Wilder - EGFR, Miljenko - Ras, and PKR
Intersting things here.
EGFR can contribute to Ras Activation. Ras activation inhibits PKR which in non-activated (normal) cells inhibits reovirus replication, reovirus replicates and causes cell death.
"The mechanism by which reovirus causes tumor cell lysis came to light serendipitously during reovirus receptor studies. These studies revealed that although the receptor for viral attachment was the ubiquitous sialic acid, not all cells bearing this moiety supported a productive infection by reovirus. Furthermore, cells with high levels of EGF-R sustained viral replication, compared with those with low levels of the receptor (14). These observations, together with the demonstration that reovirus can bind directly to the EGF-R, suggest that the receptor either served as a site of viral attachment and entry or effected a change in the intracellular environment that conferred susceptibility. Evidence for the latter possibility came from studying NIH-3T3 fibroblasts transformed with the oncogene v-erbB, which encodes a constitutively activated receptor lacking the extracellular domain. Untransformed NIH-3T3 cells were not infectible, but v-erbB–transformed cells enabled efficient replication of reovirus, demonstrating that constitutive tyrosine kinase activity of the EGF-R, but not the extracellular domain of the receptor, was required for viral replication (15).
Tyrosine kinase activity of the EGF-R is normally stimulated in response to ligand binding to the receptor’s extracellular domain (16). This in turn leads to autophosphorylation of the receptor’s cytoplasmic tail, to which phosphotyrosine-binding adapter molecules, such as Grb2, are recruited. Grb2 recruits the protein Sos to the plasma membrane, where it stimulates the exchange of GTP for GDP on the small G protein, Ras. Ras-GTP can then activate myriad signaling pathways important in such cellular processes as differentiation and proliferation. Constitutive activation of signaling pathways downstream of Ras, such as the mitogen-activated protein kinase (MAPK) and the stress-activated, c-Jun NH2-terminal protein kinase (SAPK/JNK) cascades, are implicated in cellular transformation and progression toward cancer.
As described earlier here, reovirus can take advantage of the constitutive tyrosine kinase activity of the truncated EGF-R. Transformation by oncogenes downstream of the EGF-R, such as sos or ras, has also been found to render a cell susceptible to reovirus infection. Ras signals may sensitize cells to reovirus by blocking cellular defenses against viral infection (Figure 1). Comparison of susceptible ras-transformed fibroblasts with resistant NIH-3T3 fibroblasts has given some insight into where the differences lie in the antiviral response of these two cell types.
Usurpation of the Ras signaling pathway by reovirus. In untransformed, reovirus-resistant cells, double-stranded RNA structures in reovirus transcripts activate PKR, which subsequently phosphorylates eIF-2, inhibiting translation initiation of viral genes. In cells with an activated Ras signaling pathway, however, PKR phosphorylation in response to viral transcripts is inhibited and viral translation proceeds unimpeded."
jci.org
"Background: Reovirus is a naturally occurring oncolytic virus that usurps activated Ras-signaling pathways of tumor cells for its replication. Ras pathways are activated in most malignant gliomas via upstream signaling by receptor tyrosine kinases. The purpose of this study was to determine the effectiveness of reovirus as an experimental treatment for malignant gliomas. Methods: We investigated whether reovirus would infect and lyse human glioma cell lines in vitro. We also tested the effect of injecting live reovirus in vivo on human gliomas grown subcutaneously or orthotopically (i.e., intracerebrally) in mice. Finally, reovirus was tested ex vivo against low-passage cell lines derived from human glioma specimens. All P values were two-sided. Results: Reovirus killed 20 (83%) of 24 established malignant glioma cell lines tested. It caused a dramatic and often complete tumor regression in vivo in two subcutaneous (P = .0002 for both U251N and U87) and in two intracerebral (P = .0004 for U251N and P = .0009 for U87) human malignant glioma mouse models. As expected, serious toxic effects were found in these severely immunocompromised hosts. In a less immunocompromised mouse model, a single intratumoral inoculation of live reovirus led to a dramatic prolongation of survival (compared with control mice treated with dead virus; log-rank test, P<.0001 for both U251N and U87 cell lines). The animals treated with live virus also appeared to be healthier and gained body weight (P = .0001). We then tested the ability of reovirus to infect and kill primary cultures of brain tumors removed from patients and found that it killed nine (100%) of nine glioma specimens but none of the cultured meningiomas. Conclusions: Reovirus has potent activity against human malignant gliomas in vitro, in vivo, and ex vivo. Oncolysis with reovirus may be a potentially useful treatment for a broad range of human cancers."
