Myxoma virus attenuates expression of activating transcription factor 4 (ATF4) which has implications for the treatment of proteasome inhibitor-resistant multiple myeloma

Abstract

The recent development of chemotherapeutic proteasome inhibitors, such as bortezomib, has improved the outcomes of patients suffering from the plasma cell malignancy multiple myeloma. Unfortunately, many patients treated with these drugs still suffer relapsing disease due to treatment-induced upregulation of the antiapoptotic protein Mcl1. We have recently demonstrated that an oncolytic poxvirus, known as myxoma, can rapidly eliminate primary myeloma cells by inducing cellular apoptosis. The efficacy of myxoma treatment on proteasome inhibitor-relapsed or -refractory myeloma, however, remains unknown. We now demonstrate that myxoma-based elimination of myeloma is not affected by cellular resistance to proteasome inhibitors. Additionally, myxoma virus infection specifically prevents expression of Mcl1 following induction of the unfolded protein response, by blocking translation of the unfolded protein response activating transcription factor (ATF)4. These results suggest that myxoma-based oncolytic therapy represents an attractive option for myeloma patients whose disease is refractory to chemotherapeutic proteasome inhibitors due to upregulation of Mcl1.

Keywords: drug resistance; oncolytic.

Figure 1

MYXV eliminates bortezomib-resistant human MM cells. Dox40 or Dox40^BTZ cells were infected with MYXV at the indicated MOIs. After 24 hours, cellular viability was measured using the MTT assay. No significant differences in the killing of Dox40 or Dox40^BTZ were observed. Abbreviations: Dox40, bortezomib-sensitive human multiple myeloma cells; Dox40^BTZ, bortezomib-resistant human multiple myeloma cells; MM, multiple myeloma; MOI, multiplicity of infection; MYXV, myxoma virus; NS, not significant.

Figure 2

MYXV prevents expression of CHOP. U266 cells were either mock-infected or infected with MYXV at MOI =10 and subsequently incubated with 1 µM BreA. Six hours after infection, cells were harvested and analyzed for either (A) CHOP and actin protein expression via immunoblot or (B) expression of CHOP mRNA using qPCR. ***P<0.005 using Student’s t-test. Abbreviations: BreA, brefeldin-A; MOI, multiplicity of infection; mRNA, messenger RNA; MYXV, myxoma virus; qPCR, quantitative real-time polymerase chain reaction.

Figure 3

MYXV differentially activates each arm of the UPR. MYXV-treated U266 cells were harvested 6 hours postinfection. (A) RNA extracted from U266 cells was subjected to qPCR. The resulting PCR product was then digested with Pst1, loaded onto an 8% DNA acrylamide gel, and stained with SYBR^® Gold. PCR products derived from XBP1 mRNA, which had undergone IRE1-mediated splicing, contain a Pst1-resistant band of higher molecular weight (arrow). (B) U266 lysate was subjected to treatment with 50 mM DTT prior to loading onto a Laemmli gel. Samples were then immunoblotted for ATF6. Full-length ATF6 (arrow) is lost following activation. (C) U266 lysate was separated on a Laemmli gel and then immunoblotted with antibodies against PERK. Phosphorylated PERK is observed as a high-molecular-weight band (arrow). Abbreviations: ATF, activating transcription factor; BreA, brefeldin-A; DTT, dithiothreitol; IRE1, inositol-requiring protein-1; mRNA, messenger RNA; MYXV, myxoma virus; PCR, polymerase chain reaction; PERK, protein kinase RNA-like endoplasmic reticulum kinase; qPCR, quantitative real-time polymerase chain reaction; UPR, unfolded protein response.

Figure 4

Phosphorylation of eIF2alpha: (A) U266 cells were either mock-infected or infected with MYXV at MOI =10 and subsequently incubated with 1 µM BreA. Six hours after infection, phosphorylation of eIF2α was analyzed using immunoblot. (B) U266 cells were pretreated for 30 minutes with the PERK-specific inhibitor GSK2606414 at a final concentration of 10 nM. Cells were mock-infected or infected with MYXV at MOI =10. At the indicated time points, cells were harvested, and the phosphorylation of eIF2α was analyzed using immunoblot. (C) Densitometry quantitation of data shown in (B). Abbreviations: BreA, brefeldin-A; GSK, GSK2606414; hpi, hours postinfection; MOI, multiplicity of infection; MYXV, myxoma virus; p, phosphorylated; PERK, protein kinase RNA-like endoplasmic reticulum kinase.

Figure 5

MYXV inhibits expression of ATF4. U266 cells were either mock-infected or infected with MYXV at MOI =10 and subsequently incubated with 1 µM BreA. Six hours after infection, expression of ATF4 was analyzed using immunoblot. Abbreviations: ATF, activating transcription factor; BreA, brefeldin-A; MOI, multiplicity of infection; MYXV, myxoma virus.

Figure 6

MYXV prevents translation of ATF4 mRNA. (A) U266 cells were either mock-infected or infected with MYXV at MOI =10 and subsequently incubated with 1 µM BreA or 10 nM MG132. Cells were then either mock-infected or infected with MYXV at MOI =10. Six hours after infection, the expression of ATF4 was analyzed using immunoblot. (B) U266 cells were either mock-infected or infected with MYXV at MOI =10 and subsequently incubated with 1 µM BreA. After 6 hours, RNA was harvested, cDNA synthesized, and the expression of ATF4 mRNA was determined using qPCR. (C) Quantitation of qPCR normalized to 18s RNA. (D) U266 cells were either mock-infected or infected with MYXV at MOI =10 and subsequently incubated with 1 µM BreA. Twelve hours postinfection, translation of ATF4 or UBCH5 mRNAs was analyzed using polysome profiling. ***P<0.005 and *P<0.05 using Student’s t-test. Abbreviations: ATF, activating transcription factor; BreA, brefeldin-A; cDNA, complementary DNA; MOI, multiplicity of infection; mRNA, messenger RNA; MYXV, myxoma virus; qPCR, quantitative real-time polymerase chain reaction.

Figure 7

MYXV prevents expression of Mcl1 during the UPR. (A) U266 cells were either mock-infected or infected with MYXV at MOI =10. At the indicated times postinfection, cells were harvested, and the expression of Mcl1 was analyzed using immunoblot. (B) U266 cells were either mock-infected or infected with MYXV at MOI =10 and subsequently incubated with 10 nM MG132. Six hours postinfection, expression of ATF4 and Mcl1 was analyzed using immunoblot. Abbreviations: ATF, activating transcription factor; MOI, multiplicity of infection; MYXV, myxoma virus; UPR, unfolded protein response.

Figure 8

Model of MYXV modulation of the UPR. The UPR is primarily comprised of three distinct arms mediated by the ER stress sensors IRE1, ATF6, and PERK. Our results indicate that MYXV infection activates both the ATF6 and PERK arms of the UPR (shown in blue) while having virtually no effect on the IRE1 arm. After activation of PERK, however, MYXV infection subsequently blocks the downstream signaling from this arm by actively inhibiting the translation of ATF4 (shown in red). Abbreviations: ATF, activating transcription factor; ER, endoplasmic reticulum; IRE1, inositol-requiring protein-1; MYXV, myxoma virus; PERK, protein kinase RNA-like endoplasmic reticulum kinase; UPR, unfolded protein response.