Autophagy Is a Key Myeloma Survival Pathway That Can Be Manipulated Therapeutically to Enhance Apoptosis

Abstract

4083 In normal cells, autophagy is up-regulated under conditions of stress ensuring cell survival, suggesting that if it is inhibited cell death will ensue. In some cancer settings however it has been shown to enhance cell death. Therefore if autophagy is to be a target for anti-cancer therapy, it is important to determine whether to promote or inhibit the process. Cellular entry to autophagy can be manipulated via the PI3K/AKT/mTOR pathway, a pathway known to be important for myeloma cell growth and survival. In this study we investigated the effects of PI-103, a dual Class I PI3K and mTOR inhibitor, which activates autophagy, and used it as a tool to investigate the interaction of autophagy with other myeloma therapies. As the PI3K/AKT/mTOR pathway has been suggested to be a central pathway controlling entry into autophagy, we first determined the basal expression of key members of the pathway in a panel of myeloma cell lines. PI3K alpha, beta delta and gamma isoforms were expressed to varying degrees and constitutive activation of the pathway, (pAKT or pMTOR), was seen in the majority of cell lines. As predicted, treatment with PI-103 induced autophagy in myeloma cells as demonstrated by an increase in cellular inclusions staining positively with acridine orange, cleavage of the autophagosome marker, LC3, and a decrease in p62. PI-103 was shown to inhibit proliferation of all the myeloma cell lines and patient cells tested to varying degrees, although 100% growth inhibition was not seen. Bone marrow stromal cells were unaffected. The main mode of action of PI-103 was autophagy activation, and in keeping with this the extent of cell death measured by Annexin V/PI binding and trypan blue exclusion, was minimal. Cell cycle analysis demonstrated an increase in G0-G1 phase. The unfolded protein response (UPR) is important in myeloma cells and UPR activation and autophagy have been reported to be interlinked. Following exposure to PI-103, splicing of XBP1 mRNA to its active form, XBP1s, was seen, CHOP and ATF4 mRNA levels were also increased, consistent with activation of at least two branches of the UPR in response to PI3K/mTOR inhibition and autophagy induction. As myeloma cells activate autophagy as a pro-survival pathway following PI3-kinase inhibition, we were interested to understand the effect of blocking autophagy in this context. When PI-103 was combined with the autophagy inhibitor, Bafilomycin greatly enhanced apoptosis was seen. This increased apoptosis was seen in cells constitutively expressing p-AKT, with a complete loss of both phospho- and total levels of AKT and mTOR, an increase in the cleaved forms of caspase 3 and Bcl2, and massive activation of the IRE1 and PERK branches of the UPR. This effect was not seen in cells lacking p-AKT, a phenomenon described as ‘context-dependent oncogene addiction' suggesting that measurement of p-AKT may be a useful predictive marker for response to joint PI3K/autophagy inhibition. Importantly the pro-apoptotic effects of the combination are not overcome by the presence of bone marrow cytokines, a more representative model of the physiological situation in vivo. In conclusion our data highlights the interplay between known myeloma growth and survival pathways and autophagy and suggests that combining PI3K inhibitors, with agents that target autophagy, may be beneficial for the treatment of myeloma, particularly in >50% of patients that express p-AKT. Disclosures: No relevant conflicts of interest to declare.

The Genetic Architecture of Multiple Myeloma

“…AKT then subsequently activates several downstream targets including mTOR, GSK-3B and FKHR which influence many processes including cell proliferation and apoptosis resistance. Deregulation of the PI3K pathway is thought to be important in myeloma as phosphorylated AKT, an indicative marker of pathway activity, is observed in approximately 50% of cases [ 101 ]. Additionally, DEPTOR, a positive regulator of the PI3K pathway is commonly upregulated in myeloma, especially in those with MAF translocations [ 102 ], further demonstrating pathway activity.…”

The novel autophagy inhibitor elaiophylin exerts antitumor activity against multiple myeloma with mutant TP53 in part through endoplasmic reticulum stress-induced apoptosis

“…15,16 High levels of autophagy have been observed in both MM cell lines and primary MM samples. 17,18 However, too little or too much autophagy may be deleterious. This biologic process, which makes MM stronger, can also kill it.…”

Modulation of Apoptosis Pathways in the Biology and Treatment of Multiple Myeloma

“…42 Deregulation of the PI3K pathway is thought to be important in myeloma as phosphorylated AKT, an indicative marker of pathway activity, is observed in approximately 50% of cases. 48 High basal level endoplasmic reticulum (ER) stress in multiple myeloma induces downstream targets of AKT pathways, like NF-kB and phosphorylation of MDM2, which inhibits nuclear localization and degradation of p53 with a related decrease in expression of p53 target genes, such as BAX. 17 AKT activation also negatively regulates proapoptotic proteins, BAD; caspase 9; forkhead family members, which induce CD95L like FKHR, FKHRL-1 and AFX; and glycogen synthase kinase 3.…”