"Glio Cell Lines Susceptable to Reoviris Infection"
U87, U87lacZ, U251N, 251lacZ, 9L, 9LlacZ, RG2, A172, U563, C6, C6lacZ, SNB19, UC10, UC12, UC13, UC17, UC29, and SF188 SF126 and U373
Nonsusceptible U118, U343, U178, and UC18"
"Malignant Glioma Surgical Specimens
To determine whether reovirus oncolysis also occurred in primary cultures from brain tumor surgical specimens, we tested 16 ex vivo brain tumor surgical specimens (Fig. 6) derived from four glioblastoma multiformes, three anaplastic astrocytomas, one astrocytoma, one oligodendroglioma, and seven meningio-mas. Reovirus infected and killed all nine (100%) primary glioma cultures but had no effect on cultured meningiomas. Viral proteins were detected in live-virus-treated glioma cells with the use of indirect immunofluorescence microscopy. Glial fibrillary acidic protein staining of cultured gliomas detects glial
lineage. Dead-virus-treated tumor specimens remained healthy and continued to proliferate. The number of specimens was small, but this evidence suggests that reovirus oncolysis might be effective in a substantial portion of gliomas."
jncicancerspectrum.oupjournals.org
The Phase I trial was designed as a dose escalation study to determine the safety and tolerance of REOLYSIN® in late-stage cancer patients who have failed all other treatment options. None of the patients to date have experienced any serious adverse events related to the virus nor were there any dose limiting toxicities detected in any patient. As secondary endpoints, Oncolytics measured tumour response at both the treated lesion as well as remote metastatic sites. Evidence of viral activity in tumours was observed, which ranged from changes in tumour structure to partial and complete tumour regression in the injected tumours. 50% of injected tumours in the first four of six groups (twelve of the eighteen patients) demonstrated evidence of viral activity. Preliminary evidence of remote tumour responses were also noted in the first four groups.
The Phase I study enrolled a total of 18 patients in six groups of three patients each and examined intratumoural (into the tumour) administration of REOLYSIN®. The patients had a variety of primary cancers including breast, head and neck, melanoma, non-AIDS Kaposi’s sarcoma, and others. REOLYSIN® was administered directly into a subcutaneous (underneath the skin) tumour. None of the patients were screened for RAS activation of their tumours before entry into the trial.
Each group received increasing dosages of the virus. Dosages examined included single injections of 107, 108, 109, and 1010 PFU (plaque forming units, a measure of live virus particles), and multiple injections of 108and 109 PFU. The maximum dose tested in the trial was a single injection of 1010 PFU (virus particles). Both 108 PFU groups and the multiple 109 PFU group were added to the original study design when tumour activity was noted in the 107 PFU group, which was unexpected during the design of the study. Oncolytics had originally anticipated conducting groups at greater dosages using multiple injections of 1010 PFU but Oncolytics determined that it would not provide any more useful information for the future development of REOLYSIN®.
Detailed final results of this study are expected to be presented next year at an international cancer conference, and will include final safety, efficacy, and immune data on all the patients.
The final design of this clinical program evaluating the safety and efficacy of intracerebral administration of REOLYSIN® will be disclosed after it has been discussed with and approved by Health Canada and the FDA. In the dose escalation or Phase I portion of the study, patients with a variety of recurrent malignant gliomas will be enrolled. In the Phase II portion of the program, patients with recurrent glioblastoma multiforme, the most aggressive glioma, will be treated at the dose selected on the basis of the dose escalation study. |
