Identification of Novel Markers and Molecular Mechanisms with Diagnostic, Prognostic, and Therapeutic Potential in Brain Tumors

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DEPARTAMENTO DE BIOLOGÍA CELULAR, FISIOLOGÍA E INMUNOLOGÍA

PROGRAMA DE DOCTORADO - BIOMEDICINA

Identification of Novel Markers and Molecular Mechanisms with Diagnostic, Prognostic, and

Therapeutic Potential in Brain Tumors

Identificación de Nuevos Marcadores y Mecanismos Moleculares con Potencial Diagnóstico, Pronóstico y

Terapéutico en Tumores Cerebrales

Antonio Carlos Fuentes Fayos

Directores

Raúl M. Luque Huertas Justo P. Castaño Fuentes

Córdoba, 28 enero 2022

Catedrático de Biología

Universidad de Córdoba Catedrático de Biología Universidad de Córdoba

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TITULO: Identification of Novel Markers and Molecular Mechanisms with Diagnostic, Prognostic, and Therapeutic Potential in Brain Tumors

AUTOR: Antonio Carlos Fuentes Fayos

https://www.uco.es/ucopress/index.php/es/

[email protected]

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TÍTULO DE LA TESIS: Identification of Novel Markers and Molecular Mechanisms with Diagnostic, Prognostic, and Therapeutic Potential in Brain Tumors

DOCTORANDO/A: Antonio Carlos Fuentes Fayos

INFORME RAZONADO DEL/DE LOS DIRECTOR/ES DE LA TESIS

Durante el desarrollo de la presente Tesis Doctoral, en el periodo comprendido entre octubre 2017 y enero 2022, el doctorando Antonio Carlos Fuentes Fayos no solo ha superado con creces los objetivos planteados al comienzo de esta, sino que ha desarrollado y validado técnicas experimentales novedosas de una gran utilidad para el grupo de investigación, que le han permitido obtener resultados muy relevantes en el campo del estudio del proceso de splicing y del metabolismo en tumores cerebrales.

El doctorando ha realizado dos estancias internacionales en centros de referencia en el área de neuro-oncología ambas en Estados Unidos de América [Cedars-Sinai Medical Center (Los Ángeles, CA) y Massachusetts General Hospital-Harvard Medical School (Boston, MA)]. Concretamente, como fruto de su trabajo durante este periodo, ha publicado 3 trabajos directamente relacionados con su Tesis Doctoral en revistas de referencia dentro del área de neurociencias y oncología molecular (“Brain”, “Journal of Experimental & Clinical Cancer Research” and “International Journal of Molecular Sciences”. Adicionalmente, el trabajo realizado en este periodo ha dado lugar a dos artículos uno actualmente bajo revisión y otro en preparación. Además, el doctorando ha colaborado con diferentes investigadores de su ámbito de forma fructífera publicando 4 revisiones, 1 comentario y 11 publicaciones relacionadas con su área de estudio.

Por último, el doctorando ha presentado los resultados de su Tesis en diferentes congresos de ámbito nacional e internacional, así como ha participado en el establecimiento de 2 patentes.

Por todo ello, se autoriza la presentación de la tesis doctoral.

Córdoba, 28 de enero de 2022

Firma de los directores

Fdo.: Dr. Raúl M. Luque Huertas Fdo.: Justo P. Castaño Fuentes

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DEPARTAMENTO DE BIOLOGÍA CELULAR, FISIOLOGÍA E INMUNOLOGÍA

D. Raúl Miguel Luque Huertas y D. Justo Pastor Castaño Fuentes, Catedráticos de Biología Celular del Departamento de Biología Celular Fisiología e Inmunología de la Universidad de Córdoba,

INFORMAN

Que D. Antonio Carlos Fuentes Fayos, Graduado en Bioquímica, ha realizado bajo nuestra dirección el trabajo titulado “Identification of Novel Markers and Molecular Mechanisms with Diagnostic, Prognostic, and Therapeutic Potential in Brain Tumors” y que, bajo nuestro juicio, reúne los méritos suficientes para optar al Grado de Doctor en Biomedicina.

