Roskilde Universitet / Roskilde University

Den Naturvidenskabelige bacheloruddannelse / The Natural science bachelor programme

Targeting G-quadruplex structures in

c-Myc oncogenes of cancer cells
An investigation on how targeting c-Myc oncogenes, and particularly targeting a specific DNA
configuration within a promotor region of a c-Myc oncogene, may provide a potential promising
therapy for treating cancer

Figure 1: A G-quadruplex structure[100]

Group (1.B) members: Maria Abboud – 61324

Laura Martinenghi – 59788

Project Supervisor: Ole Skovgaard

Semester: First Semester

House: 14.2

Year: 2016/2017
1. Abstract:

c-Myc is a nuclear transcription factor that controls around 15% of the human genome
and plays a vital role in regulating the different mechanisms going on within the cell,
however, it is found deregulated and overexpressed in wide range of malignancies.
Furthermore, its deregulation and overexpression is shown to be essential for cancer cell
survival, growth and proliferation. For that reason, being able to target c-Myc´s gene
expression is believed to put the cancer cell under stress, forcing it to commit apoptosis.
A nuclease hypersensitivity element III1, located within a promotor region of the c-Myc
gene, controls 85–90% of its transcriptional activation. The sequence of this element involves
repetitive Guanine tracts, that have shown to fold into a higher-order four-stranded DNA
structure, known as G-quadruplexes, when subjected to transcriptional stress (negative
superhelicity). This DNA secondary structure was first discovered to form and inhibit
telomerase activity in telomeric regions, but was lately discovered in the promotor region of
c-Myc genes functioning as a transcriptional repressor element.
This exciting discovery opened the door for the development of ligands with high
affinity, selectivity and efficacy in stabilizing and maintaining these structures in c-Myc
oncogenes rather than telomeric regions, for a better approach to treat cancer cells.
Throughout this paper, targeting cancer cells through targeting G-quadruplex structures
in c-Myc oncogenes using small molecules ligands (Quarfloxin and TMPyP4), will be studied
in the light of the research question:
Is it possible to induce cancer cell apoptosis through targeting G-quadruplex structures
in c-Myc oncogenes?

I
2. Acknowledgements

We would like to thank the following people for their help in the production of this project:

First of all, our Project Supervisor Ole Skovgaard, for guiding us through our work on the
investigation. Secondly, we gratefully acknowledge Henning Bjerregaard for helping us with
specific explanations concerning cancer cells. We would also like to thank the members of
our opponent group and their Supervisor Lauren Seaby for the useful discussions regarding
improvements that can be done in our investigation. And finally, we would like to thank our
previous group members for taking part in the project and helping improving it.

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3. Table of contents
Section number and headline Page
1. Abstract I

2. Acknowledgements II

3. Table of contents III

4. Standard Abbreviations and Acronym IV

5. Investigation 1

5.1 Introduction 1

5.2 Theory/background information 2

5.2.1 The Cellular Myelocytomatosis proto-oncoprotein(c-Myc protein) 2

5.2.1.1 Significance of c-Myc´s structure on its functional activity 3

5.2.1.2 Effect of mutation on c-Myc´s function in the cell 5

5.2.2 Causes of c-Myc gene mutation 7

5.2.3 The Cellular Myelocytomatosis proto-oncogene (c-Myc gene) 8

5.2.3.1 Human c-Myc gene structure 8

5.2.3.2 Regulation of the c-Myc proto-oncogene 10

5.2.4 The G-quadruplex structures 13

5.2.4.1 G-quadruplex structures and telomerase activity in telomeric regions 15

5.2.4.2 G-quadruplexes structures in NHE III1 of the c-Myc gene 18

5.2.5 Targeting c-Myc oncogene through G-quadruplex structures 19

5.2.5.1 Inhibition of telomerase by stabilizing G-quadruplexes 20

5.2.5.2 Stabilization of the G-quadruplex structures in c-Myc oncogene 22

5.2.5.3 Tables with ligands developed to target G-quadraplexes 26

5.3 Discussion/Conclusion 29

6. Bibliography V

7. Glossary X

III
 4. Standard Abbreviations and Acronym

ALT Alternative Lengthening of Telomeres Sp1 Specificity protein

APBs ALT-associated PML bodies TBP TATA binding protein

bHLH-LZ basic helix-loop-helix-leucine zipper TERT Telomerase reverse transcriptase protein

c-Myc Cellular Myelocytomatosis oncogene TFIIH Transcription factor II Human

CNBP Cellular nucleic acid binding protein TMPyP4 Cationic porphyrin 5,10,15,20-tetra(N-

C-rich sequence DNA sequence rich in Cytosine bases methyl-4-pyridyl)porphin

E-box Enhancer box sequence TOPOI Topoisomerase I

FBP FUSE-binding protein TOPOII Topoisomerase II

FIR FBP-interacting repressor protein TOPOIII Topoisomerase III

FUSE Far Upstream Sequence Element

G-rich sequence DNA sequence rich in Guanine bases

G-quadruplexes Guanine quadruplexes

hnRNP K heterogeneous nuclear ribonucleoprotein K

hTERT Telomerase reverse transcriptase gene

MB(I, II,III or IV) Myc Box (I, II, III or IV)

mRNA Messenger RNA

NM23-H2 Non-metastatic 23 isoform 2

NHE III1 Nuclease hypersensitive element III1

P0, P1, P2 and P3 Promotor (0,1,2 and 3)

PDI Protein-DNA interaction

PML bodies Promyelocytic leukaemia bodies

Pol II RNA polymerase II

PPI Protein-Protein Interaction

Pu27 27 base-pair guanine-rich NHE III1 sequence

RAS Recepter-associated protein

rDNA Ribosomal DNA

IV
5. Investigation
5.1 Introduction
Cancer is a disease known for being the leading cause of morbidity and mortality worldwide.
Moreover, it is one of the most difficult diseases to be treated. According to the World Health
Organization, the number of new cancer cases is expected to rise up to about 70% over the
next 2 decades, where 23.6 million new cancer cases worldwide are estimated by 2030[101].
For that reason, many researches have been done and many experiments have been conducted
in the past decades, to get a better approach in targeting this disease and providing a more
effective therapy for treating it.

Cancer is caused by the gradual and progressive accumulation of random and epigenetic
changes in a single lineage of one cell[88]. It is a disease characterized by its ability of
growing and proliferating out of control through escaping control mechanisms that normally
limit its growth. For that reason, mutations that cause loss of function in transcription factors
that limits cell growth, such as tumor suppressors, is a necessity for cancer cell survival.
Furthermore, mutations in transcription factors that promote tumorigenesis, such as
oncogenes, will allow cancer cells to grow and proliferate with no restraints. In an abnormal
cell, which has lost the function of its tumor suppressors, preventing cell growth and
proliferation can be achieved by targeting the oncogenes responsible for this growth and
proliferation, such as the mutated c-Myc oncogene.

c-Myc is a proto-oncogene that encodes for the transcription of c-Myc proto-oncoproteins.

These c-Myc proteins are nuclear transcription factors that bind a multitude of genomic sites,
estimated to be over 10–15% of all promoter regions[1]. c-Myc plays a vital role in the
regulation of the different mechanisms going on within the cell. It transduces intracellular
signals to the nucleus that result in the regulation of cell cycle progression, cell proliferation,
cell differentiation and metabolism[2]. However, the expression of the c-Myc proto-oncogene
was found to be overexpressed and deregulated in a wide range of malignancies, including
carcinomas of the breast, colon, cervix, small cell lung cancer, osteosarcomas, glioblastomas,
and myeloid leukemia, giving rise to the formation of a c-Myc oncogene, which is the
mutated form of c-Myc proto-oncogene[3]. This gene is among the most frequently affected
genes in human cancer[4]. Therefore, being able to target the c-Myc oncogene may possibly
lead to the discovery of a potential new therapy in treating cancer. This is due to the fact that,
cancer cells cannot survive without growing and proliferating, and will thereby commit
apoptosis.

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Recently, a specific secondary DNA structure, called G-quadruplex, was shown to form in
promotor regions of many proto-oncogenes. This structure was first identified 55 years ago
and initially discovered in telomeric regions of cellular DNA[5]. In the case of a c-Myc
oncogene, this structure was shown to form in a nuclease hypersensitive element, NHE III1, in
the promotor region of the c-Myc gene. This structure is expected to have functional
significance in inhibiting the transcription of the c-Myc gene, as it had in inhibiting
telomerase activity and disrupting telomeric ends, when it was targeted with small
molecule[6].

