Browsing by Author "Gromeier, Matthias"

Cancer-specific cytopathogenicity of oncolytic viruses is often defined by viral

sensitivity to innate antiviral immune responses, e.g. type I Interferons (IFNs), limiting

cytotoxicity to cells lacking these responses. However, recent work suggests some cancer

cells inhibit IFN-sensitive oncolytic viruses, preventing efficacy. IFNs are also antiproliferative in cancer and activate anti-tumor immunity.

In this work I show that the recombinant poliovirus PVSRIPO, currently in

clinical trial as a treatment for glioblastoma, induces and evades IFN-β signaling in

cancer cell lines infected at low doses. Likewise, IFN-α treatment of cancer cells

inhibited PVSRIPO less than on the related encephalomyocarditis virus (EMCV).

Antibody blockade of the IFN-α/β receptor had no effect on either virus in IFN-secreting

melanoma cell lines. Depletion of the pattern recognition receptor MDA5 or inhibition of

TBK1/IKKε eliminated IFN responses to PVSRIPO or EMCV and promoted EMCV, but

not PVSRIPO, replication. The Toll-like receptor 3 (TLR3) agonist poly(I:C) suppressed

EMCV (semi-independently of type I IFN signaling) but not PVSRIPO. Thus, MDA5 and

TLR3 provoke type I IFN-dependent and -independent antiviral effects, likely involving

upregulation of genes downstream of TBK1/IKKε. PVSRIPO subverts anti-viral

immunity in cancer cells at low doses and activates type I IFNs through MDA5,

supporting its oncolytic and immunotherapeutic use even in IFN-competent cancers.

Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.

Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.

In the following document, I will describe two distinct strategies that poliovirus

(PV) deploys to manage host antiviral responses. In the first section, I report on a role of

the constitutive repressor of eIF2α phosphorylation (CReP) in translation of PV and the

endoplasmic reticulum (ER)-resident chaperone binding immunoglobulin protein (BiP)

at the ER. Functional, proximity-dependent labeling and cell fractionation studies

revealed that CReP, through binding of the eukaryotic translation initiation factor eIF2α,

anchors translation initiation machinery at the ER and enables protein synthesis in this

compartment. This ER site was protected from the suppression of cytoplasmic protein

synthesis by acute stress responses. I propose that partitioning of translation initiation

machinery at the ER enables cells to maintain active translation of PV during stress.

In the second section, I report that PV 2A protease cleaves all three members of

the YTHDF protein family, cytosolic N6-methyladenosine (m6A) ‘readers’ that regulate

target mRNA fate. These cleavages occurred early during infection, and preemptive

YTHDF3 depletion enhanced viral replication. This corresponded with diminished type-

I interferon (IFN) receptor (IFNAR) expression and IFN-stimulated gene induction,

while IFN production was not significantly changed. I propose that 2A protease cleaves

YTHDF proteins, in part, to interfere with IFNAR expression and antagonize the host

antiviral response.

Signaling pathways converge on the translation machinery and influence protein synthesis globally or specifically on certain classes of transcripts. The experiments described in this thesis focus on regulation of translation initiation through the cap-binding protein eIF4E.

Aberrant regulation of eIF4E has important roles in several pathologies and, most notably, in tumorigenesis. Nevertheless, the understanding of the molecular con-sequences of changes in eIF4E activity remains incomplete. We employ a cell-free system to demonstrate that eIF4E function is required for efficient cap-dependent translation but inhibitory for translation of both cellular and viral RNAs relying on cap-independent mechanisms. Furthermore, we show that phosphorylation of eIF4E favors cap-independent translation in vitro.

To verify that our findings in the cell-free system are representative of an in vivo system, we also analyzed growth of an oncolytic poliovirus, relying purely on cap-independent translation, in the context of varying activity of signaling pathways. Data obtained from this virus helps to confirm that phosphorylation of eIF4E does indeed result in increased cap-independent translation. Additionally, these experiments provide important information for the clinical application of this oncolytic poliovirus, as they help to explain virus specificity and might allow for rational patient selection.

Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses can provide a context optimal for generating antigen-specific CD8 T cells, as they have natural tropism for dendritic cells, preeminent inducers of CD8 T cell immunity; elicit Th1-promoting inflammation; and lack interference with innate or adaptive immunity. However, notorious genetic instability and underlying neuropathogenicity has hampered poliovirus-based vector applications. We devised a strategy based on the polio:rhinovirus chimera PVSRIPO, devoid of viral neuropathogenicity after intracerebral inoculation in human subjects, for stable expression of exogenous antigens. PVSRIPO vectors infect, activate, and induce epitope presentation in DCs in vitro; recruit and activate DCs with Th1-dominant cytokine profiles at the injection site in vivo. They efficiently prime tumor antigen-specific CD8 T cells in vivo, induce CD8 T cell migration to the tumor site, delay tumor growth and enhance survival in murine tumor models.

Background

Methods

Results

Conclusions

Malignant glioma formation involves an abundant inflammatory infiltrate dominated by glioma-associated macrophages and microglia (GAMM). GAMM constitutes a large portion of the glioma mass and tumor microenvironment. They are actively involved in tissue repair and immune surveillance, however in the tumor microenvironment (TME), they are subverted to promote tumor progression. The human poliovirus receptor, hCD155h, is constitutively expressed in members of the mononuclear phagocytic system and is upregulated ectopically in the neoplastic compartment of malignant gliomas (and solid cancers in general). Intratumor treatment with the highly attenuated rhino:poliovirus chimera, PVSRIPO, has a dual effect of releasing neoantigens by oncolysis and activating the GAMM component via sublethal infection, leading to a substantial but transient immune therapy effect. In a phase I clinical trial, PVSRIPO treatment resulted in 21% long-term survival with durable radiographic responses in patients with recurrent glioblastoma (Desjardins et al. New England Journal of Medicine, 2018). Therefore, studying the mechanisms of PVSRIPO immunotherapy in mouse brain tumor models to decipher contributions of viral infection to GAMM vs. malignant cells is critical to improving the therapeutic efficacy in ongoing clinical trials. We recapitulated the clinical trial scenario in an immunocompetent intracerebral mouse tumor model (CT2A-CD155) and obtained baseline and post treatment brain in a time series. Histopathology studies, combined with detailed multiplex IHC/IF and RNAseq were performed on tumor bearing brains. We found the PVSRIPO therapy induced intense engagement of the GAMM infiltrate accompanied by substantial, but transient tumor regression. There were extensive microglia activation and proliferation in adjacent brain parenchyma and even part of the contralateral cortex. This occurred against a backdrop of sustained innate antiviral inflammation and is associated with an induction of the PD-L1 immune checkpoint on GAMM. In contrast to transient antitumor effects observed after PVSRIPO monotherapy, combining PVSRIPO with PD1/PD-L1 blockade led to durable remission. Our work implicates GAMM as active drivers of inflammation and reveals broad neuroinflammatory activation of the CNS-resident myeloid compartment upon polio virotherapy of malignant glioma.

Eukaryotic translation initiation is a rate-limiting step of protein synthesis and is controlled by signal transduction in response to various extracellular cues and stresses. This thesis is focused on the eukaryotic initiation factor 4G (eIF4G), which participates in multiple steps of initiation: (i) eIF4G interaction with polyA-binding protein (PABP) links the 5' pre-initiation complex to the 3' poly(A) tail of mRNAs; (ii) eIF4G recruits 40S ribosomal subunit to mRNAs through interactions with the m7-G cap binding protein eIF4E; (iii) eIF4G binds to the eIF4E kinase, mitogen activated protein kinase (MAPK) interacting kinase 1 (Mnk1), modulating eIF4E phosphorylation. I studied how eIF4G function is affected by viral infection, mitogenic stimulation and during mitosis.

First, I reported that herpes simplex virus 1 (HSV-1) infection leads to re-localization of PABP to the nucleus, dissociating it from translation initiation machinery. Next, I showed that MAPK-mediated phosphorylation of Mnk1 leads to Mnk1 conformational changes, enhancing its binding to eIF4G and, hence, increasing phosphorylation of its substrate, eIF4E. Finally, I demonstrated that Cdk1/cyclin B1 directly phosphorylates eIF4G in mitosis and speculated on the role of this phosphorylation event in mitotic translation. In summary, my work demonstrated that eIF4G plays key roles in the regulation of the translational response to viral infection, growth signaling and cell cycle progression.