Y para que conste, firmamos la presente en Córdoba, a 28 de enero de 2022.

Fdo.: Raúl M. Luque Huertas Fdo.: Justo P. Castaño Fuentes

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Esta Tesis Doctoral ha sido realizada en el Departamento de Biología Celular, Fisiología e Inmunología de la Universidad de Córdoba y en el Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), bajo la dirección de los Dres. Raúl M. Luque Huertas y Justo P. Castaño Fuentes.

Dicho trabajo fue subvencionado mediante proyectos/ayudas del MINECO (PID2019-105564RB-I00, PID2019-105201RB-I00), Junta de Andalucía (P20_00442; PEER-0048-2020, BIO-0139) y del Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y Nutrición (CIBERobn), con la financiación para el doctorando de una Ayuda para la Formación del Profesorado Universitario (FPU) del Ministerio de Educación, Cultura y Deporte (referencia FPU16/05059). Durante el transcurso de la presente Tesis Doctoral se han realizado dos estancias de tres meses cada una de ellas justificando la obtención de la Mención Internacional en el Título de Doctor de la Universidad de Córdoba:

- Departamento de Ciencias Biomédicas del Centro de Neurociencias en Medicina/Samuel Oschin Comprehensive Cancer Center del Cedars-Sinai Medical Center en Los Angeles (CA, EEUU) bajo la supervisión del Dr. Josh J. Breunig (mayo-julio, 2018) financiada por la ayuda de movilidad internacional de la Universidad de Córdoba y co-financiada por el Instituto Maimónides de Investigación Biomédica de Córdoba.

- Departamento de Patología Molecular en el Massachusetts General Hospital-Harvard Medical School en Boston (MA, EEUU) bajo la supervisión del Prof. Mario L Suvà (marzo-mayo, 2021) financiada por la ayuda complementaria para estancias breves y traslados temporales (EST21/00229), destinadas a beneficiarios del Subprograma de Formación del Profesorado Universitario del Ministerio de Universidades del Gobierno de España.

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List of Publications

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This Doctoral Thesis is founded on the scientific articles listed below, cited in the text as:

Article 1

Antonio C. Fuentes-Fayos, Mari C. Vázquez-Borrego, Juan M. Jiménez-Vacas, Leire Bejarano, Sergio Pedraza-Arévalo, Fernando L-López F, Cristóbal Blanco-Acevedo, Rafael Sánchez-Sánchez, Óscar Reyes, Sebastián Ventura, Juan Solivera, Joshua J. Breunig, María A. Blasco, Manuel D. Gahete, Justo P. Castaño, Raúl M. Luque.

Splicing machinery dysregulation drives glioblastoma development/aggressiveness:

oncogenic role of SRSF3.

2020. Brain. 143(11):3273-3293. DOI:10.1093/brain/awaa273.

[IF: 13.501 (2020); 6/208 (D1) Clinical Neurology (JCR); 10/273 (D1) Neuroscience (JCR)]

Article 2

Antonio C. Fuentes-Fayos, Miguel E. G-García, Jesús M. Pérez-Gómez, Annabel Peel, Cristóbal Blanco-Acevedo, Juan Solivera, Alejandro Ibañez-Costa, Manuel D. Gahete, Justo P. Castaño, Raúl M. Luque.

Somatostatin receptor splicing variant sst5TMD4 overexpression in glioblastoma is associated to poor survival, increased aggressiveness features, and somatostatin analogs resistance.

2022. International Journal of Molecular Sciences. 23(3), 1143. DOI:10.3390/ijms23031143 [IF: 5.924 (2020); 67/313 (Q1) Biochemistry & Molecular Biology (JCR)].

