Abstract SY32-04: The translation initiation factor elF2α regulates cytoprotective autophagy in Myc-dependent lymphoma.
Authors: Koumenis Constantinos , Hart Lori S. , Cunningham Tom , Dey Souvik , Tameire Feven , Mills Ian , Ruggero Davide , Diehl Alan J. , Fuchs Serge 
Published: August 2014

The Unfolded Protein Response (UPR) is a cellular homeostatic program initiated by an excess of unfolded/misfolded client proteins in the ER lumen, with a primarily cytoprotective effect. The accumulation of unfolded proteins results in the dissociation of the chaperone GRP78/BiP from three ER-resident proximal sensors of unfolded proteins and ER stress: PKR-like ER stress kinase (PERK/EIF2AK3), activating transcription factor 6 (ATF6), and inositol requiring transmembrane kinase and endonuclease 1 (IRE1). This results in subsequent activation of these transducers. Active PERK phosphorylates the eukaryotic initiation factor 2-alpha (eIF2α) at serine 51, preventing the exchange of GDP for GTP, effectively blocking the initiation of protein synthesis during cellular recovery from ER stress. While translation of the vast majority of mRNAs is limited, the translation of specific mRNAs is induced, including that of the transcription factor ATF4.

A substantial amount of evidence supports a major role of the UPR in tumor progression and response to therapy.We and others previously showed that stresses in the tumor microenvironment (e.g. low oxygen, decreased amino acid availability and low glucose) activate components of the UPR, the abrogation of which leads to cell death, decreased angiogenesis and inhibition of tumor progression.

Cell-autonomous insults, including oncogenic activation, are also known to activate cellular stress responses, including metabolic stress, apoptosis, DNA damage responses, and cellular senescence. Therefore, transformation often requires the concurrent activation of pro-survival pathways. In particular, the myc oncogene, which is the target of chromosomal translocation and gene amplification during the development of many human cancers, is known to induce both increased proliferation and apoptosis depending on the cellular context. Overactivation of the myc proto-oncogene family (c-Myc, N-Myc and L-Myc) drives the development of several human malignancies, including lymphoma, neuroblastoma, and colorectal carcinoma and contributes to treatment resistance.

Based on the ability of MYC to induce considerable increases in both protein content and rates of ribosomal biosynthesis, we hypothesized that oncogenic activation of c-MYC during transformation would inflict ER stress and elicit UPR activation. Moreover, we reasoned that such an induction of the UPR would be cytoprotective and thus promote MYC-dependent tumorigenesis. Using several inducible MYC cell models as well as genetic and pharmacologic tools, we showed that MYC induction leads to activation of the PERK-eIF2α-ATF4 axis of the UPR, and enlargement of the endoplasmic reticulum. Amelioration of ER stress by chemical chaperones resulted in blunting of the UPR. Moreover, PERK ablation or use of a non-phosphorylatable allele of eIF2α (ser51ala) in transformed MEFs resulted in substantially lower levels of clonogenic survival and increased apoptosis upon c-MYC induction. Similarly, PERK ablation in mouse hematopoetic progenitor cells isolated from PERKfl/fl mice resulted in increased apoptosis and lower clonogenicity in methylcellulose upon c-MYC activation.

Mechanistically, we showed that the MYC-induced, PERK-dependent UPR pathway in multiple cell lines including human lymphomas, led to increased levels of autophagy and protection against ER-dependent apoptosis, since inhibition of autophagy by pharmacologic agents (i.e., spautin-1 or bafilomycin A1) or genetic means (siRNAs against atg7 or ulk1) resulted in significant increases in cell death following MYC activation. Moreover, samples of human lymphoma and mouse lymphoma models display robust upregulation of UPR markers and genes compared to normal B cells suggestive of a role for UPR activation in the pathogenesis of disease. Finally, we demonstrated an important link between MYC-dependent increases in protein synthesis and UPR activation. By employing a mouse minute, which results in wild-type levels of protein synthesis and attenuation of MYC-induced lymphomagenesis, we show that MYC-induced UPR activation and induction of autophagy in B cells is reversed.

To translate these findings into novel therapeutic strategies, we employed a recently described, highly specific and potent inhibitor of the PERK kinase (Exten et al, J Med Chem. 2012) to test the role of PERK activity in tumor growth. Using p493-6 human lymphoma cells implanted into NOD/SCID mice, we showed that oral administration of GSK2606414A twice a day for a period of 3 weeks resulted in significant inhibition of tumor growth. This inhibition was accompanied by potent decrease in the levels of phospho-PERK, phospho-eIF2α and ATF4 in the GSK2606414A treated vs. vehicle-treated tumors. We are currently evaluating the effects of this PERK inhibitor in conjunction with ionizing radiation and genotoxic chemotherapy in multiple mouse tumor models including the Eμ-Myc model of lymphoma. We are also extending these studies to elucidate the potential role of the UPR effector ATF4 in this process. Preliminary data strongly suggest that ATF4 plays a major role in MYC-induced activation of autophagy downstream of PERK and eIF2α and we are identifying mRNAs whose levels and/or translation is differentially impacted upon MYC induction in an ATF4-dependent manner.

In summary, we have identified a novel cell autonomous role for PERK as a molecular switch regulating autophagy and apoptosis in response to MYC activation, and have uncovered the PERK-eIF2α-ATF4 pathway as a therapeutic target for the treatment of MYC-associated malignancies.

This work was supported by NIH R01CA094214 and NIH R01CA139362 (to C.Koumenis); ACS 121364-PF-11-184-01-TBG (to J.T. Cunningham); NIH R01CA140456 (to D. Ruggero); by Commonwealth of Pennsylvania Universal Research Enhancement Program Funds and P01CA104838 to J. Alan Diehl.

Citation Format: Constantinos Koumenis, Lori S. Hart, Tom Cunningham, Souvik Dey, Feven Tameire, Ian Mills, Davide Ruggero, Alan J. Diehl, Serge Fuchs. The translation initiation factor elF2α regulates cytoprotective autophagy in Myc-dependent lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY32-04. doi:10.1158/1538-7445.AM2013-SY32-04