Oncolytic Viruses I - Specificity

Question 1

what is an oncogene vs tumor suppressory gene?

Answer 1

oncogene: normally a “go” step that is “controlled” - mutations leads to “uncontrolled go”

tumor supressor: normally a “stop” step to stop “uncontrolled go” - mutations lead to “uncontrolled go”

Question 2

Describe the CDK, Ras, and E2F oncogene and p53 tumor suppressor pathway

Answer 2

Ras (a mitogenic signal) activates CDK which phorsphorylates E2F-Rb –> release of E2F from Rb –> E2F is a TF that turns on S-phase genes and activates p53 –> p53 inhibits this cycle

Question 3

Describe the general features of the tumor microenvrionment

Answer 3

• cytokines
• enzyme/proteases –> chew up the ECM to make room for cells to grow
• blood vessels
• growth factors
• chemokines –> recruits and supports growth of all cells
• many cell types
• endothelial cells –> supplies nutrients and growth factors

Question 4

what 3 types of cells support cancer progression and how?

Answer 4

1. adipocytes: release free fatty acids (FFAs) –> used by cancer cells for ATP and growth
2. mesenchymal stem cells: multipotent adult stem cells –> release cytokines and angiogenic factors
3. cancer associated fibroblasts: transfer protein and lipids to cancer cells through ectosomes, secrete growth factors like TGF-B (immune suppression), MMP, VEGF

Question 5

describe how cancer requires immune suppression

Answer 5

• immunosuppressor cells such as MDSCs, iDCs, Tregs and M2 macrophages suppress activity of helper and cytotoxic T cells and NK cells
• tumor supporting cells secrete cytokines that inhibit immune cells and support immunosuppressor cells
• tumor cells upregulate PDL1 – an inhibiitory receptor that binds PD on Tcells –> inhibit T cell activation

Question 6

how are antiviral/anti-damage pathways dampened in cancer?

Answer 6

• DAMPs such as ATP, HMGB1, S100A are released from damaged and dying cells – frequent collateral of a fast growing tumor environment
• similar to PAMPs which turn on IFNs during virus infection, DAMPs stimulate IFN expression
• IFNs have negative effects on cell growth and cancers (don’t want to support growth of virus), and prompt anti-tumoral cells
• this pressure selects for dysregulated antiviral/anti-damage signaling pathways

Question 7

what do oncoyltic viruses target/do in general?

Answer 7

• kill cancer cells
• dismantle tumor microenvironments
• stimilate anti tumoral immunity

Question 8

how do OVs promote anti-tumoral immunity?

Answer 8

• increase PAMPs/DAMPs –> immune cell stimulation
• increase cytokines –> more recruitment of immune cells
• increase tumor debris/Ags –> DCs now present and activate T cells

Question 9

what are three ways OVs are specific to tumors, give examples for each category?

Answer 9

1. re-targeting by modulation – adenovirus
2. re-targeting by depletion – HSV, vaccinia virus
3. wrong host or niche – VSV, newcastle disease virus, reovirus

Question 10

what is needed for adenovirus infection and replication? which genes are essential for turning on the cell cycle, how?

Answer 10

• fiber needed for Ad infection
• E2F and cell cycle needed for Ad replication
• E1A phosphorylates Rb –> E2F is released –> turns on cell replication
• E1B activates cyclines and inhibits p53 (must inhibit p53 because cyclins will upregulate p53)

Question 11

what does adenoviruses fiber interact with? why is this problematic when trying to target cancer cells

Answer 11

fibre interacts with CAR, which is low on cancer cells

Question 12

what is biochemical targeting vs genetic targeting? in adenovirus

Answer 12

biochemical targeting: viruses can be directed to specific receptors with “adaptors” – sCAR-ligand; biotin-avidin-ligand; bivalent Ab

genetic targeting: fiber knob domain can be substituted with receptor binding domains from: other human serotypes, xenotype switching, pseudotyping, add binding domains (modify fibre)

Question 13

describe transcriptional targeting using adenovirus

Answer 13

making a virus selective by driving expression of virus’ essential genes (like E1A/B) by TFs expressed in cancer cells

the oncogene Survivin (BIRC5), a member of the inhibitor of apoptosis gene family, is overexpressed in most cancers, but not in normal cells –> make an adenovirus where the transcription of E1 is controlled by the survivin promoter

Question 14

what are advantages/disadvantages of biochemical vs genetic transductional targeting?

