BIO 483-FINAL HOMEWORK

RETROELEMENT
REACTIVATION THROUGH
EPIGENETIC THERAPIES CAN
INDUCE CANCER REGRESSION
BY MODULATING THE
UNDERLYING ENERGY
METABOLISM OF TUMOR CELLS
Epigenetic Therapies can reactivate heterochromatin
endogenous retroviruses, kind of TE, which are known to
induce antiviral responses in tumor cells that facilitate their
ultimate demise. A novel effect of the therapy is now found,
targeting the energy metabolism of the tumor cells,
contributing to necroptosis induced tumor regression.

Tumor therapies have come a long way from its chemotherapy and radiotherapy,
breaking of DNA by radio waves, which include are long, sensitive and tiring
processes that have different success rates depending on the type of tumor and
the type of body. New therapies have been coming along to improve the success
rate of this disease but most of them include novel techniques which are under
the title of “epigenetic therapies” that still need some time for their approval.
Some of the therapies focus on damage reversel of these mutations by changing
the expression levels of the genes, muting some and overexpressing some.
These therapies can also achive what radiotherapy intends with the breaking of
the DNA into double or single stranded fragments which drive the cell into
apoptosis, either through p53 dependent or independent pathways such as Bcl-2/
BAX, both of them work through proteases called caspasesand lead to the
degredation of the cell[4]. So instead of radiotherapy we can use epigenetic
drugs to induce these DNA damages and lead the cell into apoptosis dependent
on the characteristics and the abilites of the tumor cell. Recent studies have
shown that a subset of Transposable Elements called “Endogenous
Retroviruses(ERV)”, which are integrated viral DNA that once invaded the host
DNA, can get activated and express dsRNAs and can trigger another pathway
which is the interferon dependent immune pathway, and it starts with the
recognition of these dsRNA transcriptomes by RIG-I and MAD5 cytosolic viral
RNA sensing proteins. However the tumor regression and cell death induced by
epigenetic treatments that target HDAC, DNMT and HMTs, epigenetic regulators
that methylate DNA or histone proteins, and change the epigenome of these
ERVs, are thought to occur not because of the completed viral IFN response due
to some change that occurs in the cell during the process which underlies this
review[1].

In the research conducted by Vicente et al. CM272, HMT and DNMT inhibitor,
and Vorinostat, HDAC inhibitor, drugs were used for ERV reactivation which is a
widespread whole genome encompassing effect and doesn’t only activate ERV
and these dsRNA transcriptomes aren’t sequenced so there is no proof of them
beinf ERVs in this paper but the paper goes on and shows that while
proapoptotic proteins of caspases 3,8 and 9 were seen only at high amounts of
the drug usage, the phosphorylation of necroptosis-related proteins like RIP were
detected in abundance in western blot analysis. Knocking down IRF (Interferon
related factors) and downstream proteins of viral immune response pathway
revealed that tumor cell death rates depend on RIG-I and MAD5 recognition of

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the retroelement transcripts and not on proteins downstream of the immune
pathway. Vicenete et al. considers that the high ATP consuming reaction of
dsRNA recognition from RIG-I and MAD5 helicases could lead to the changes
that drive the cell to its death and the addition of glucose into tumor cell cultures
decreased cell death rates supporting this idea[2].

It is known that tumor cells experience what is called the “Warburg Effect” that
explains the metabolic shift from oxidative phosphorylation to fermentation which
is required for tumor cells due to their “rapid” need of energy. SeahorseFX
technology was used to measure the OCR(oxygen consumption rate) and
ECAR(extracellular acidi cation rate) levels of the tumor cell upon epigenetic
treatment. Heightened OCR levels were observed in epigenetically treated cells
reversing the Warburg Effect. Upon knockout analysis of RIP proteins,
epigenomic treatment is shown to reverse the Warburg Effect through RIP
mediated Necroptosis. Because the oxidative phosphorylation pathway depends
on the TCA cycle for the production of its electron donors the TCA cycle rate was
measured by LC/MS C13 labeled glucose and glutamine which also are the
substrates for glycolysis in tumor cells[2].

While most TCA component levels increased upon epigenetic treatment

succinate levels were shown to decrease. This effect might be explained by two
things: either the heightened activity of the ETC which is debunked by the lack of
increase of the enzymatic activity of the other component in the ETC even
though SDH(succinate dehydrogenase) activity had increased, another possible
explanation is the heightened activity of SDH due to its re-routed electron
transfer to other substances which produces mitochondrial ROS(reactive oxygen
species) which is shown to increase with treated cells and the loss of RIP1
decreased this effect on tumor cells. Inhibition of the ATPase further indicated

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 that the production of ROS elements through the ETC component and especially
through the SDH induces these tumor cell deaths upon applying epigenetic
drugs[2].