However, all this raises questions concerning the importance of c-Myc in cancer cells, the
effect of G-quadruplex structures on c-Myc expression, the possibility of stabilizing G-
quadruplex structures using small ligands and the efficiency of ligands in stabilizing G-
quadruplex structures and inducing cancer cell apoptosis. Answering these questions will
provide us with an answer for the main focus of our investigation, which is:

Is it possible to induce cancer cell apoptosis through targeting G-quadruplex structures

in c-Myc oncogenes?

5.2 Theory/background information:

5.2.1 The Cellular Myelocytomatosis proto-oncoprotein (c-Myc):

c-Myc is a gene that was first identified 35 years ago as a cellular homologue of a viral
oncogene present in chicken retrovirus. c-Myc has been intensively studied in the past years,
and was initially discovered to be mutated and overexpressed in human Burkitt’s
lymphoma[7]. The c-Myc gene encodes for the transcription of c-Myc proteins that belong to
a Myc transcription factor family with distinct members. Even though, the Myc family is
comprised of three types of Myc proteins, c-Myc, l-Myc and n-Myc proteins, it displays
notable differences in potency between its member.[8] This is due to the fact that, somatic
cells are mainly dependent on the c-Myc proteins to regulate and coordinate transcription of
their different proliferative programs[90].

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 5.2.1.1 Significance of c-Myc´s structure on its functional activity:

The c-Myc protein is a gene regulatory protein, also known as a transcription factor, that has
the ability to activate, turn on, or repress, turn off, the transcription of different genes by
directly modulating their transcription[9].
This happens through the recognition of the c-Myc protein to a defined DNA sequence in a
regulatory region of the DNA and binding to it. The structure of the c-Myc protein is an
essential determinate of its function in recognizing the information of the DNA sequence.
This is because the c-Myc protein has to make specific contacts with the DNA through a set
of DNA-binding structural motifs that can read DNA sequences, in order for it to recognize
the defined DNA sequence of the target gene[88].
The c-Myc protein undergoes two types of interaction in order to control the transcription of
its target genes, a Protein-Protein Interaction (PPI) and a Protein-DNA Interaction (PDI).
Furthermore, it consists of two domains and a central region, that control these interactions
and are crucial for all its biological functions. The two domains are, the N-terminal domain
(NTD) related to the amino-terminal end of the c-Myc protein, and the C-terminal domain
(CTD) related to the carboxy-terminal end of the c-Myc protein[10]. (Figure 2)
The N-terminal domain of the c-Myc protein consists of two regulatory motifs, termed as
Myc Box I and II (MBI, MBII) necessary for its transactivation function[2]. These two boxes
are involved in regulating c-Myc´s biological activities. They interact with some major c-Myc
interacting proteins, such as coactivator proteins that can cause histone acetylation, and
thereby, the opening of chromatin structures promoting transcription activation[7].
On the other hand, the central region is the least characterized region among the three.
However, recent studies have started to reveal the importance of this region as it contains
highly conserved Myc Box III (MBIII) within it. MBIII is important for the transcription
repression activity of the c-Myc protein through causing histone deacetylation and chromatin
structure closing.[1] Furthermore, another conserved Myc Box, Box IV (MBIV), has been
studied and shown to take part in transcription activation and repression, in addition to being
necessary for c-Myc´s pro-apoptotic function, and for c-Myc´s recognition of its target
genes[94].
The C-terminal domain (CTD) of c-Myc protein is a is a basic helix-loop-helix-leucine zipper
region (bHLH-LZ), that is essential for its dimerization with its partner bHLH-LZ protein
Max and for its DNA-binding[11]. The Myc/Max dimerization requires the interaction of the
HLH-LZ regions of both proteins to allow the formation of a heterodimer[12]. (Figure 2)
During this protein-protein interaction, it is the leucine zipper motif of both proteins that drive
the dimerization of the two proteins. This happens by forming a coiled-coil structure at the C-

3
terminals of both proteins[88]. On the other hand, the basic regions of these proteins are
specified in interacting with the major groove of the DNA allowing the c-Myc protein to
recognize the information of the DNA sequence[13]. These interactions between the two
partner proteins and between the proteins and the DNA are seen to resemble the grip of a
clothespin on a clothesline[88]. (Figure 2)

Figure 2*: The c-Myc/Max heterodimer interact and bound together through their HLH-LZ regions,
and bound to the DNA at the E-Box through their Basic region, resembling the grip of a clothespin on
a clothesline[7][100].( Figure modified with a photo program “Paint”)

The function of the c-Myc protein is dependent on its dimerization with its partner protein
Max. This has been confirmed by experiments that have provided evidence for the occurrence
of c-Myc/Max interaction at all the genes, c-Myc binds to, across the genome[11]. Once the
c-Myc/Max complex is formed; this dimer acts as a positive transcription factor and activate
the transcription of specific E-box-containing genes. This happens by the binding of the
complex to the DNA through an enhancer box sequence, E-Box motif (5’-CACGTG-3’), and
thereby, stimulating the transcription of its downstream genes.

*
The figure only shows the regions of c-Myc/Max interaction, while the central region and the NTD domain are
not shown.

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This shows that, all these motifs within the c-Myc protein gives it a unique structure that
allows it to interact, either with the DNA or with proteins of major functions in the cell
mechanisms, which, in return, gives it a wide spectrum of target genes. And thus, provides the
c-Myc protein with the facility to fulfill its job as transcriptional factor with a unique ability in
influencing multiple biochemical pathways within the cell.

5.2.1.2 Effect of mutation on c-Myc´s function in the cell

Due to the ability of the c-Myc proteins to influence multiple biochemical pathways within
the cell, it is obvious that these proteins are regularly subjected to control. Due to the fact that,
maintaining cellular hemostasis and stability requires a stable and controlled activity of
critical transcriptional factors in the cell. Since the c-Myc protein is an essential part of the
transcriptional regulator network, promoting and regulating fundamental cellular processes,
such as, growth, proliferation, differentiation, metabolism, angiogenesis and apoptosis[14],
any mutation that disconnect the c-Myc protein from signaling processes, that keep it in check
and restrain its activity, will have viral effects on the activity of the cell and will push it
toward tumorigenesis.
In most of the cancer cases, mutation in c-Myc has been a gain in function mutation; in other
words, it has led to an enhanced activity of the c-Myc protein in the cell. This enhanced
activity of the c-Myc proteins and their ability to activate genes that influences glucose
metabolism, mitochondrial homeostasis, amino acid metabolism, DNA repair, protein
synthesis, ribosomal biogenesis and cell cycle progression[88], make the c-Myc proteins
sufficient in promoting tumorigenesis, and capable of inducing changes in the cell that are
critical for cancer cell survival.(Figure 3)
The c-Myc protein has the ability to influence cell growth by enhancing protein synthesis. It
does so, by activating the transcription of genes that code for proteins involved in ribosomal
biogenesis, protein translation and other process that will lead to an increased amount of
protein present within the cell, which will, in return, boost cell growth, causing increase in
cell size[7]. Furthermore, it influences metabolism by reprogramming its metabolic capacity
through enhancing glycolysis and inducing changes to its amino acid metabolism. This helps
the cell to grow and survive under conditions of deficit oxygen supply and makes it more
addict to glucose and specific amino acids for its survival, which, in return, support the rapid
expansion of the cell[15].
These changes in cell growth and metabolism, back up c-Myc´s ability in forcing cell cycle
progression in the absence of mitogenic signals or growth stimuli. This is because each cell

5
needs to increase in size in order to be able divide and proliferate. c-Myc has the ability to
force cell replication, by inhibiting cell cycle checkpoints and activating transcription of
cyclin proteins, which are proteins that help progression from one stage in the cell cycle to the
next, allowing the cell to divide and exit the cell cycle[16].
No wonder that c-Myc can cause genomic instability with its ability to force cell replication
and inhibit cell cycle checkpoints. The process of chromosome duplication is a very tightly
coordinated process, however with c-Myc´s ability in causing replication stress, c-Myc can
induce several DNA damages and chromosomal
c- abnormalities, which are essential incidents
for cancer progression. Furthermore, c-Myc can also influence the development of tumor
environments, by taking part in inducing tumor metastasis through influencing
angiogenesis[17], plus activating the expression of factors that take part in enhancing tumor
invasion[11].
On the other hand, c-Myc is also characterized by it ability of causing cell apoptosis as a safe
response to prevent tumorigenesis when being overexpressed. This can happen through
repressing the transcription of anti-apoptotic proteins, while activating the expression of pro-
apoptotic proteins, or through inducing the expression of other proteins that activate and
increase the expression of tumor suppressor proteins[18]. However, this process must be
overcome in cancer cells in order for c-Myc to cause oncogenesis. Indeed, this function (or
pathway) is shown to be lost in almost all cancer cells allowing the overexpressed c-Myc
proteins to drive oncogenesis[88].

c-

Figure 3: The most tumor relevant processes influenced by the overexpression and deregulation of the
c-Myc protein[7].