Answer 14

biochemical

• what if the adaptor doesn’t bind to the receptor and the virus infects healthy cells
• easier
• as virus replicates, adaptors decreases so it’s only good for one hit

genetic

• harder
• have to watch carefully when giving gain of functions
• works on incoming and progeny virus

Question 15

what are challenges of using transcriptional/transductional targeting the major basis for specifity to cancer cells?

Answer 15

• cancers are very different from each other, there are not many “common” receptors and/or TFs
• cancer cells mutate their receptors or TFs to resist infection

Question 16

describe the general prinicple of “by deletion” method of creating OVs

Answer 16

if you delete these virual regulatory genes, the virus will not replicate in a “normal” cell, but will replicate in a cancer cell where the process in circumnavigated already by the cell

Question 17

how can we “by deletion” adenovirus?

Answer 17

disable adenovirus E1A, so it cannot turn on cell cycle through Rb and p53 modulation, this will disable adenovirus from replicating in normal cells, but since most cancers already have this pathway dysregulated, adenovirus will still replicate in the cancer cells

however, E1A has many essential roles – can’t just delete the whole protein –> delete the RB interaction domain of E1A

Question 18

what do large dna viruses (e.g. HSV and VV) express because they need large pools of deoxynucleotides for DNA synthesis? if we KO these genes, how would a cancer cell provide a good environment for these viruses?

Answer 18

HSV

• RR (ribonucleotide reductase) subunit: UL39 gene = ICP6 protein
• TK (thymidine kinase): UL23 gene

VV

• RR subunit: F4L gene
• TK: J2R gene

Cancer via E2F converts NDP –> dNDP and dNTP –> dTTP

Question 19

how is VV modified to be a “by deletion” OV?

Answer 19

VV is normally lethal to mice, deletion of RR F4L from VV makes the virus non-pathogenic, but it still replicates efficiently in tumors and prolongs survival in mice with bladder cancer

Question 20

describe the antiviral signaling pathway activated by viral PAMPs

Answer 20

TBK1 is a kinase activated by cytoplasmic receptors that detect viral PAMPs. TVK1 phosphorylates the TF IRF3 that stimulates the expression of interferons (IFNs). IFNs induce an antiviral state in neighboring cells by turning on ISGs, which include PKR. When PKR binds dsRNA from viruses, it auto-phosphorylates to become active. Then it stops virus replication by phosphorylating eIF2a and stopping translation imitation.

Question 21

how can we “by deletion” herpesvirus to make it an OV?

Answer 21

g34.5 gene recruits phophotase PP1a to dephosphorylate and inactivate TBK1 and PKR –> remove g34.5

Question 22

how can we use non-human (animal) viruses as OVs (“by insufficiency”)?

Answer 22

don’t naturally infect humans –> don’t inhibit humans IS or human pathways –> no modification to virus genes and yet specific

Question 23

what is the function of the viral protein “V” in NDV? is it species specific?

Answer 23

stops IRF3 nuclear translocation and prevent IFN production

species specific and can overcome IFN production in chicken cells but human cells: in chickens V is necessary for virus replication, in humans NDV with V doesn’t replicate well –> insufficient

Question 24

what does adding influenza’s NS1/2 (cross-species IFN inhibitor) to deltaV NDV do in chickens and humans?

Answer 24

deltaV can now replicate in chickens and NDV can now replicate in human cells

Question 25

when well plates containing normal human cells and pancreatic cancer cells are treated with NDV, which are susceptible and why?

Answer 25

takes a lot of PFU virus to kill normal cells, takes a little to kill IFN-deficient cancer cells

Question 26

why is reovirus – a virus that infects humand and most mammals – a “by deficiency” OV?

Answer 26

• very stable in environment
• non-pathogenic virus that infects most mammals –> many hosts
• naturally infects replicating gut enterocytes where it rapidly sheds 10000s new viruses into envrionment
• naturally cleared by the IS rapidly – didn’t bother collectging virus genes to modulate antiviral signalling –> cancer being immunosuppressed (less IFN, less cellular immunity) permits reovirus infection
• didn’t bother collecting genes to modulate cell signaling, since gut cells provide Ras signaling, p38 signaling, and P13K signaling…to mediate various stages of virus replication –> cancer cells also have these signaling pathways “on” while normal cells do not have these pathways constitutively and strongly active