibition of autophagy on DNA damage-induced apoptosis. Furthermore, the antimyeloma activity of autophagy inhibitor in combination with DNA-damaging agents was evaluated in MM xenograft models.Results: We showed that DNA-damaging drugs, doxorubicin and melphalan, induce caspase-dependent apoptosis and concurrently trigger Beclin 1-regulated autophagy in human MM cell lines H929 and RPMI 8226. Mechanistically, association of autophagy execution proteins Beclin 1 with class III phosphoinositide 3-kinase, which is inhibited by Bcl-2 recruitment, contributes directly to the autophagic process. Importantly, targeting suppression of autophagy by minimally toxic concentrations of pharmacologic inhibitors (hydroxychloroquine and 3-methyladenine) or short hairpin RNAs against autophagy genes, Beclin 1 and Atg5, dramatically augments proapoptotic activity of DNA-damaging chemotherapy both in vitro using MM cell lines or purified patient MM cells and in vivo in a human plasmacytoma xenograft mouse model.Conclusion: These data can help unravel the underlying molecular mechanism of autophagy in DNAdamaged MM cells and also provide a rationale for clinical evaluation of autophagy inhibitors in combination with DNA-damaging chemotherapy in MM. Clin Cancer Res; 17(10); 3248-58. Ó2011 AACR.

Autophagy in drug resistance of the multiple myeloma cell line RPMI8226 to doxorubicin

“…In MM cells, hypersecretion of monoclonal immunoglobulin caused aggregation of numerous unfolded proteins, leading to endoplasmic reticulum stress and autophagy activation (Yang et al, 2011). In addition, a previous study (Pan et al, 2011) confirmed that DNAdamaging reagents such as DOX and melphalan can mediate the activation of autophagy. Based on this information, autophagy may be of great significance in the drug resistance of MM cells.…”

“…Autophagy inhibitors (8 mM hydroxychloroquine or 10 mM 3-methyladenine (Pan et al, 2011) were added to RPMI8226/DOX cells with DOX for 24 h to observe the effect of combined treatment on cells. The cells in each group were collected and adjusted to a density of 1 μM/mL.…”

“…In autophagy, cytoplasmic proteins and organelles that are injured or aged undergo degradation and are reconstructed as intracellular nutrients or macromolecules, such that the substances are recycled and organelles are renewed. Meticulous regulation of this process is very important for maintaining the balance of protein metabolism and intracellular homeostasis (Pan et al, 2011). Recent studies examining the relationship between autophagy and tumors have revealed some of the details of this process.…”

CD20 positive cells are undetectable in the majority of multiple myeloma cell lines and are not associated with a cancer stem cell phenotype

“…It is well-known that RPMI-8226 cells promote plasmacytomas in SCID mice. 23 However, whether this is due to a haematologica | 2012; 97(7) 0 1E3 1E4 1E5 0 1E3 1E4 1E5 CD19 CD27 CD38 CD45 CD56 CD138 CD19 CD27 CD38 CD45 CD56 CD138 +DEAB -DEAB CD20 -CD20 unfractionated (n=4) or CD20 depleted (n=4) RPMI-8226 cells into 8 CB17-SCID mice. We found that 100% of the mice in each group developed plasmacytomas without any difference in the growth pattern ( Figure 2C), suggesting that CD20 dim+ cells are not essential for tumor formation.…”

Targeting NAD+ salvage pathway induces autophagy in multiple myeloma cells via mTORC1 and extracellular signal-regulated kinase (ERK1/2) inhibition

“…7,8 Although apoptosis induction has been the major focus of research in novel MM therapies, a recent study documented a pivotal role for autophagy as a prosurvival mechanism in MM cells, suggesting its potential as an additional target for novel therapeutics. 9,10 Intracellular nicotinamide adenine nucleotide (NAD ϩ ) plays a major role in the regulation of several cellular processes. 11,12 In mammals, NAD ϩ is replenished from nicotinamide (Nam), tryptophan or nicotinic acid (NA), with Nam as the most important and widely available precursor.…”