6
 5.2.2 Causes of c-Myc gene mutation

Now that we have considered the effect of mutation on the oncogenic activity of the c-Myc
protein, knowing what causes the overexpression of c-Myc, and the way it causes this
mutation (molecular cause) will provide us with a better insight into understanding how to
target its expression.
The change in the c-Myc gene can be due to random spontaneous mutation. It may also be a
result of environmental influences, such as high energy radiations, chemical carcinogens or
due to some retroviruses[92]. These mutations can result in accidents that may rise genetic
changes and may convert a c-Myc proto-oncogene to a hyper activated c-Myc oncogene, by
three mechanisms:
Firstly, by deletion or point mutation in the proto-oncogene or a control element, causing a
small change in the DNA within a protein-coding sequence in the regulatory region of the
gene, by which a single nucleotide is substituted, inserted or even deleted[93]. (Figure 4)
Secondly, by chromosomal translocation, causing the movement of the DNA within the
genome, where the gene moves to a new locus under new controls. This happens when the
breakage and rejoining of the DNA helix is incorrect and a fragment from one chromosome is
translocated to another, leading to a change in the protein-coding region.(Figure 4) This
situation is mainly observed in Burkitt’s lymphomas, however are less common in other
cancer types[7].
Thirdly, by gene amplification, causing gene duplication, due to error in the replication of the
DNA[92].(Figure 4) This type of mutation is the main type of mutation observed in the
different cancer types, including small-cell lung cancer, breast, ovarian and some types of
squamous cell carcinomas cancers[93].

Figure 4: The different genetic changes that can convert a proto-oncogene into an oncogene[92].

7
If the occurrence of any of these accidents led to the mutation of a single allele (a single copy)
of the c-Myc proto-oncogene, this proto-oncogene will get overexpressed and converted into
an oncogene[89]. Thereby, enhancing the activity of c-Myc and promoting tumorigenesis.
Furthermore, the coding sequence of the c-Myc gene does not need to altered in order for it to
show its oncogenic side. It can be deregulated due to extrinsic changes that damage critical
regulatory mechanisms and disconnect it from important signaling process that normally
keeps it under control and regular check[7].

5.2.3 The Cellular Myelocytomatosis proto-oncogene (c-Myc gene)

5.2.3.1 Human c-Myc gene structure

In the process of targeting the c-Myc gene, taking a closer look into its composition and the
elements that control and regulate its transcription, will provide us with a better insight into
the approach that can be taken to target c-Myc oncogenes.
The c-Myc gene is located on chromosome 8 q24.1, consisting of three exons, 2 introns and
four promotors of distinct strengths (P0, P1, P2 and P3),from which the c-Myc gene is
transcribed[2]. (Figure 5.A) Among the four promotors, P1 and P2, located within the first
exon, mainly control the transcription of the c-Myc gene, while P0 and P3 play an inferior
role in c-Myc transcription[91][19].
Regardless of promotor usage, a Nuclease Hypersensitive Element III1 (NHE III1) located
upstream to the P1 controls up to 80-95% of the total c-Myc gene transcription and is
considered c-Myc´s essential element[20].(Figure 5.A) In this region of the c-Myc gene, the
nucleosomal structures are less compacted, allowing the binding of transcription factors to the
DNA. With an usual asymmetry, the NHE III1 element is comprising a guanine-rich, G-rich,
(purine-rich) noncoding strand (antisense strand) and a cytosine-rich, C-rich (pyrimidine-rich)
coding strand (sense strand)[21], which contains six runs of three to four neighboring
guanines and cytosines each[5].
It was recently discovered that specific intramolecular secondary DNA structures, that were
initially discovered in the telomeric regions of cellular DNA, can, likewise, be formed in
promotor regions[22].These intramolecular structures motifs are shown to form in the NHE
III1 element of the c-Myc gene, specifically through its single G-rich strand and its
complementary single C-rich strand. (Figure 5.B) These two secondary DNA structures are
called G-quadruplexes and i-motif respectively[2][20][23].

8
The formation of these secondary DNA structures (G-quadruplex/i-motif structures) have
shown to silence the expression of the c-Myc gene and cause transcription repression, thus,
allowing the NHE III1 to form a transcriptionally active or silent mode[16]. This means that,
the transcription of the c-Myc gene will be turned off due to the formation of these structures,
however, will first be turned on and transcriptionally activated once these secondary DNA
structures are converted back to unstructured G-rich and C-rich single strands.

Once unstructured single strands are formed, DNA binding proteins, such as a cellular nucleic
acid binding protein, CNBP, and a heterogeneous nuclear ribonucleoprotein K, hnRNP K,
bind to the G-rich and C-rich single strands respectively and act as transcriptional
enhancers[24]. hnRNP K, which is an RNA binding protein interact with the RNA
polymerase II (Pol II) transcription machinery via interacting with a TATA binding protein,
TBP, thus stimulating the activation of gene transcription[25].(Figure 5.C)

A)
)

C) B)
) )

Figure 5: Firstly, the c-Myc gene is located on chr8. Secondly, the c-Myc gene consists of 4
promotors, 3 extrons, 2 introns and a NHE III1 element. Thirdly, the NHE III1 Element is a GC-rich
sequence that that can form a transcriptionally silent mode when secondary DNA structures are
formed, and a transcriptionally active form, when unstructured single strands are formed[2].( Figure
modified with the photo program Paint)

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 5.2.3.2 Regulation of the c-Myc Proto-oncogene

The regulation of c-Myc gene transactivation or repression is mainly controlled by two

regulatory elements, the Nuclease Hypersensitive Element, NHE III1, and the Far Upstream
Sequence Element, FUSE, which both require torsional stress(negative supercoiling) for this
regulation. These two elements regulate c-Myc transcription by shifting from B-DNA
arrangement to non-B-DNA arrangement and vice versa, when subjected to torsional stress
during transcription[20].

When there are no growth factor stimuli, both elements are double stranded, however the
FUSE element is nucleosome bound (Figure 6.A-7.A), while the NHE III1 element is
nucleosome-free.(Figure 7.A) Once growth factor stimuli are received, c-Myc transcription
gets activated, and a transcription factor Sp1, called Specificity protein, binds to the GC-rich
repeats of the double strand within the NHE III1 element and initiate the transcription of the
c-Myc gene.(Figure 7.B) During transcription, the RNA polymerase II (Pol II) causes strand
separation at NHE III1, by creating negative supercoiling stresses at the promotor region as it
passes along the DNA; It generates positive supercoiling as it translocates downstream the
gene, while it generates negative supercoiling upstream the gene[26].

These stresses cause the destabilization of the FUSE element, allowing the formation of a
nucleosome-free single stranded FUSE that will increase promotor firing and the rate of
transcription.(Figure 6.B-Figure 7.B) It binds to a FUSE-binding protein, FBP, capable of
influencing c-Myc transcription by direct binding to a general transcription factor that makes
up the RNA polymerase II complex, known as Transcription factor II Human (TFIIH), in
addition to DNA helicases[27].(Figure 6.C-D) This protein-protein bridge between FBP and
TFIIH causes the formation of a loop between the FUSE and the promotor. The supercoiling
in the c-Myc gene is not immediately relieved by the topoisomerases I and II causing the
formed loop to get supercoiled during transcription, and melt the FUSE elements[20].(Figure
6.E) When this happens, the FUSE element loses the FBP protein, but then associates with an
FIR (FBP-interacting repressor protein) that binds to it and inhibits transcription, thereby,
reducing torsional stress, allowing the FUSE element to go back to its initial state.(Figure
6.F-B-A) These two proteins simply control the rate of transcription through binding to the
FUSE[28].

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Figure 6: The Fuse-FBR-FIR-TFIIH governor for transcription of the c-Myc gene. It is only shown at
promotor region 2 for clarity. And GTF refers to general transcription factors including (Pol II) [27].

On the other hand, when this negative supercoiling stress generated behind the transcription
bubble causes denaturation and opening of the double stranded DNA at NHE III1, two
possible outcomes can arise[27]. Depending on the further signals that NHE III1 receive, it
either allows the DNA binding proteins, CNBP and hnRNP K to stick separately to each
single strand and stabilize it, thus increasing its rate of transcription under appropriate
conditions(Figure 7.C) or it forms non-B-DNA configurations(G-quadruplex/i-motif
structures), through absorbing the torsional stress into it, preventing the CNBP, hnRNP K and
Sp1 to bind to their binding site in the NHE III1 element. Thus, destabilizing the FUSE-FIR-
TFIIH association and silencing gene transcription[24][27].(Figure7.D).

