MINISTRY OF EDUCATION AND RESEARCH University of Medicine and Pharmacy of

Craiova DOCTORAL SCHOOL

Phd THESIS

ABSTRACT

NEW TREATMENT STRATEGIES FOR

GLIOBLASTOMA MULTIFORME IN VITRO

SCIENTIFIC COORDINATOR:

PROF.UNIV.DR. DRICU ANICA

PhD student:

FOLCUȚI CĂTĂLIN-ALEXANDRU

CRAIOVA

2020

TABLE OF CONTENTS

1. BRAIN TUMORS. OVERVIEW .................................................................................. 1

2. GLIOBLASTOMA ..................................................................................................... 1

2.1. Tumor heterogeneity and recurrence (classical, proneural, neural and mesenchymal subtypes) ........................................................................................ 1

2.2. The genetic and molecular factors in glioblastoma ........................................ 1

2.2.1. Epithelial growth factor receptor (EGFR) ................................................. 1

2.2.2. PDGFR ...................................................................................................... 2

2.2.3. PTEN ......................................................................................................... 2

2.2.4. MGMT ...................................................................................................... 2

2.2.5. IDH1/2 ...................................................................................................... 2

2.2.6. β-arrestin .................................................................................................. 2

2.3. GBM therapy ................................................................................................... 2

3. PERSONAL CONTRIBUTION .................................................................................... 3

3.1. WORK HYPOTHESIS AND GENERAL OBJECTIVES ............................................. 3

3.2. RESULTS AND DISCUSSION .............................................................................. 4

3.2.1. Meta-analytic investigation of dendritic cell vaccination and viral therapy in patients with malign glioma. ............................................................ 4

3.2.2. The effect of EGFR inactivation on malign glioma cells viability ............. 4

3.3. STUDY OF Β-ARRESTINE 1 TRANSFECTION INFLUENCE ON MALIGN GLIOMA CELL PROLIFERATION AND ON THE RESPONSE OF E TREATMENT ....................... 5

4. CONCLUSIONS ........................................................................................................ 9

BIBLIOGRAPHY ……………………………………………………………………………………………………..9

1

KEY WORDS: glioblastoma, therapy, arrestin, temozolamide

1. BRAIN TUMORS. OVERVIEW

Brain tumors are the most severe form of tumors, considering the fact they are

malign cell masses that affect vital systems in neurological balance regulation (1). They

are considered to be primary, classified according to the type of cell that generated them

and secondary – cerebral metastases (2).

Gliomas are the most frequent type of central nervous system tumors, representing

almost 80% of primary brain tumors. These types of tumors can develop at any age, with a

peak in the 5th-6th decade. Glial tumors are characterized by an important intratumoral

heterogeneity, proliferative potential and tumor recurrence (1). Taking into consideration

these properties, the overall survival is less that 15 months, despite the latest therapeutic

approaches (3).

The study of histological characteristics of gliomas is crucial in identifying the

molecular mechanisms underlying the actual limitations of therapy and their resistance to

chemotherapy and radiotherapy.

2. GLIOBLASTOMA

The overall survival in glioblastoma, the most aggressive, invasive and

undifferentiated brain tumor, is 14,6 months (4), despite modern multimodal therapeutic

modalities.

2.1. Tumor heterogeneity and recurrence (classical, proneural, neural and

mesenchymal subtypes)

Glioblastoma is known for its’ heterogeneity regarding signaling pathways, tumor

site, phenotype, genetics, epigenetics and molecular properties.

2.2. The genetic and molecular factors in glioblastoma

2.2.1. Epithelial growth factor receptor (EGFR)

Specialized studies from the last decade have shown that over 50% of GBM present

EGFR mutations. The most common is EGFR amplification (approximately 40% of EGFR

amplification are EGF mutant) (3).

2

2.2.2. PDGFR

PDGFR overexpression is present in ~23% cases of GBM, having unfavorable

repercussions on patients prognosis and survival with IDH1 mutation.

2.2.3. PTEN

40% of GBM cases have shown an early diminution of PTEN expression (5-7), and

in 50-70% of recurrent GB, respectively in 50-90% of primary GB was found a loss of

chromosome 10 heterozygosis (8-9).

