Translational Profiling Reveals The eIF2 Kinase Pathway As a Mediator Of Dexamethasone Induced Apoptosis In Multiple Myeloma

Dexamethasone has been used to treat Multiple Myeloma (MM), a plasma cell cancer, for over forty years. Nonetheless, dexamethasone toxicity is a source of morbidity for many patients and drug resistance invariably develops. A better understanding of the mechanisms of dexamethasone activity could provide novel avenues for drug development. To gain new insights into dex activity in MM, we started with the clinical observation that dex is an effective treatment for both MM and the ribosomal disorder Diamond Blackfan anemia (DBA). Interestingly, dex has opposite effects in these two disorders, promoting apoptosis in MM and inducing an erythroid response in DBA. Given these opposing responses to dex, we hypothesized that dex disrupts ribosomal profiles in MM, altering the translation of specific mRNAs to promote apoptosis. To address this hypothesis, we performed an unbiased assessment of mRNA translation in MM +/- dex, using translational (polysome) profiling.

We show that dex disrupts ribosomal profiles in sensitive (MM1.S), but not resistant (MM1.R) myeloma cell lines as early as 4 hours, well-preceding dex-induced apoptosis. These effects are dose and time dependent. We next treated the MM1.S and MM1.R cell lines with 1μM dex or control for 4 hours. Cells were lysed and separated by ultracentrifugation. Polysome gradients were obtained, and the gradients were fractionated into two pools: (A) non-polysome associated RNA and (B) polysome associated RNA. Pool B is enriched for actively translated mRNA. A translation coefficient for each condition was defined as the amount of mRNA in polysome/non-polysome pools. Unfractionated total cellular RNA was used to assess transcriptional changes. RNA was analyzed using human gene arrays.

The results demonstrate that dex alters a group of translational networks that are distinct from its effect on transcriptional networks. The top translational network modulated by dex was eIF2 signaling (p-value 1.4 E-09). The eIF2 kinase pathway regulates protein translation, and repression of this pathway paradoxically increases the translation of the transcription factor ATF4. We show that dex increases ATF4 translation. In addition dex up-regulates transcription of the ATF4 target gene DDIT4 (REDD1). Dex also represses the translation of ribosomal protein genes downstream of REDD1, including RPS6, RPS19 and RPS24. The large ribosomal protein genes RPL3 and RPL10 were unchanged, suggesting that dex represses the translation of a subset of ribosomal protein genes. Changes in protein expression were validated by immunoblot.

Finally, we tested whether specific inhibition of the eIF2 kinase pathway could overcome dex resistance. Using an MTS assay we demonstrate that BTdCPU, a specific eIF2 kinase inhibitor, induces cytotoxicity in dex sensitive (MM1.S, RPMI8266) and dex resistant (MM1.R, U266) cell lines. Using flow cytometry, BTdCPU also induces apoptosis in both dex sensitive and resistant cell lines. Furthermore, BTdCPU and dex have additive cytotoxicity/apoptosis in the MM1.S cell line. Lastly, we show that BTdCPU specifically up-regulates the ATF4 target genes REDD1 and CHOP. Up-regulation of the pro-apoptotic gene CHOP by BTdCPU may explain the additive cytotoxicity in conjunction with dex.

In summary, 1) The eIF2 kinase pathway is modulated by dexamethasone at the level of mRNA translation in multiple myeloma. 2) Direct targeting of the eIF2 kinase pathway with BTdCPU overcomes dex resistance.

We show that dexamethasone alters the translation of specific pathways in MM (independent of transcription), including eIF2, revealing potential new avenues for drug development.