Critical Role for Cap-Independent c-MYC Translation in Progression of Multiple Myeloma.

Dysregulated c-myc is a determinant of multiple myeloma progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5'UTR of mRNA, termed the internal ribosome entry site (IRES), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. Thus, we studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in multiple myeloma lines was mediated by c-myc itself, suggesting a positive feedback circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted multiple myeloma cells demonstrated downregulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor, which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human multiple myeloma lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically reexpressed in A1-deleted multiple myeloma cells, tumor growth was reestablished. These results support the critical role of A1-dependent myc IRES translation in myeloma.

A Novel Therapeutic Induces DEPTOR Degradation in Multiple Myeloma Cells with Resulting Tumor Cytotoxicity.

Prior work indicates DEPTOR expression in multiple myeloma cells could be a therapeutic target. DEPTOR binds to mTOR via its PDZ domain and inhibits mTOR kinase activity. We previously identified a drug, which prevented mTOR-DEPTOR binding (NSC126405) and induced multiple myeloma cytotoxicity. We now report on a related therapeutic, drug 3g, which induces proteasomal degradation of DEPTOR. DEPTOR degradation followed drug 3g binding to its PDZ domain and was not due to caspase activation or enhanced mTOR phosphorylation of DEPTOR. Drug 3g enhanced mTOR activity, and engaged the IRS-1/PI3K/AKT feedback loop with reduced phosphorylation of AKT on T308. Activation of TORC1, in part, mediated multiple myeloma cytotoxicity. Drug 3g was more effective than NSC126405 in preventing binding of recombinant DEPTOR to mTOR, preventing binding of DEPTOR to mTOR inside multiple myeloma cells, in activating mTOR and inducing apoptosis in multiple myeloma cells. In vivo, drug 3g injected daily abrogated DEPTOR expression in xenograft tumors and induced an antitumor effect although modest weight loss was seen. Every-other-day treatment, however, was equally effective without weight loss. Drug 3g also reduced DEPTOR expression in normal tissues. Although no potential toxicity was identified in hematopoietic or hepatic function, moderate cardiac enlargement and glomerular mesangial hypertrophy was seen. DEPTOR protected multiple myeloma cells against bortezomib suggesting anti-DEPTOR drugs could synergize with proteasome inhibitors (PI). Indeed, combinations of drug NSC126405 + bortezomib were synergistic. In contrast, drug 3g was not and was even antagonistic. This antagonism was probably due to prevention of proteasomal DEPTOR degradation.

A cell-penetrating bispecific antibody for therapeutic regulation of intracellular targets.

The therapeutic use of antibodies is restricted by the limited access of antibodies to intracellular compartments. To overcome this limitation, we developed a cell-penetrating monoclonal antibody, mAb 3E10, as an intracellular delivery vehicle for the intracellular and intranuclear delivery of antibodies constructed as bispecific single-chain Fv fragments. Because MDM2 is an important target in cancer therapy, we selected monoclonal antibody (mAb) 3G5 for intracellular transport. mAb 3G5 binds MDM2 and blocks binding of MDM2 to p53. Here, we show that the resulting 3E10-3G5 bispecific antibody retains cell-penetrating and MDM2-binding activity, increases tumor p53 levels, and inhibits growth of MDM2-addicted tumors. The use of cell-penetrating bispecific antibodies in targeted molecular therapy will significantly broaden the spectrum of accessible intracellular targets and may have a profound impact in cancer therapy.

Oncogenic EGFR signaling activates an mTORC2-NF-κB pathway that promotes chemotherapy resistance.

UNLABELLED: Although it is known that mTOR complex 2 (mTORC2) functions upstream of Akt, the role of this protein kinase complex in cancer is not well understood. Through an integrated analysis of cell lines, in vivo models, and clinical samples, we demonstrate that mTORC2 is frequently activated in glioblastoma (GBM), the most common malignant primary brain tumor of adults. We show that the common activating epidermal growth factor receptor (EGFR) mutation (EGFRvIII) stimulates mTORC2 kinase activity, which is partially suppressed by PTEN. mTORC2 signaling promotes GBM growth and survival and activates NF-κB. Importantly, this mTORC2-NF-κB pathway renders GBM cells and tumors resistant to chemotherapy in a manner independent of Akt. These results highlight the critical role of mTORC2 in the pathogenesis of GBM, including through the activation of NF-κB downstream of mutant EGFR, leading to a previously unrecognized function in cancer chemotherapy resistance. These findings suggest that therapeutic strategies targeting mTORC2, alone or in combination with chemotherapy, will be effective in the treatment of cancer. SIGNIFICANCE: This study demonstrates that EGFRvIII-activated mTORC2 signaling promotes GBM proliferation, survival, and chemotherapy resistance through Akt-independent activation of NF-κB. These results highlight the role of mTORC2 as an integrator of two canonical signaling networks that are commonly altered in cancer, EGFR/phosphoinositide-3 kinase (PI3K) and NF-κB. These results also validate the importance of mTORC2 as a cancer target and provide new insights into its role in mediating chemotherapy resistance, suggesting new treatment strategies.

IL-6-induced stimulation of c-myc translation in multiple myeloma cells is mediated by myc internal ribosome entry site function and the RNA-binding protein, hnRNP A1.

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-three-hybrid 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.

AKT activity determines sensitivity to mammalian target of rapamycin (mTOR) inhibitors by regulating cyclin D1 and c-myc expression.

Prior work demonstrates that AKT activity regulates sensitivity of cells to G(1) arrest induced by mammalian target of rapamycin (mTOR) inhibitors such as rapamycin and CCI-779. To investigate this, a novel high-throughput microarray polysome analysis was performed to identify genes whose mRNA translational efficiency was differentially affected following mTOR inhibition. The analysis also allowed the assessment of steady-state transcript levels. We identified two transcripts, cyclin D1 and c-myc, which exhibited differential expression in an AKT-dependent manner: High levels of activated AKT resulted in rapamycin-induced down-regulation of expression, whereas low levels resulted in up-regulation of expression. To ectopically express these proteins we exploited the finding that the p27(kip1) mRNA was efficiently translated in the face of mTOR inhibition irrespective of AKT activity. Thus, the p27(kip1) 5'-untranslated region was fused to the cyclin D1 and c-myc coding regions and these constructs were expressed in cells. In transfected cells, expression of cyclin D1 or c-myc was not decreased by rapamycin. Most importantly, this completely converted sensitive cells to a phenotype resistant to G(1) arrest. Furthermore, the AKT-dependent differential expression patterns of these two genes was also observed in a mouse xenograft model following in vivo treatment with CCI-779. These results identify two critical downstream molecular targets whose expression is regulated by AKT activity and whose down-regulation is required for rapamycin/CCI-779 sensitivity.