Also Pro Apoptotic drugs and Epigenetic drugs are found to have a synergistic
effect on tumor cell death in different Tumor types. In AML, B-cell leukemia and
lymphoma a BCL2(anti-apoptotic protein) inhibitor venetoclax and epigenetic
drugs in combination increased tumor cell deaths whilst other tumor cells types
which don’t rely on BCL-2 but rely on BCL-XL or MCL1 are resistant to
venetoclax but positively react to BH3-like drugs which antagonize the BCL-XL or
MCL1 and promote apoptosis. The synergistic mechanism arise from the
hyperpolarization of the mitochondrial membrane and is measured by the Time-
course TMRE assays that prevents the mitochondrial apoptosis of the cell, and is
reversed by the depolarization of the membrane by venetoclax leading to
caspase mediated apoptosis. The inhibition of SDH reduces the
hyperpolarization of the membrane and inhibits the release of cytochrome c
through the co-translocation of anti-apoptotic BCL-2 and proapoptotic BAX
proteins[2].

These ndings show that the synergistic treatment of epigenetic drugs like
CM272 and pro-apoptotic drug venetoclax induce necroptosis and apoptosis in
increasing the effectiveness of tumor cell death in common cancer illnesses[3].
Although deemes successful its important to recognize that epigenetic treatments
are not localized and effect the whole epigenome and also considering the
unknown potentşal of the reactivation of these ERVs maybe this way of inducing
immune response related cell necrotpsis is not the best idea. It’s known that
ERVs can also cause diseases in human tissues like MS and ALS, considering
these, maybe CRISPR/Cas9 mediated double stranded breaks can be

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 considered for a radiotherapy like effect with less physiological side effects or
ERVs can be reactivated through CRISPR/dCAS9CM272 type models where the
speci c location of the ERVs are found and their epigenome changed by
modi ed deactivated Cas9 enzymes[5].

Figure 1:The schematic of the epigenetic and proapoptotic therapy. Mitochondrial

hyperpolarization of the membrane due to the epigenetic therapy induced by the high energy
demanding recognition of dsRNA through the ERV reactivated viral immune response elements
RIG-I and MAD5.The switch in this metabolic pathway from glycolysis to OXOPHOS(oxidative
phosphorylation) doesn’t change the catalytic activity of the ATP forming unit of the ETC but
promotes the catalytic activation of the ROS generating components of the ETC which are
increased after the treatment of tumor cells with CM272 and are shown to participate into RIP
mediated Necroptosis of the cell. Epigenetic treatment alone doesn’t show major cell death
rate since the hyperpolarized mitochondria membrane inhibits apoptosis, a pro-apoptotic
drug inhibiting anti-apoptotic factors increases cell death rate through the synergetic activity
of the two.

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REFERENCES

1. Baba AI, Câtoi C. Comparative Oncology. Bucharest (RO): The

Publishing House of the Romanian Academy; 2007. Chapter 3,
TUMOR CELL MORPHOLOGY. Available from: https://
www.ncbi.nlm.nih.gov/books/NBK9553/
2. Fresquet V, Garcia-Barchino MJ, Larrayoz M, et al. Endogenous
Retroelement Activation by Epigenetic Therapy Reverses the
Warburg Effect and Elicits Mitochondrial-Mediated Cancer Cell
Death. Cancer Discovery. 2021 May;11(5):1268-1285. DOI:
10.1158/2159-8290.cd-20-1065. PMID: 33355179.
3. Giménez-Orenga K, Oltra E. Human Endogenous Retrovirus as
Therapeutic Targets in Neurologic Disease. Pharmaceuticals. 2021;
14(6):495. https://doi.org/10.3390/ph14060495
4. Little JB. Principal Cellular and Tissue Effects of Radiation. In: Kufe
DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei
Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003.
Available from: https://www.ncbi.nlm.nih.gov/books/NBK12344/

5. Pulecio, J., Verma, N., Mejía-Ramírez, E., Huangfu, D., & Raya, A.
(2017). CRISPR/Cas9-Based Engineering of the Epigenome. Cell stem
cell, 21(4), 431–447. https://doi.org/10.1016/j.stem.2017.09.006