Article 3

Antonio C. Fuentes-Fayos, Jesús M. Pérez-Gómez, Miguel E. G-García, Juan M. Jiménez- Vacas, Cristóbal Blanco-Acevedo, Rafael Sánchez-Sánchez, Juan Solivera, Joshua J.

Breunig, Manuel D. Gahete, Justo P. Castaño, Raúl M. Luque.

SF3B1 inhibition disrupts malignancy and prolongs survival in glioblastoma patients through BCL2L1-splicing and mTOR/ß-catenin pathways imbalances.

2022. Journal of Experimental & Clinical Cancer Research. 41:39. DOI:10.1186/s13046- 022-02241-4

[IF: 11.16 (2020); 25/242 (Q1) Oncology (JCR)].

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Article 4

Antonio C. Fuentes-Fayos, Miguel E. G-García, Jesús M. Pérez-Gómez, Julia Martín- Colom, Carlos Doval-Rosa, Cristóbal Blanco-Acevedo, Encarnación Torres, Álvaro Toledano-Delgado, Juan M Jimenez-Vacas, Justo P. Castaño, Manuel D. Gahete, Juan Solivera, Raúl M. Luque.

Metformin and simvastatin exert additive antitumor effects in glioblastomas via senescence-state and telomere-lengthening: Clinical and translational evidence.

Manuscript in preparation for Metabolism - Clinical and Experimental.

Article 5

Antonio C. Fuentes-Fayos, et al.

RNA-exosome machinery is dysregulated in glioblastoma: DIS3 as a novel diagnostic and prognostic biomarker and a potential therapeutic target.

Manuscript is being prepared for submission soon.