2.2.4. MGMT

MGMT methylation determine a growth in tumor cell sensitivity to the action of

alkylant agents like TMZ. In low grade gliomas, the role and clinical impact of MGMT

methylation status is still in scientific debate.

2.2.5. IDH1/2

Recent studies have shown that the most frequent mutation in gliomas is IDH1

R132H (10). Also, there have been observed IDH mutations in 80-90% of grade II and III

gliomas (10-11).

2.2.6. β-arrestin

A recent in vitro study conducted on GBM tumor cells has shown a growth of their

sensitivity to TMZ through β-arrestine1 gene activation (12). The exact role of β-arrestine

in gliomagenesis, proliferation and therapeutic response are still insufficient.

2.3. GBM therapy

GBM is still a therapeutic challenge despite multimodal and interdisciplinary

approaches (13). The classic therapeutic methods are surgery, chemotherapy and

radiotherapy. The new generation of therapeutic modalities consists in molecular target

therapy, immunotherapy, viral therapy, adoptive therapy, dendritic cell vaccination and

genetic therapy.

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3. PERSONAL CONTRIBUTION

3.1. WORK HYPOTHESIS AND GENERAL OBJECTIVES

OBJECTIVE NO. 1 Meta-analytic investigation of viral therapy effect comparing to immune therapy

with dendritic cells in malign gliomas.

OBJECTIVE NO. 2

In this study we analyzed the potential of EGFR-targeting on malign glioma cells

viability.

OBJECTIVE NO. 3 Study of β-arrestine 1 transfection influence on cell proliferation and on the response

of malign gliomas to the e treatment.

3.2. RESULTS AND DISCUSSION

3.2.1. Meta-analytic investigation of dendritic cell vaccination and viral therapy

in patients with malign glioma.

3.2.1.2. OS and PFS meta-analysis in dendritic cell vaccination

Our systematic analysis integrated 9 specialized studies. In these studies, were

included 357 patients, from which 104 patients were in experimental groups and 253 in

control groups. Of these, 207 patients were newly-diagnosed with HGG (70 patients in

experimental group and 137 patients in control group) and 150 patients were diagnosed

with recurrent HGG (34 patients in experimental group and 116 in control group).

Regarding therapy combined with dendritic cell vaccination, a PFS improvement was

noticed (HR 0,49), with CI 95% 0,21-1,16. Statistically, results of dendritic cell

vaccination therapy of HGG patients was not significant (p=0,10).

3.2.1.3. OS and PFS meta-analysis of viral therapy

In the four studies integrated in the systematic analysis of viral therapy we identified

642 newly-diagnosed HGG patients, the experimental group included 237 patients, and

control group included 405 patients. In total, the OS meta-analysis integrated 4 studies,

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and PFS meta-analysis included 3 studies. In three studies we observed an OS progress,

the most notable one being described by Wheeler et al (14) (HR 0,72, 95% CI: 0,54-0,97).

On the other hand, Rainov (15) reported a benefit for the patients from the control group

(HR 1.08, 95% CI: 0,81-1,46).

3.2.2. The effect of EGFR inactivation on malign glioma cells viability

3.2.2.1. The growth pattern of untreated HGG cells

In this study we analyzed the proliferation of 11 HGG cell line in 7 days.

Fig. 1. The growth pattern of HGG cells

3.2.2.2. The effect of EGFR inactivation on HGG cells

In this study we investigated the effect of AG556 on 11 HGG cells in order to

conclude on the cytotoxicity of this agent. HGG cells were exposed to different

concentrations of AG556: 10 µM, 20 µM and 30 µM. The cell proliferation rates were

measured in day 3 and day 7. We observed that the most significant cytotoxic effect was

reached at the concentration of 30 µM, with a diminish of survival malign cells with

approximately 17% in day 3 (Fig 2). This value was constant and there were no significant

changes the rest of the experiment, including day 7 of AG 556 treatment (Fig.3).

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Fig. 2, 3. The effect of AG556 inhibitor on EGFR inactivation in 11 HGG cell line at

3, respectively 7 days of treatment.

3.3. STUDY OF Β-ARRESTINE 1 TRANSFECTION INFLUENCE ON

MALIGN GLIOMA CELL PROLIFERATION AND ON THE RESPONSE OF E

TREATMENT

3.3.1. The TMZ treatment effect on malign glioma cells

Our experiment tested the cytotoxic effect of the alkylating agent TMZ on two

different malign cell lines: 18 HGG and U-343MGa. We analyzed the decrease of malign

cell proliferation at 24h, 48h and 72h after treating them with different concentrations of

the alkylating agent (200 µM, 250 µM and 300 µM).