Figure 7: Models of the different promoter forms within the NHE III1 of the c-Myc gene[29].

11
If further signals that stimulate transactivation are not provided, or if other signals that
stimulate c-Myc transcription repression are received, due to the activation of a tumor
suppressor, secondary DNA structures get formed and repress the transcription of the c-Myc
gene[30]. This formation takes place by the movement of a nuclear protein, called nucleolin,
into the nucleoplasm and consequently stabilizing the secondary DNA G-quadruplex
structures formed in the prmotor region of c-Myc[27]. (Figure 8.A-8.D)
These secondary DNA structures have a very high melting point compared to the helical DNA
structure and are, hence, in a more stable state. Therefore, it is expected that these structures
probably need the help of an enzyme to resolve them into the single stranded form[31].
A transcription regulator protein NM23-H2 has been recognized as an activator of c-Myc
transcription through interacting with the NHE III1 element[32]. (Figure 8.B) It was found
that NM23-H2 proteins have high affinity to single strands of DNA, and will, hence,
preferentially bind to single C and G-rich DNA strands, rather than duplex DNA. Therefore, it
was proposed that this protein is involved in resolving the two secondary DNA structures by
binding and wisely unfolding them, allowing the access and the takeover of single-strand-
specific transcription factors, such as hnRNP K and CNBP[33].(Figure 8.C)

Figure 8:NM23-H2, nucleolin and G-quadruplex-interactive compounds are involved in modulating

the activation and silencing of the NHE III1 element in the c-Myc promoter region[34]

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 5.2.4 The G-quadruplex structures

The tetrameric arrangement of guanines in G-quadruplex structures was first discovered in the
human telomeric regions of the DNA in 1962 by Gellert and co-workers, although the
capacity of a guanine rich DNA sequence to form aggregates was first reported in 1910 by
Bang, who reported that a solution of guanylic acid formed a gel[35]. The biological role of
G-quadruplexes has been controversial and has raised a quite number of skeptical opinions
concerning the biological function of these secondary structures in DNA. Lately, there have
been compelling evidences for the crucial biological roles of G-quadruplexes in, for instance,
initiation of DNA replication, telomere maintenance, control of gene expression and gene
stability[36]. G-quadruplexes are also present in the gene of a number of viruses, including
viruses with replication schemes based on RNA (e.g., flaviviruses), DNA (e.g.,
papillomavirus) or intermediates thereof (e.g., retroviruses, ribovirus). Furthermore, G-
quadruplexes are part of their life cycle, where the formation of these structures is crucial for
regulating important replication steps[37][38].
Ever since the discovery of G-quadruplex structures in telomeric DNA caught interest,
understanding their functional significance in telomeres kept growing till investigations
showed that the formation of these structures in telomeres was capable of causing telomerase
inhibition leading to disruption in telomeric ends, when these structures were targeted with
small molecules. This fact opened new doors toward finding potential therapies for treating
cancer. Following this discovery, investigation showed the presence of these structures within
promotor regions of some genes (including the promotor region of the c-Myc gene), which
raised questions concerning their ability of repressing the transcription of the genes present in,
as they were able to prevent the activity of the telomerase enzyme in telomeres[39]. Indeed,
experiments performed in vitro showed that the formation and stabilization of G-quadruplexes
in the promotor region of c-Myc had the ability of silencing the transcription of that
gene[19][2][5].
Due to the fact that, the NHE III1 element, present within the c-Myc promotor region, controls
up to 80-95% of the total c-Myc gene transcription, many investigations to understand how
this element control c-Myc transcription have been done. Latest discoveries have revealed the
presence of a targetable secondary DNA structure can form within this element.
Understanding the structure of this secondary DNA structure, G-quadruplexes, will provide us
with a closer insight into the way small ligands can interact with them and target the c-Myc
oncogene in cancer cells.

13
G-quadruplexes can be found in DNA or in RNA. They are structures that form in sequences
where the guanosines in oligo or polynucleotides are able to aggregate. Furthermore, Adjacent
runs of guanines are essential for the formation of the secondary DNA structure.

A) B)

A)
11 C)
A)
11

Figure 9:A)The different edges of interaction in the Guanine[40]. B) N1 and N2 from one side of the
guanosine bind to the O6 and N7 of the other side of another guanosine respectively[41].
C) G-quadruplexes planar structure of one of the G-tetrads. Four guanines and a monovalent cation. G-
quadruplexes can form a structure of three g-tetrads upon each other, forming a very stable structure [42].

Four guanines connect together through eight hydrogen bonds to form a square planar
structure, known as a quartet or a G-tetrad[43]. (Figure 9.B) The four guanines are bonded
through Hoogsteen hydrogen bondings. In Hoogsteen hydrogen bonding the base is “flipped”,
and since base pairs are attached to the backbone of the DNA, the flipping effect will change
the configuration of the DNA. In G-tetrads, the Hoogsteen hydrogen bond of one guanine
pairs to the Watson Crick face of the other one[44].(Figure 9.A-B)
In the G-tetrad, each nucleic acid base is a donor and acceptor of a hydrogen bond, where the
N1 and N2 from one side of the guanosine bind to the O6 and N7 of the other side of another
guanosine respectively[45].(Figure 9.B). Within the G-quadruplex structures, these planar or
G-tetrads are located one above the other, and since each tetrad consist of four O6 atoms, the
tetrad forms a negatively charged space that needs to be stabilized by a cation, a positively
charged ion, such as K+ or Na+[43].(Figure 9.C) However, potassium cations form more
relevant structures, due to their higher concentration in the nucleus.

14
a)

b)

Figure 10: a) A schematic view of some examples of different G-quadruplex conformations formed
from different adopted guanine conformations[46]. b) G-quadruplexes structures. Intramolecular G-
quadruplexes are formed with only one strand of DNA. There 6 kinds of structures that can be formed,
antiparallel, parallel and mixed. Intermolecular G-quadruplexes are made of two or more strands of the
DNA. Dimer can be also parallel, antiparallel and mixed, but tetramer can only have a parallel structure.
Under the picture it is notated the cation that participate in the formation of the G-quadruplex and the DNA
sequence[47]. (Figures modified with the photo program Paint)

G-quadruplexes can be formed by two or more tetrads upon each other, they can have
different strand polarity and can exhibit extensive structural polymorphism[48]. This is
because, in G-tetrads, the guanines can adopt a syn or an anti-conformation with respect to the
glycosidic bond and can thus provide many different guanine combinations. (Figure 10.a.A-
D) Anti-conformation extends the base and pentose rings in opposite directions , while a syn-
conformation orients the base and the pentose in the same direction[41]. Moreover, G-
quadruplex structures can be formed by one(mono), two(dimer) or four strands(tetra) of the
same DNA strand and these can all be parallel, three parallel and one antiparallel (mixed),
depending on the direction of their strands[47].(Figure 10.b)

5.2.4.1 G-quadruplex structures and telomerase activity in telomeric regions

Telomeres are nucleoprotein structure that play a vital role in the human genome and are
placed at the end of the chromosomes[49]. They are non-coding compounds composed of
tandem repeats of around 500–3000 hexanucleotide, 5′-TTAGGG-3′, with a 3′ single-stranded

15
extension (G-overhang) associated with six protein factors, that form a t-loop protecting
complex (shelterin), which protects the genes from degradation and is essential for genome
stability[50]. In each cell replication, somatic cells loose between 50-200 bases from its
telomeres. [51] After reaching a certain shortening, due to aging and certain life styles, the
cell will activate cellular stress responses that cause cell apoptosis before important genetic
information are lost from its chromosome ends[52].
Ironically, during early tumorigenesis, cancer cells often contain shortened telomeres because
of their uncontrolled cell division, but the cell does not activate the apoptosis signals, on
contrary, they activate an enzyme called telomerase, which is a specialized reverse
transcriptase, responsible for maintaining the length of telomeric DNA.(Figure 11) This
enzyme is capable of extending the 3′ end of chromosomes by adding TTAGGG repeats and,
thereby, allow cancer cell proliferation. Telomerase consists of two components, a reverse
transcriptase protein TERT encoded by the hTERT gene (one of c-Myc´s target genes) and an
RNA template, which together allow the activation of the telomerase[53]. These two
components allow the cancer cell to maintain their chromosome ends while continuing their
fast division. Furthermore, the activation of the telomerase enzyme is observed in around 80-
90% of cancer cells[54]. This has made telomerase a target for cancer drug devolopment.
Even though telomerase does not drive the oncogenic process, it is permissive and required
for the sustain growth of most cancers[55].
But that is not the only mechanism that prevent the shortening of telomeric sequences, a
mechanism known as, Alternative Lengthening of Telomeres (ALT) involves specific nuclear
bodies called ALT-associated PML bodies(APBs). These bodies concentrate telomeric DNA,
shelterin components and factors associated with telomere recombination[56].

