Because accumulation of potentially toxic malfolded protein may be extensive in immunoglobulin-producing multiple myeloma (MM) cells, we investigated the phenomenon of autophagy in myeloma, a physiologic process that can protect against malfolded protein under some circumstances. Autophagy in MM cell lines that express and secrete immunoglobulin and primary specimens was significantly increased by treatment with the endoplasmic reticulum stress-inducing agent thapsigargin, the mammalian target of rapamycin inhibitor rapamycin, and the proteasome inhibitor bortezomib. Inhibition of basal autophagy in these cell lines and primary cells by use of the inhibitors 3-methyladenine and chloroquine resulted in a cytotoxic effect that was associated with enhanced apoptosis. Use of small interfering RNA to knock down expression of beclin-1, a key protein required for autophagy, also inhibited viable recovery of MM cells. Because the data suggested that autophagy protected MM cell viability, we predicted that autophagy inhibitors would synergize with bortezomib for enhanced antimyeloma effects. However, the combination of these drugs resulted in an antagonistic response. In contrast, the autophagy inhibitor 3-methyladenine did synergize with thapsigargin for an enhanced cytotoxic response. These data suggest that autophagy inhibitors have therapeutic potential in myeloma but caution against combining such drugs with bortezomib.

Although preclinical work with rapalogs suggests potential in treatment of multiple myeloma (MM), they have been less successful clinically. These drugs allostearically inhibit the mammalian target of rapamycin kinase primarily curtailing activity of the target of rapamycin complex (TORC)1. To assess if the mammalian target of rapamycin within the TORC2 complex could be a better target in MM, we tested a new agent, pp242, which prevents activation of TORC2 as well as TORC1. Although comparable to rapamycin against phosphorylation of the TORC1 substrates p70S6kinase and 4E-BP-1, pp242 could also inhibit phosphorylation of AKT on serine 473, a TORC2 substrate, while rapamycin was ineffective. pp242 was also more effective than rapamycin in achieving cytoreduction and apoptosis in MM cells. In addition, pp242 was an effective agent against primary MM cells in vitro and growth of 8226 cells in mice. Knockdown of the TORC2 complex protein, rictor, was deleterious to MM cells further supporting TORC2 as the critical target for pp242. TORC2 activation was frequently identified in primary specimens by immunostaining for AKT phosphorylation on serine 473. Potential mechanisms of up-regulated TORC2 activity in MM were stimulation with interleukin-6 or insulin-like growth factor 1, and phosphatase and tensin homolog or RAS alterations. IntroductionPreclinical data with mammalian target of rapamycin (mTOR) inhibitors such as rapamycin, temsirolimus, and everolimus suggest these drugs may have therapeutic potential in multiple myeloma (MM). [1][2][3] These mTOR inhibitors associate with the FKBP12 protein and together they bind to mTOR adjacent to its kinase domain. At this site, rapamycin allostearically inhibits the kinase, primarily that which is functional within the multiprotein complex kinase called target of rapamycin complex (TORC)1. 4 The TORC1 complex consists of mTOR associated with mLST8 and Raptor. 4 TORC1 phoshorylates the p70S6kinase (p70) and factor 4E binding protein 1 (4E-BP1) translational repressor and both these events stimulate translation of cell cycle proteins, thus promoting cell cycle transit. [5][6][7] By inactivating TORC1, these first generation mTOR inhibitors prevent cell cycle protein translation and induce G1 arrest. 8 Although some early results of phase I/II trials that use these mTOR inhibitors in combination with other anti-MM agents suggest modest efficacy, 9,10 use of tensilorimus as a single agent was relatively ineffective in MM patients. 11 Some possible reasons for these disappointing results are suggested by previous mechanistic studies. In particular, treatment of MM cells with rapamycin or temsilorimus only induces cell cycle arrest without induction of apoptosis. 1 Thus, as treated MM cells maintain viability, they may resume tumor growth during the time intervals between drug administration. One potential reason for lack of apoptosis is that there is a feedback activation of AKT when MM cells are treated with mTOR inhibitors. 12 Activated AKT could serve as an antiapoptotic pr...

Prior work indicates that c-myc translation is up-regulated in multiple myeloma cells. To test a role for interleukin (IL)-6 in myc translation, we studied the IL-6-responsive ANBL-6 and IL-6-autocrine U266 cell lines as well as primary patient samples. IL-6 increased c-myc translation, which was resistant to rapamycin, indicating a mechanism independent of mammalian target of rapamycin (mTOR) and cap-dependent translation. In contrast, the cytokine enhanced cap-independent translation via a stimulatory effect on the myc internal ribosome entry site (IRES). As known IRES-trans-activating factors (ITAF) were unaffected by IL-6, we used a yeast-threehybrid screen to identify novel ITAFs and identified hnRNP A1 (A1) as a mediator of the IL-6 effect. A1 specifically interacted with the myc IRES in filter binding assays as well as EMSAs. Treatment of myeloma cells with IL-6 induced serine phosphorylation of A1 and increased its binding to the myc IRES in vivo in myeloma cells. Primary patient samples also showed binding between A1 and the IRES. RNA interference to knock down hnRNP A1 prevented an IL-6 increase in myc protein expression, myc IRES activity, and cell growth. These data point to hnRNP A1 as a critical regulator of c-myc translation and a potential therapeutic target in multiple myeloma. [Cancer Res 2008;68(24):10215-22]

Background: Although the active site mTOR inhibitor pp242 overcomes feedback activation of AKT, it may still be complicated by feedback ERK activation. Results: In myeloma cell models, pp242 was more potent than rapamycin for activating ERK, causing resistance. Conclusion: Activation of ERK is a complication of pp242. Significance: PP242 would be more effective if used in combination with inhibitors of the ERK pathway.