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List of Abbreviations

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3'ss

3' splice site 5'ss

5' splice site A3SS

Alternative 3' Splice Site A5SS

Alternative 5' Splice Site AC-like

Astrocyte-like ARE

AU-Rich Element ASOs

AntiSense Oligonucleotides ATRX

Alpha Thalassemia/mental Retardation syndrome X BRAF

B-RAF proto-oncogene serine/threonine kinase CBTRUS

Central Brain Tumor Registry of the United States

CDKs

Cyclin-Dependent Kinases CGGA

Chinese Glioma Genome Atlas CNS

Central Nervous System CNVs

Copy Number Variations CPTAC

Clinical Proteomic Tumor Analysis Consortium

EGFR

Endothelial Growth Factor Receptor EMA

European Medicines Agency EPed-glioma model

ElectroPorated-glioma model ES

Exon Skipping ESE

Exonic Splicing Enhancers ESFs

Exosome-Specificity Factors ESS

Exonic Splicing Silencer

FDA

Food and Drug Administration GBM

GlioBlastoMa GDSC

Genomics of Drug Sensitivity in Cancer GSCs

Glioma Stem-Cells HFD

High-Fat Diet HGAs

High-Grade Astrocytomas hnRNPs

heterogeneous nuclear RiboNucleoProteins IDH1

Isocitrate DeHydrogenase (NADP(+)) 1

IR

Intron Retention ISE

Intronic Splicing Enhancer ISS

Intronic Splicing Silencer LITT

Laser Interstitial Therapy MAPKs

Mitogen Activated Protein Kinases MES-like

MESenchymal-like MGMT

O-6-MethylGuanine-DNA MeThyltransferase MR

Magnetic Resonance MRI

Magnetic Resonance Imaging mut

mutant MXE

Mutually Exclusive Exons NF1

NeuroFibromin 1 NGD

Non-Go Decay NGS

Next-Generation Sequencing

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NMD

Nonsense-Mediated Decay NPC-like

Neural Progenitor Cell-like NSD

Non-Stop Decay OPC-like

Oligodendrocyte Progenitor-like OS

Overall Survival PDGFA

Platelet-derived Growth Factor Subunit A PDGFDD

Platelet-Derived Growth Factor DD

PDGFRA

Platelet-Derived Growth Factor Receptor Alpha

PDGFRB

Platelet-Derived Growth Factor Receptor Beta

PI3K

PhosphoInositide 3-Kinase PSI

Percent Spliced-in Index PTCs

Premature Termination Codons PTEN

Phosphatase and TENsin homolog RBM

RNA-Binding Motifs RBPs

RNA-Binding Proteins ROC-curve

Receiver Operating Characteristic-curve SASP

Senescence-Associated Secretory Phenotype

SF3B1

Splicing-Factor 3B Subunit-1 SFs

Splicing Factors SMD

Staufen1-Mediated mRNA Decay

snRNA

small nuclear RNA snRNPs

small nuclear RiboNucleoProteins SR proteins

Serine-arginine-Rich proteins SREs

Splicing Regulatory Elements SRSFs

Serine/arginine-Rich Splicing Factors SSAs

SomatoStatin Analogs sst5TMD4

somatostatin receptor subtype 5 Transmembrane Domain 4 SST5TMD5

somatostatin receptor subtype 5 Transmembrane Domain 5 SSTRs

SomatoStaTin Receptors STAU1

STAUfen1 TCGA

The Cancer Genome Atlas TERT

TElomerase Reverse Transcriptase TTF

Tumor-Treating Field TMZ

TeMoZolomide TP53

Tumor Protein P53 TP73

Tumor Protein P73 TTF

Tumor-Treating Field VEGF

Vascular Endothelial Growth Factor

VEGFR

Vascular Endothelial Growth Factor Receptor

WHO

World Health Organization;

OMS in Spanish.

wt wild type

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Figures Index

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Figure 1: Summary of brain tumors statistics from CBTRUS Statistical Report 2020

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Figure 2: Criteria of 2016 WHO Glioma Classification

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Figure 3: Survival rates from CBTRUS Statistical Report 2020

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Figure 4: Inter- and intra-tumor heterogeneity of glioblastoma at the molecular level

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Figure 5: Alternative splicing events

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Figure 6: Spliceosome structure

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Figure 7: Spliceosome cycle dynamics and catalytics reactions.

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Figure 8: Regulation of the splicing process: trans- and cis-acting elements

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Figure 9: Alternative splicing dysregulation in cancer

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Figure 10: Schematic representation of the RNA metabolism and surveillance pathways

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Figure 11: Graphical Abstract of Article I

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Figure 12: Graphical Abstract of Article II

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Figure 13: Graphical Abstract of Article III

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Figure 14: Graphical Abstract of Article IV

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Figure 15: RNA-exosome machinery is dysregulated in HGAs/GBM

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Figure 16: DIS3 overexpression is associated with poor OS in patients with glioblastomas

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Figure 17: DIS3 silencing in vitro decreases critical functional parameters of aggressiveness in GBM cells

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Figure 18: Venn-diagram of RNA-exosome targets to predict candidates which could drive the tumorigenic role of DIS3 in malignant brain tumors

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Resumen

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3 Los tumores cerebrales primarios representan un grupo muy variado y heterogéneo de neoplasias (~150 tipos diferentes) que se dividen en tumores no malignos y malignos. Los tumores cerebrales malignos más frecuentes son los gliomas (representando el 80,8 %), que constituyen un grupo diverso de patologías tumorales que pueden clasificarse según tres criterios: 1) tipo celular de origen, 2) grado tumoral y 3) características genéticas moleculares.