Figure 11: Increase in cell replication in tumor cells due to the high levels of telomerase
In tumor cells[87](Figure modified with the photo program Paint)

16
As mentioned earlier, human telomeric regions have a repetitive DNA motif
(TTAGGG/CCCTAA)n. This G-rich telomeric sequence allows the formation of secondary
DNA G-quadruplex structures. Optimal telomerase activity requires the non-folded single-
stranded telomere overhang. The formation of G-quadruplexes in these 3′ overhangs has been
shown to decrease the activity of telomerase and prevent DNA elongation, thus controlling
cell proliferation. That is the main reason why G-quadruplex structures were seen as attractive
anti-cancer drug targets[57][58].
In telomeres, telomeric K+ dependent G-quadruplexes form a hybrid-type, mixed
parallel/antiparallel-stranded G-quadruplex structure, with the first, second and fourth G-
strands being parallel with each other, and the third G-strand being antiparallel with respect to
the other three. These hybrids (categorized as anti-parallel G-quadruplexes) contain guanines
with both syn- and anti-conformations. Moreover, the telomeric G-quadruplexes are always in
dynamic equilibrium between these two conformations, the hybrid-1 and hybrid-2
structures.(Figure 13.A-B). The Hybrid-type G-quadruplex structure can fold stack end to
end in the elongated telomeric DNA strand, thanks to the opposite direction of the 5´and
3´ends of this type. The capping (at the end of the telomeres) can also stack to the adjacent
telomeric G-quadruplexes, but most importantly can provide specific binding sites for drug
targeting[59][60].

Figure 12: A) Hybrid 1 and 2 structure of telomeric G-quadruplexes. B) A model for hybrid G-
quadruplex structures placed in the telomeric DNA[61].

17
 5.2.4.2 G-quadruplexes structures in NHE III1 of the c-Myc gene

Ever since the discovery of G-quadruplex structures in promotor regions, and specially in
c-Myc promotor regions, the light has been shifted toward investigating G-quadruplex
structures in c-Myc genes rather than telomeric regions. This is due to the fact that, one of the
main components needed for the activation of telomerase, TERT, is encoded by the hTERT
gene, which the c-Myc protein is a transcription factor of. So, being able to target G-
quadruplex structures in c-Myc oncogenes will also target the activity of telomerase in cancer
cells. For that reason, investigations were performed to understand the structures of the G-
quadruplexes formed in c-Myc promotor regions and the way they regulate c-Myc gene
transcription.
As, discussed earlier, Putative G-quadruplex structures form in the NHE III1 elements of the
c-Myc genes. This NHE III1 is a 27 base-pair guanine-rich sequence, also called Pu 27, that
consist of 6 runs of 3 or more guanines[5]. These has been shown to fold into multiple G-
quadruplex structures in solutions, in vitro, adopting various topologies, and displaying a high
degree of polymorphisms compared to telomeric sequences[43]. Due to the complexity of
these structures, steps that include reduction in structural polymorphism were applied to
study the G-quadruplex-forming sequence[20]. This was performed through sequence
modifications, which demonstrated that the major and most stable structure among the G-
quadruplex structures formed was a parallel intramolecular structure of 4 guanine runs, three
stacking tetrads with a short loop size of 1:2:1 arrangement and with all guanine bases
adopting an anti-conformation with respect to the glycosidic bond[43][62][41].(Figure 13,
Figure 10.a.A) Furthermore, experiments, in vitro, have verified that the parallel-stranded G-
quadruplex structure is more preferentially and likely to be formed in the promotor region of
the c-Myc gene[5].

Figure 13: The 27 base-pair guanine-rich sequence fold into a parallel G-quadruplex structures[34].

18
But, how important is the presence of G-quadruplexes in c-Myc promotor region on its
transcriptional activity?
In order to gain a better understanding of how the presence of G-quadruplex structures can
affect the transcriptional activity of the c-Myc gene, experiments have been conducted, in
vitro, where sequence mutations have been performed to destabilize the G-quadruplex
structure within the NHE III1 of the c-Myc gene followed by measuring its transcriptional
activity after such mutation[63]. This was performed by causing specific point mutation in the
sequence of the G-quadruplex, where the G-quadruplex structure was mutated with a single
G → A or G→T transition in one of the guanines taking part in forming the G-quartet[19].
The measurements of c-Myc promotor´s transcriptional activity, showed a 3-fold increase in
its transcriptional activity[64]. This confirmed that the presence of this secondary DNA
structure within the c-Myc promotor region regulates the transcription of the c-Myc promotor,
by acting as a transcriptional silencing element[65]. Furthermore, it showed that it is a
negative regulator of c-Myc, because one single base mutation was capable of causing such
an increase in its transcriptional activity.

5.2.5 Targeting c-Myc oncogene through G-quadruplex structures using

Ligands.

In order to have relevance in biological systems, it is important that the duplex DNA and the
G-quadruplex forms are in equilibrium. The interconversion between double and single
stranded DNA is important, in order to switch mechanisms in control of gene expression and
telomerase activation on or off[66]. Furthermore, the polymorphism of the G-quadruplex
structures, which give rise to G-quadruplex structural diversity, also contributes in making G-
quadruplexes molecular switches.

The process of folding and unfolding G-quadruplexes structures in DNA is a very controlled
process, and is mainly kept in equilibrium by the control of specific proteins. Different
folding and unfolding proteins and molecules are in charge of this function, facilitating either
the formation or the resolution of these secondary DNA structures. The proteins that are in
charge of DNA folding are called chaperone molecules and are essential for the formation of
G-quadruplexes, such as nucleolin, RAP-1 and TBP-B. On the other hand, NM23-H2,
topoisomerases , yeast and helicases are essential for unfolding the DNA[48].
As a matter of fact, there are three main approaches that can help the formation or prevalence
of G-quadruplexes, in order to silence the expression of a c-Myc gene, when it is mutated or

19
overexpressed. Firstly, there are several experiments conducted to target chaperone proteins
via drug molecules, in order to accelerate G-quadruplex formation[60]. Secondly, there are
several studies and clinical trials of small molecules that bind the G-quadruplex structure, in
order to disrupt signal mechanisms and extend the preformed structure or facilitate the
formation of it[67]. And a third approach to controlling the formation of these secondary
DNA structures has studied the inhibition of helicases unwinding enzymes[68][69].
Even though, G-qaudruplexes are highly polymorphic, different G-quadruplex conformations
have several structural features in common that can be controlled in order to develop ligands
and optimize their affinity to these structures, however, this does not increase the selectivity
of these ligands to a particular G-quadruplex structure. On contrary, it is the diversity between
the different G-quadruplex structures that makes structural selectivity achievable[70]. The
focus of the present investigation is to point out the development of some small molecules,
called ligands, that can help the formation and stabilization of secondary DNA structures, and
possibly induce cancer cell apoptosis.

5.2.5.1 Inhibition of telomerase by stabilizing G-quadruplexes

After the discovery of G-quadruplex structures in telomeric regions, a number of small

molecules that act as ligands have been synthesized, developed and evaluated as possible
drugs for maintaining secondary DNA structures. The strategy of binding small ligands to the
G-quadruplex structure formed in telomeric regions, was used to inhibit and deactivate
telomerase activity in telomeric region, to inhibit the functions of telomeres and to prevent
cancer cell growth[71]. The main two ligands developed to target telomeric G-quadruplexes
so far, are Telomestatin and the cationic porphyrin TMPyP4.