Prior work indicates that IL-6 can stimulate c-Myc expression in multiple myeloma (MM) cells, which is independent of effects on transcription and due to enhanced translation mediated by an internal ribosome entry site in the 5-UTR of the c-Myc RNA. The RNA-binding protein hnRNP A1 (A1) was also critical to IL-6-stimulated translation. Because A1 shuttles between nucleus and cytoplasm, we investigated whether the ability of IL-6 to enhance Myc translation was mediated by stimulation of A1 shuttling. In MM cell lines and primary specimens, IL-6 increased A1 cytoplasmic localization. In contrast, there was no effect on the total cellular levels of A1. Use of a dominant negative A1 construct, which prevents endogenous A1 from nucleus-to-cytoplasm transit, prevented the ability of IL-6 to enhance Myc internal ribosome entry site activity, Myc protein expression, and MM cell growth. IL-6-stimulated cytoplasmic localization was mediated by alterations in the C-terminal M9 peptide of A1, and this correlated with the ability of IL-6 to induce serine phosphorylation of this domain. A p38 kinase inhibitor prevented IL-6-induced A1 phosphorylation. Thus, IL-6 activates c-Myc translation in MM cells by inducing A1 phosphorylation and cytoplasmic localization in a p38-dependent fashion. These data suggest A1 as a potential therapeutic target in MM.Our previous work (1) demonstrated that IL-6 could enhance c-Myc protein expression in multiple myeloma (MM) 2 cells independent of any effect on Myc transcription. This was not due to altered c-Myc protein stability or the mammalian target of rapamycin-dependent stimulation of cap-dependent translation. In contrast, it resulted from an up-regulation of Myc IRES activity and cap-independent translation. IRESes are structures in the 5Ј-UTR of transcripts that can promote translation and are especially important when RNA leaders are highly structured or when cap-dependent translation is inhibited (2, 3). The three-dimensional IRES structure recruits the 40 S ribosomal subunit to the mRNA for translation initiation. The Myc IRES is well characterized (4, 5). In addition, there are trans-acting proteins that facilitate Myc IRES function via binding to the RNA at sites in the 5Ј-UTR and inducing conformational changes that facilitate recruitment of the IRES to the ribosome (4). Several publications (6, 7) document a myeloma-specific up-regulation of Myc IRES function.The ability of IL-6 to stimulate Myc IRES function in MM cells, up-regulate rapamycin-resistant Myc translation and expression, and stimulate MM cell proliferation were all prevented by knockdown of hnRNP A1, an RNA-binding protein (1). Because other studies (8 -10) demonstrate that hnRNP A1 (A1) can function as an IRES-trans-activating factor (ITAF), regulating Myc IRES activity, these results collectively indicate that IL-6-up-regulated c-Myc translation in MM cells was dependent on enhanced A1 ITAF function.hnRNP A1 is a shuttling protein with movement from nucleus to cytoplasm (11), and a recent study (12) demonstrated ...

<div>Abstract<p>Because accumulation of potentially toxic malfolded protein may be extensive in immunoglobulin-producing multiple myeloma (MM) cells, we investigated the phenomenon of autophagy in myeloma, a physiologic process that can protect against malfolded protein under some circumstances. Autophagy in MM cell lines that express and secrete immunoglobulin and primary specimens was significantly increased by treatment with the endoplasmic reticulum stress–inducing agent thapsigargin, the mammalian target of rapamycin inhibitor rapamycin, and the proteasome inhibitor bortezomib. Inhibition of basal autophagy in these cell lines and primary cells by use of the inhibitors 3-methyladenine and chloroquine resulted in a cytotoxic effect that was associated with enhanced apoptosis. Use of small interfering RNA to knock down expression of beclin-1, a key protein required for autophagy, also inhibited viable recovery of MM cells. Because the data suggested that autophagy protected MM cell viability, we predicted that autophagy inhibitors would synergize with bortezomib for enhanced antimyeloma effects. However, the combination of these drugs resulted in an antagonistic response. In contrast, the autophagy inhibitor 3-methyladenine did synergize with thapsigargin for an enhanced cytotoxic response. These data suggest that autophagy inhibitors have therapeutic potential in myeloma but caution against combining such drugs with bortezomib. [Mol Cancer Ther 2009;8(7):1974–84]</p></div>

Supplementary Fig. S2 from Effect of autophagy on multiple myeloma cell viability

Supplementary Fig. S2 from Effect of autophagy on multiple myeloma cell viability