0

50

100

150 11

7days

050

100150

U-343MGa

24h0

50100150

U-343MGa

48h

6

Fig. 4, 5, 6. The effect of TMZ treatment on U-343MGa cell line at 24h, 48h and 72h

24 hours from the beginning of the treatment with 200 µM, we observed a decrease

of the cell viability with 17,32%. 250 µM concentration determined a 17,88% decrease

and the maximum concentration determined the highest cytotoxicity of 20,17% (Fig.4).

Regarding the prolonged treatment at 48h, we recorded a significant increase of the

cytotoxic effect in U-343MGa cell line, as follows: at 300 µM we observed the most

important cytotoxic effect (45,3%) (Fig 5). At 72 h from the TMZ treatment initiation,

tumor cells cytotoxicity was reduced comparing with the one recorded at 48 hours (Fig 6).

Regarding the second cell line, line 18 HGG, treated with the same concentrations of

alkylating agent for 24, 48 and 72 hours, we observed similar values with U-343MGa cell

line (Fig 7, 8, 9).

0

50

100

150U-343MGa

72h

0

50

100

150

18HGG

48h

7

Fig. 7, 8, 9. Efectul tratamentului cu TMZ la 24, 48, 72 h în linia celulară 18 HGG

3.3.2. Study of β-arrestine-1 transfection influence on the response of TMZ

treatment in U-343MGa cell line

Fig. 10, 11, 12. β-arrestine 1 transfection influence on the response of temozolomide treatment in U-343MGa cell line at 24, 48 and 72 hours

After 24 hours of treatment, β-arr1 transfection countered by ~4% the effect of 200

µM TMZ treatment, but this result cannot be considered statistically significant (p>0.05)

(Fig. 10). The results showed that U-343MGa cell line proliferation recorded a significant

0

50

100

15018HGG

72h

-20

30

80

130

180

Control TMZ200µM

TMZ250µM

TMZ300µM

U-343MGa 24h treatment

Untransf…

-20

30

80

130

180

Control TMZ200µM

TMZ250µM

TMZ300µM

U-343MGa 48h treatment

Untransf…

0

50

100

150

200

Control TMZ200µM

TMZ250µM

TMZ300µM

U-343MGa 72h treatment

Untransfe…

8

growth after 48 hours from β-arr1 transfection (26%), comparing with the control group.

(p≤0.05) (Fig. 11). 72 hours from the beginning of TMZ treatment in high dose, the β-arr1

did not prevent cytotoxicity (p≥0.05) (Fig. 12).

3.3.3. Study of β-arrestine 1 transfection influence on the response of

temozolomide treatment in 18 HGG cell line

β-arrestine 1 transfection caused the decrease of 18 HGG cell viability and presented

a moderate influence on the cytotoxic effect of TMZ treatment.

Fig. 13, 14, 15. The influence of β-arr transfection on TMZ treatment at 24, 48 and 72 hours

020406080

100120 18HGG 24h treatment

020406080

100120

Control TMZ200µM

TMZ250µM

TMZ300µM

18 HGG 48h treatmentUntransfected

020406080

100120

Control TMZ200µM

TMZ250µM

TMZ300µM

18 HGG 72h treatment

Untransfected

β1-Arestin transfected

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4. CONCLUSIONS

Our analysis demonstrated that the therapeutic approach based on dendritic cell

vaccination shows an improvement of patients with newly-diagnosed (HR=0.65) and

recurrent (HR=0.63) high grade glioma overall survival and PFS (HR=0.49) comparing to

viral therapy.

In our experiment, the EGFR inactivation with cytotoxic agent AG556 determined a

proportional decrease of cell proliferation with the inhibitor dose. This effect was

observed at 3 days of treatment, with no significant changes at the prolonged 7 days of

treatment.

Β-arr1 transfection determined a growth of cell proliferation in U-343MGa and

countered the temozolomide cytotoxic effect.

Β-arr1 transfection also determined the decrease of cell viability in 18 HGG cell line

and presented a moderate influence on the temozolomide cytotoxic effect.

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