Telomestatin is a natural occurring product that has been used as a ligand to target telomeric
G-quadruplexes and has shown to be a potent telomerase inhibitor. Telomestatin has shown
high selectivity for G-quadruplex structures over duplex DNA. It has shown the ability to
stabilize G-quadruplex structures in the absence of monovalent cations, where it can replace
the need of positively charged ions, which is a unique characteristic among G-quadruplex
targeting ligands[72]. The ability of telomestatin in forming or capturing and stabilizing
telomeric G-quadruplex structures is owed to its structure that resemble the structure of a G-
tetrad.( Figure 14.B) This ability allows it to utilize the same DNA strand d[T(2) AG(3)](n)
required for telomerase activity, but with an opposite functionality. It has large 𝜋-planar core
that allows it to stabilize the G-quadruplex structures by interacting with the carbonyl oxygen

20
atoms that are present in the plane of the G-tetrads, and thereby engage in 𝜋-𝜋 stacking
interactions with the two tetrads[73]. Treatment with telomestatin has shown to prevent
proliferation and induce apoptosis of various cancer cells at noncytotoxic concentrations, with
relatively less of an effect on somatic cells[74].
A) B)

Figure 14: Structure of the two telomeric ligands TMPyP4 and Telomestatin[95].

on the other hand, the cationic porphyrin TMPyP4 is another molecule with an inhibitory
function on telomerases that has been extensively studied, in order, to promote or maintain the
G-quadruplexes structures in telomeric sequences of DNA. This molecule has the same
function as telomestatin, but with a distinct structure[75][76]. (Figure 14.A) One of the
structural characteristics of this molecule is the presence of four cationic functional groups
that allows it to bind to telomeric G-quadruplex structures through interacting and binding to
the stacked G-tetrads via electrostatic attraction and interaction[47]. However, after many
studies, it has been concluded that telomestatin is more effective in stabilizing G-
quadruplexes both in single and in duplex telomeric sequences of the DNA. This is because it
has a higher affinity and selectivity to telomeric G-quadruplexes than the interactive
compound TMPyP4, which can undergo non-specific interaction with double-stranded DNA
due to the positive charges that it possesses[77].
Telomestatin has been shown great results in suppressing telomerase-positive cells by
inducing the formation of G-quadruplexes and stabilizing them within very short periods.
While TMPyP4 facilitates preferentially the formation or stabilization of intermolecular
(several strands) G-quadruplex structures and suppresses both the proliferation of telomerase-
positive cells and the Alternative Lengthening of Telomeres (ALT-positive) cells but within
longer periods[75].
Accordingly, Telomestatin proved to be more efficient to inhibit telomerase activity in
telomeric sequence of the DNA[78], while TMPyP4, for its greater interaction with the

21
hTERT gene (gene that encode the TERT protein, one of the main components needed for the
activation of telomerase) and its transcription factor c-Myc, it has been further considered for
targeting G-quadruplexes in other parts of the DNA, as promoter regions[75].
Telomestatin ligand has an effective inhibition of the telomerase, suppressing proliferation
with no growing effect, but mostly showed in vitro, although there is in vivo evidence of
positive responses in leukemia cell. Telomestatin is, among other similar ligands, in pre-
clinical trials[73].(Figure 15)

Figure 15: Schema that models the G-quadruplex´s formation in telomeric DNA. Telomestatin
ligands can inhibit helicases, induce the formation of new G-quadruplexes and stabilize the formed G-
quadruplexes through binding to it from above and below the tetrads [79].( The figure has been
modified)

5.2.5.2 Stabilization of the G-quadruplex structures in c-Myc oncogenes

Stabilization of secondary structures of DNA has been the subject of studies in the past two
decades. As mentioned earlier, there has been conducted a number of experiments in vitro, in
order to promote or maintain G-quadruplex structures in telomeric sequences of the DNA.
The importance and the effect of two ligands, Telomestatin and TMPyP4, on the activity of
the telomerase enzyme, which is usually activated in most cancer cells, has been discussed.
As discussed earlier in this investigation, the latter discovery of these secondary structures in
promoter regions of the DNA, has opened new discussions concerning which region of the
two, telomeric or promotor region, will it be more effective to target G-quadruplexes in. For
several reasons, including the fact that telomerases are not yet confirmed to be a clinically
validated target, even though their activation is observed in a big percentage of cancer cells,
the formation and maintenance of G-quadruplex structures have a significantly bigger

22
importance in promoter areas of oncogenes[80]. For that reason, stabilization of G-
quadruplexes has been a main target for cancer drug development, and since then, a number
of small molecules have been developed to inhibit transcription in promoter regions of the
DNA.
Some of the common features among these small molecules-ligands is a fused-ring system
with a flat aromatic surface, which has the ability to cable to the terminal G-tetrads and some.
Another feature is that some of them possess positively charged side-chains, that replaces the
need of monovalent cations in that region and facilitate the formation of G-quadruplexes.

One of the positive qualities concerning the structure of G-quadruplexes is its polymorphism,
it gives easier access to a variety of ligands. Moreover, different ligands have different
affinities to some kinds of G-quadruplex structures. As reviewed before in Telomestatin and
TMPyP4, telomestatin, for example, induces and facilitates the formation or stabilization of
G-4 both in single (intra molecular) and in duplex (inter molecular) telomeric sequence of the
DNA, also in absence of potassium cations. While TMPyP4 facilitates preferentially the
formation or stabilization of intermolecular (several strands) G-quadruplex structures,
preferentially at promoter regions. Summing up, it appears to be relevant that different small
molecules has a different affinity to a specific structure of G-quadruplex, which is highly
related to its biological function.

Consequently, there has been developed a number of G-quadruplexes-targeting-small-

molecules-ligands for targeting G-quadruplexes in promotor regions, taking as a reference,
the earlier studies in telomeric region, and the ligands developed to target them.

For targeting G-quadruplexes in promotor regions of cancer cells, Quarfloxin is the first drug
in Phase II in clinical trials as a therapy for neuroendocrine/carcinoid cancer tumors[70].
Laurence Hurley and his colleagues developed a G-quadruplex-interactive drug that can
interact with duplex DNA-Topoisomerase II complex.(Figure 16.a) This compound called
QQ58 was created from a ligand called norfloxacin. After this contribution, another
laboratory, Cylene Pharmaceuticals, optimized the functionality of the ligand QQ58 by
making some modifications to its structure that can increase its selectivity to a particular G-
quadruplex structure.(Figure 16.b) Thus, they were able to develop fluoroquinolone CX-
3543, called Quarfloxin, having higher selectivity for G-quadruplexes(particularly parallel G-
quadruplexes) and the ability to eliminate topoisomerase II activity[5].

23
 b

Figure 16: Hurley Laboratory created QQ58(an intermediate compound)to target G-quadruplexes,
however, with the contribution of Cylene Pharmeaceuticals, the small molecule Quarfloxin, formed of
a system of rings, was developed and entered phase II trials[70].(The figure has been modified)

Quarfloxin was shown to inhibit topoisomerase II and I activity, it has been shown to be a
potentially tumor selective compound both in vivo and in vitro[81]. Moreover, Quarfloxin has
high selectivity for parallel G-quadruplexes, which is the structure formed in promotor
regions of c-Myc genes, and was, therefore, shown to selectively target c-Myc G-
quadruplexes, in vitro, by interacting with the site between the π-π patterns of the G-
tetrads[2]. Furthermore, Quarfloxin was found interacting with G-quadruplex structures in
ribosomal DNA (rDNA), where some of these structures have high affinity to nucleolin.
Nucleolin, as mentioned earlier, facilitates the folding of the single-stranded DNA form into
secondary DNA structures in c-Myc genes, and then stabilizes them upon binding. Quarfloxin
disrupts the binding of rDNA quadruplexes and nucleolins, redistributes the nucleolins and
inhibit ribosome biogenesis. The redistribution of nucleoulin allows them to bind to G-
quadruplexes formed in c-Myc promotor regions, which, in return, stresses the cancer cells,
inducing cancer cell apoptosis[30]( Figure 17)This characteristic only occurs in cancer cells,
but not in normal cell, displaying low toxicity and has shown positive response in patients[5].

24
Figure 17: Schema modelling the mechanism where nucleolin is replaced in the nucleoplasm due to
the binding of the ligand Quarfloxin and a G-quadruplex structure [70].(The figure has been modified)

On the other hand, the small molecule TMPyP4, as mentioned before, has a great affinity to a
variety of G-quadruplexes. In telomeres, TMPyP4 facilitates preferentially the formation or
stabilization of intermolecular (several strands) G-quadruplex structures and suppresses both
the proliferation of telomerase-positive cells and alternative lengthening of telomeres of
ALT-positive cells[76]. However, further investigations showed that TMPyP4 also
downregulates c-Myc expression, and this contributed to its observed effect on telomerase
activity, where the expression of hTERT is downregulated, since it is a downstream target
gene of the transcription factor c-Myc[82]. So, since c-Myc regulates the gene hTERT that
activate the telomerase enzyme, this means that via this path TMPyP4 can become a great
inhibitor of telomerase from the promoter region. From this approach, a number of
experiments have been made, in order to increase the affinity and binding of this ligand to
non-telomeric G-quadruplexes.