Death associated protein 5 (DAP5) is an atypical isoform of the translation initiation scaffold eukaryotic initiation factors 4GI and II (eIF4GI/II), which recruit mRNAs to ribosomes in mammals. Unlike eIF4GI/II, DAP5 binds eIF2β, a subunit of the eIF2 complex that delivers methionyl-tRNA to ribosomes. Despite extensive work describing eIF4GI, an understanding of DAP5 activation is yet to be described.

Here I describe our discovery that DAP5:eIF2β binding is regulated by DAP5 phosphorylation and can be stimulated by specific stimuli including protein kinase C PKC-Raf-ERK1/2 signals, mitosis and hypoxia, wherein DAP5:eIF2β binding determines DAP5’s influence on global and template-specific translation. However, DAP5 depletion causes an unanticipated surge of hypoxia-inducible factor 1α (HIF-1α), the transcription factor and master switch of the hypoxia response. The hypoxia response is tempered through HIF-1α hydroxylation by the oxygen-sensing prolyl hydroxylase-domain protein 2 (PHD2), and subsequent ubiquitination and degradation. Furthermore, we found that DAP5 regulates HIF-1α abundance and Akt signaling through DAP5:eIF2β-dependent translation of PHD2. DAP5:eIF2-induced PHD2 translation occurs during hypoxia-associated protein synthesis repression, indicating a role as a safeguard to reverse HIF-1α accumulation and curb the hypoxic response implying that DAP5:eIF2β binding may describe a conserved mechanism for selective stress induced translation.

Multiple signaling pathways control protein synthesis by modulating translation initiation factors. Map Kinase Integrating Kinase 1 (Mnk1) relays signals to its major downstream target eIF4E. Activation of Mnk1 and subsequent phosphorylation of eIF4E results in changes in translation rates for subsets of mRNAs. Both the Erk1/2 and p38 MAPK pathways activate Mnk1 meaning that Mnk1 responds to growth signals through Erk1/2 and stress signals through p38 MAPK. However, it is not clear how Mnk1 mediates translational changes specific to each pathway. We investigated the activation of Mnk1 by stress and cytokines through the p38 MAPK pathway. We found that of the four different p38 MAPK isoforms, p38α alone controls acute stress and cytokine signaling to translation machinery. Furthermore, this regulatory axis is greatly diminished in neurons. We discovered that p38α expression is repressed in the brain due to two neuron-selective microRNAs, miR-124 and -128. Next, we investigated the mechanism of p38α mediated Mnk1 activation to see if it differed from Erk1/2 mediated activation. Looking at the induced binding of Mnk1 to eIF4G, we found that the dissociation rate varies depending on the activating pathways. This shows that Mnk1 is not a true convergence point of p38 and Erk1/2 MAPK pathways resulting in identical downstream effects, but that Mnk1 mediates pathway specific effects on translation factors.

microRNAs (miRNAs) downregulate the expression of numerous mRNAs and are involved in almost every biological process where they have been examined. Inherent sequence or cis-elements located in mRNA termini and 5' and 3' UTRs likewise influence post-transcriptional gene regulation. We delineate the relative importance of the 5' m7G-cap, the 3' poly(A) tail, and Internal Ribosome Entry Sites (IRESs) in miRNA-mediated repression. mRNA targets must contain a m7G-cap to be repressed, are repressed to a greater extent when containing a poly(A) tail, and are not precluded from repression when translating via an IRES.

miRNAs can inhibit translation and / or induce mRNA decay. While the core effector proteins are established, mechanistic details of how miRNAs interfere with mRNA translation and stability remain elusive. Contrary to the repressive effects of miRNAs, the poly(A)-binding protein (PABP) (through binding to the poly(A) tail and eIF4G) can increase both translation and mRNA stability independently. We elucidate a functional role for the PABP in miRNA repression; manipulation of `active' PABP levels affects repression conversely in part by inhibiting miRNA-induced deadenylation. Furthermore, we find that expression changes in the PABP binding partner PABP interacting protein 2 (Paip2) modulates both miRNA repression and PABP protein complex formation. Additionally, we establish Paip2 as a bona fide miR-128 target, and demonstrate miR-128 de-repression of non-miR-128 target mRNAs through this targeting event.