This ligand´s selectivity is based on its physicals conditions. TMPyP4 presents a fused planar
ring system, it is positively charged and that facilitates its bonding to the majority of the G-
tetrads[47].(Figure 14.A) Up to the present days, TMPyP4 is known for its anti-proliferative
ability, its relatively nontoxicity (to normal and cancer cells) and its extreme affinity to bind

25
 G-quadruplexes, stacking them usually externally[81]. However, one of its major weakness is
its, previous mentioned, ability to bind to all sorts of G-quadruplexes structures, such as
duplex DNA, RNA, RNA-DNA hybrids and triplex DNA. For that reason, this ligand is in
actual studies to develop a second generation with higher order of selectivity[19][48].

5.2.5.3 Tables with ligands developed to target G-quadraplexes

m

Table 1: Up summery of the structure, affinity, selectivity, state, strengths and weakness of
three small molecules-ligands both for telomeric and non-telomeric G-quadruplexes(present
in c-Myc).

G4 ligand Structure[95] Affinity Selectivity State Discussion

Duplex DNA, Low 2nd Ability to inhibit

RNA, RNA- generation telomerase, but functioning
DNA hybrids not satisfactory.
and triplex Ability to stabilize G-4 in
TMPyP4 DNA. promoter regions and
C44H38N8+4 downregulate c-Myc.
High affinity to many
different G-4 structures and
low selectivity.

Single and High 1st generation Ability to stabilize G-4 only

double stranded in the telomeric sequence of
DNA, also in the DNA.
Telomestatin the absence of Ability to shrink tumors
C26H14N8O7S K+ and shorten telomeres
sequences.
Development is getting
slower because of the
importance of targeting
cancer cell directly from the
promoter regions.
Single strand High Phase II in Ability to stabilize G-
DNA, parallel clinical trials quadruplexes in the
G-quadruplexes promoter region.
High selectivity and affinity
Quarfloxin to parallel G-4, usually
C35H33FN6O found in c-Myc genes.
3 Anti-proliferative and
shrinking abilities.
In phase II in clinical trials
only for
neuroendocrine/carcinoid
cancer tumors.

26
Table 2: Selected data in vivo concerning the effect of the ligands TMPyP4, Telomestatin and
Quarfloxin on some variants of tumors.

G4 ligand Tumor model Initial Tumor Response Time in days

Mass/mg Size/mm3

MX-1 100 mg - Survival 60

mammary tumor increase from
TMPyP4[82] 45% to 75%
PC-3 60 mg - 60% tumor 18
human prostate shrinkage
carcinoma
Telomestatin[6] U937 - 1395 mm3 80% tumor 21
human lymphoma shrinkage
MIA PaCa-2 - > 125 mm3 59% tumor 35
human pancreatic shrinkage
Quarfloxin[83] cancer
MDA-MB-231 - > 125 mm3 50% tumor 37
human breast shrinkage
cancer

27
Table 3: Models in 3D showing the interaction between the three different ligands and G-
quadruplexes:

3D figures with Ligand/G-quadruplex Description

interactions[100][86]
Telomestatin bonds telomeric G-
quadruplexes through external
stacking, allowing two ligands to stack
externally to the G-quadruplex
structure.(Figure 15)

It also has the ability to capture a

potassium cation either from the G-
quadruplex or from the solution
surrounded[84].

TMPyP4 has high affinity to G-

quadruplexes, therefore, it interacts
and binds differently depending on the
structure of the G-quadruplex.

For example, it can stack intercalating

between stacked G-tetrads or by an
end-stacking mechanism[85].

Quarfloxin bonds selectively non-

telomeric G-quadruplexes and stack
intercalating between the G-tetrads
[39].

28
 4.3 Discussion/Conclusion

The aim of this investigation was to answer the following research question: Is it possible to
induce cancer cell apoptosis through targeting G-quadruplex structures in c-Myc
oncogenes?
In order to achieve our aim, we started our investigation by investigating the importance of
the transcription factor c-Myc in cancer cell, and in promoting tumorigenesis. According to
our findings c-Myc plays a vital role in different mechanisms that are crucial for cancer
survival. This confirmed our expectations concerning the possibility of putting cancer cells
under stress and at risk of committing apoptosis, if c-Myc oncogene was targeted. Then we
took a further step into investigating and understanding the mechanisms by which the c-Myc
gene expression is controlled, in order to approach a possible pathway that can be followed to
target and downregulate this mutated gene. We found that the secondary DNA G-quadruplex
structure that forms within the nuclease hypersensitive element NHE III1 of the c-Myc gene,
which control a very high percentage of c-Myc transcription, is capable of silencing its gene
expression. This structure was initially discovered in telomeric regions and had received a lot
of attention. Since the discovery of this structure in the DNA, many scientific advances have
been done. Then was started to investigate the possible way to target this secondary structure
in c-Myc genes.We found that the polymorphism of this structure allows a variety of ligands
to access it and change its conformation in order to have a specific functionality.
Since the main purpose for developing cancer drugs is to target the transcription factor that is
in charge of cancer cell proliferation and division, we came to the hypothesis that :
• Small molecules will stabilize G-quadruplexes in order to maintain their structure
• If G-quadruplexes get stabilized, they will inhibit overexpression of the c-Myc
oncogene, thereby controlling cell proliferation.
• If inhibited overexpression of c-Myc, apoptosis will be induced.

There have been thousands of compounds created to stabilize G-quadruplexes, where some
have shown the ability to do so, but not very effectively. Firstly in our investigation, we took
into consideration two compounds Telomestatin and TMPyP4 that has been used as ligands in
telomeric regions to target G-quadruplexes. This was taken, in order to follow the progress in
the development of G-quadruplexes-targeting-ligands and some structural features that
increased the selectivity and affinity of these ligands to specific G-quadruplex structure, in
addition to the ways ligands interact with G-quadruplexes. But, even though these two ligands
had high affinity to the some G-quadruplex structures and had the ability to inhibit telomerase
activity, which is expressed in most of cancer cells, telomerase is not yet a clinically validated

29
target and because inhibiting telomerase does not guarantee a treatment for cancer, drug
development and researches for targeting G-quadruplexes in telomeric sequences of DNA has
been replaced for researches in non-telomeric G-quadruplexes (c-Myc promotor region) .

After having the experience of telomeric G-quadruplexes, the focus changed due to the
discovery of the formation of these structures in promoter regions, especially in c-Myc.
Downregulation of expression of a target oncogene may be more important for tumor
progression in a particular tumor type than telomerase. c-Myc G-quadruplexes have been
targeted by a number of small molecules, such as a ligand called Quarfloxin and once again
the ligand TMPyP4. Stabilization was possible and, in a high degree, responses for the
inhibition of c-Myc oncogene overexpression was observed by the shrinkage of the tumors
confirming our other expectation and answers our research question, that targeting G-
quadraplexes within c-Myc oncogenes will inhibit c-Myc´s overexpression and thereby cause
cancer cell apoptosis.

However, this is not sufficient enough to be considered a therapy for treating cancer because
many difficulties concerning the selectivity of these ligands need to be sorted out. This is
because thousands of sequences in the human genome can potentially form G-quadruplex
structures, and due to lack of understanding of the binding preferences of these ligands in
addition to lack of definitive structures of the G-quadruplexes formed in these sequences
make it more difficult to increase the selectivity of these ligands. In that case, TMPyP4 could
not be a target drug because of its low selectivity, but further development is being conducted.
On Contrary, Quarfloxin showed to be a great ligand for targeting c-Myc G-quadruplexes,
inhibiting topoisomerase and inducing the cell to apoptosis with low cytotoxicity and without
affecting somatic cells.

Therefore, we can conclude that our hypothesis seems to be confirmed with some nuances,
due to the challenges found during the development of ligands-drugs. We have learnt that
ligands need high affinity, selectivity in order to proceed and enter next clinical trials, yet this
is still a challenge taking into consideration that out of thousands ligands, just one was
capable of proceeding and entering Phase II in clinical trial. Furthermore, ligands having low
toxicity is another challenge considered, where in our investigation the ligands Quarfloxin,
Telomestatin and TMPyP4 had low toxicity levels and did not affect the healthy cells, but
other ligands can be toxic at certain doses.

30
Although Quarfloxin is still in Phase II, investigations show that it is very promising and
suggests that medicinal chemistry optimization in the future could develop other similar
ligands to target different carcinomas. Moreover, the development of a second generation of
TMPyP4 also have great expectative, because if it is possible to remodel its structure for
raising its selectivity to parallel or antiparallel G-quadruplexes. It will be a great ligand for
cancer treatments, possibly even more promising than the existent one. This is believed
because, TMPyP4 is a very small molecule, that can stack the G-quadruplexes from side to
side, from external and from in between, it has great affinity to secondary structures and low
toxicity. All these qualities make this ligand a potential future drug, that could maybe target
not only one carcinoma, but several ones.
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IX
7. Glossary:

Definitions taken from online databases [102],[96],[98] and [97]

Alternative Lengthening of Telomeres:

ALT is defined as telomere length maintenance that is not dependent on telomerase
activity.
Angiogenesis:
Physiological process through which new blood vessels form from pre-existing
vessels
Basic helix-loop-helix
The basic helix-loop-helix proteins are dimeric transcription factors that are found in
almost all eukaryotes.
Burkitt’s lymphoma
A cancer of the lymphatic system
Chaperone proteins
Chaperones are a functionally related group of proteins assisting protein folding in the
cell under physiological and stress conditions.
Chromatin:
Chromatin is a complex of DNA and proteins that forms chromosomes within the
nucleus of eukaryotic cells
Chromosomes:
In the nucleus of each cell, the DNA molecule is packaged into thread-like structures
called chromosomes. Each chromosome is made up of DNA tightly coiled many times
around proteins called histones that support its structure. Each chromosome has a
constriction point called the centromere, which divides the chromosome into two
sections, or “arms.” The short arm of the chromosome is labeled the “p arm.” The long
arm of the chromosome is labeled the “q arm.” The location of the centromere on each
chromosome gives the chromosome its characteristic shape, and can be used to help
describe the location of specific genes
Dimerization:
Union of two molecules. Specific protein dimerization is integral to biological
function, structure and control, and must be under substantial selection pressure to be
maintained with such frequency throughout biology.
Downstream:
refer to a relative position in DNA or RNA
Enhancer box sequence:
An enhancer is a regulatory DNA sequence, often hundreds of bases long, which can
exert its effect over large genomic distances

FUSE element:
A far upstream element (FUSE) of c-Myc stimulates promoter activity when bound by
a newly identified trans-acting protein, which is expressed in cycling cells.

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G-rich DNA:
area of the DNA that is rich in guanine.

Gyrases:
Gyrase belongs to a class of enzymes known as topoisomerases that are involved in
the control of topological transitions of DNA
Heterodimer:
A molecule composed of paired proteins with some amino-acid sequence variations
Helicases:
Helicases are a class of enzymes vital to all living organisms. Their main function is to
unpackage an organism's genes. They are motor proteins that move directionally along
a nucleic acid phosphodiester, separating two annealed nucleic acid strands (i.e..
DNA, RNA, or RNA-DNA hybrid) using energy derived from ATP hydrolysis. Many
cellular processes
Histone acetylation:
Acetylation is the process where an acetyl functional group is transferred from one
molecule (in this case,Acetyl-Coenzyme A) to another. Acetylation removes the
positive charge on the histones, thereby decreasing the interaction of the N termini of
histones with the negatively charged phosphate groups of DNA. The condensed
chromatin is transformed into a more relaxed structure that is associated with greater
levels of gene transcription.
Hoogsteen hydrogen bonding:
A variation of base-pairing in nucleic acids such as the A•T pair. In this manner,
two nucleobases, one on each strand, can be held together by hydrogen bonds in the
major groove. A Hoogsteen base pair applies the N7 position of the purine base (as
a hydrogen bond acceptor) and C6 amino group (as a donor), which bind the Watson-
Crick (N3–N4) face of the pyrimidine base.

hTERT gene:
Expression and function of hTERT gene are known to be regulated at various
molecular levels. However, the transcription of hTERT has been suggested to be the
dominant step in the regulation of telomerase activity
Human genome:
A genome is an organism’s complete set of DNA, including all of its genes. Each
genome contains all of the information needed to build and maintain that organism. In
humans, a copy of the entire genome—more than 3 billion DNA base pairs—is
contained in all cells that have a nucleus.
MBI and MBII:
Myc boxes I and II contain sufficient information to independently direct the
degradation of otherwise stably expressed proteins to which they are fused.
MIA PaCa-2:
Human pancreatic carcinoma cell line
Mitogenic signals:
Signals to induce a cell to commence cell division.

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MDA-MB-231:
Breast cancer cell line
Mx-1 tumor cell:
Metastasis cannot be measured.
Nuclease hypersensitive element:
Genetic control elements are usually situated in local regions of chromatin that are
hypersensitive to structural probes such as DNase I
Nucleosome-free:
Nucleosome-free regions (NFRs) at the 5′ and 3′ ends of genes are general sites of
transcription initiation for mRNA and noncoding RNA (ncRNA)
Oncogenes:
Any gene that encodes a protein able to transform cells in culture or to induce cancer
in animals. Of the many known oncogenes, all but a few are derived from normal
cellular genes (i.e., proto-oncogenes) whose products participate in cellular growth-
controlling pathways
PC-3:
Human prostate cancer cell
PML bodies:
The promyelocytic leukemia (PML) body has been implicated in many different
cellular pathways and is characterized by the presence of the PML protein, first
identified in patients with acute promyelocytic leukemia
Polymorphism:
Having many different forms
Positive and Negative supercoiling:
Positive supercoiling of DNA occurs when the right-handed, double-helical
conformation of DNA is twisted even tighter (twisted in a right-handed fashion) until
the helix begins to distort and "knot." Negative supercoiling, on the other hand,
involves twisting against the helical conformation (twisting in a left-handed fashion),
which preferentially underwinds and "straightens" the helix at low twisting stress, and
knots the DNA into negative supercoils at high twisting stress.
Promotors:
Promoter sequences are DNA sequences that define where transcription of a gene by
RNA polymerase begins. Promoter sequences are typically located directly upstream
or at the 5' end of the transcription initiation site. RNA polymerase and the necessary
transcription factors bind to the promoter sequence and initiate transcription. Promoter
sequences define the direction of transcription and indicate which DNA strand will be
transcribed; this strand is known as the sense strand.
RAP1:
(Ras-proximate-1 or Ras-related protein 1) is a small GTPase , which are small
cytosolic proteins that act like cellular switches and are vital for effective signal
transduction

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Reverse transcriptase protein:
(Abbreviated to TERT, or hTERT in humans) is a catalytic subunit of the enzyme
telomerase, which, together with the telomerase RNA component (TERC), comprises
the most important unit of the telomerase complex
rDNA:
Ribosomal DNA is a DNA sequence that codes for ribosomal RNA. Ribosomes are
assemblies of proteins and rRNA molecules that translate mRNA molecules to
produce proteins.
Somatic cells:
Any biological cell forming the body of an organism; that is, in a multicellular
organisms, any cell other than a gamete, germ cell, gametocyte or undifferentiated
stem cell.
In contrast, gametes are cells that fuse during sexual reproduction, germ cells are cells
that give rise to gametes, and stem cell are cells that can divide through mitosis
and differentiate into diverse specialized cell types
Sp1
A zinc finger transcription factor that binds to GC-rich motifs of many promoters
Structural motifs:
Secondary structure of the DNA
TATA binding protein, TBP:
The TATA-binding protein (TBP) is a general transcription factor that binds
specifically to a DNA sequence called the TATA box.
Telomerase:
Telomerase is the ribonucleoprotein enzyme that catalyzes the extension of telomeric
DNA in eukaryotes.
TFIIH:
The transcription initiation factor TFIIH is a remarkable protein complex that has a
fundamental role in the transcription of protein-coding genes as well as during the
DNA nucleotide excision repair pathway.
Topoisomerase I:
In molecular biology Type I topoisomerases are enzymes that cut one of the two
strands of double-stranded DNA, relax the strand, and reanneal the strand.

Topoisomerase II:
Topo II are enzymes called DNA gyrase that have the ability to cut both strands of a
double-stranded DNA molecule, pass another portion of the duplex through the cut,
and reseal the cut in a process that utilizes ATP

Topoisomerase III:
Topoisomerases III relax negatively supercoiled DNA and also catenate/decatenate
DNA molecules containing single-stranded DNA regions.

Transcription factor:
Transcription factors are proteins involved in the process of converting, or
transcribing, DNA into RNA. Transcription factors include a wide number of proteins,

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 excluding RNA polymerase, that initiate and regulate the transcription of genes. One
distinct feature of transcription factors is that they have DNA-binding domains that
give them the ability to bind to specific sequences of DNA called enhancer or
promoter sequences

Tumorigenesis:
Carcinogenesis or oncogenesis or tumorigenesis is the formation of a cancer, whereby
normal cells are transformed into cancer cells

Tumor suppressors:

Tumour-suppressor proteins act to alleviate the potential for cancer and tumour
formation by modulating cell growth either through negative regulation of the cell
cycle or by promoting apoptosis. Mutation or dysregulation of tumour-suppressor
proteins can lead to unregulated cell growth and tumour development

Upstream:
Refer to a relative position in DNA or RNA.
U937:
A model cell line used in biomedical research

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