En concreto, según el tipo celular de origen se clasifican en: i) ependimomas, procedentes de células ependimarias del cerebro y la médula espinal; ii) oligodendrogliomas, derivados de oligodendrocitos; y, iii) astrocitomas, originados por astrocitos. En cuanto al segundo criterio, se estratifican en grado I y grado II (gliomas de bajo grado; a veces con tendencia a la progresión en pocos años) y grado III y grado IV (gliomas de alto grado). El tercer criterio [el último añadido en la clasificación de la Organización Mundial de la Salud (OMS)] constituye una serie de características genéticas moleculares que mejoran la clasificación de estos tumores. Por ejemplo, la identificación del estado de codeleción 1p/19q para identificar los oligodendrogliomas y la presencia de la mutación IDH1 (R132H) que se ha asociado a un mejor pronóstico y a tumores de bajo grado. Entre ellos, los astrocitomas representan la mayoría de los gliomas, entre lo que se encuentran el tumor cerebral maligno más agresivo y prevalente (~50 %), conocido como glioblastoma (GBM; astrocitoma de grado IV), el subtipo tumoral con la tasa de supervivencia más baja de todos los tumores cerebrales.

Cabe destacar que esta Tesis Doctoral se ha centrado en el estudio de los tumores cerebrales malignos con origen glial [astrocitoma de grado III (también denominado astrocitoma anaplásico) y, especialmente, en el astrocitoma de grado IV o GBM, ambos conocidos como astrocitomas de alto grado] debido a su alta prevalencia, disponibilidad de muestras y menor tasa de supervivencia. De hecho, uno de los principales problemas de estos tipos de tumores es la ineficacia de los tratamientos actuales. En concreto, la primera línea de tratamiento en los tumores cerebrales en adultos con comportamiento maligno suele consistir en la cirugía para la resección del tumor seguida de quimioterapia y/o radioterapia; sin

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embargo, la tasa de supervivencia de un paciente al que se le diagnostica un tumor cerebral maligno sigue siendo devastadora [ej., la supervivencia global solo ha sufrido una ligera mejora del 1 % en los últimos 30 años, lo que es muy bajo en comparación con otros tipos de cáncer como el de intestino (19 %) y el de mama (18 %)]. Por todo ello, es urgente identificar nuevos biomarcadores y dianas moleculares que permitan comprender mejor el comportamiento de los tumores cerebrales malignos, su adecuada clasificación y estratificación, y el desarrollo de nuevas estrategias terapéuticas para el tratamiento, así como para el control de estas devastadoras neoplasias.

En este contexto, una característica común del cáncer, incluyendo los tumores cerebrales, es la presencia atípica de variantes de splicing pertenecientes a determinadas vías de señalización que son importantes en el control de la fisiología normal de las células.

De hecho, un número considerable de evidencias apoya la idea de una asociación entre la presencia aberrante de variantes de splicing alternativas y el desarrollo y progresión de diferentes tipos de cáncer. Así, se ha sugerido que una desregulación en el spliceosoma, la maquinaria macromolecular que impulsa el proceso de splicing, podría estar asociada a la presencia de estas variantes de splicing en el cáncer. De hecho, se ha demostrado la existencia de diferentes alteraciones (i.e., mutación, pérdida, sobreexpresión...) así como un potencial papel oncogénico de algunos componentes del spliceosoma y factores de splicing (SFs, de sus siglas en inglés splicing factors) en numerosas patologías tumorales [i.e., PTBP1, SF3B1, proteínas ESRP1/2, RBM y hnRNPs, entre otras]. Asimismo, al igual que el proceso de splicing, el control de la calidad del ARNm y la presencia de transcritos aberrantes son procesos que están muy controlados en células en condiciones fisiológicas normales.

Concretamente, este control del metabolismo del ARNm está estrechamente supervisado por otras maquinarias celulares/complejos biológicos (i.e., non-sense mediated decay y el complejo ARN-exosoma). En consecuencia, su desregulación podría contribuir al desarrollo, progresión y/o agresividad de varias patologías humanas, incluyendo diferentes tipos de tumores. Sin embargo, aunque existen numerosas evidencias que indican que la

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5 desregulación de las maquinarias celulares que controlan los procesos de splicing y el control de calidad del ARNm podría ser importante en el desarrollo, progresión y/o agresividad del cáncer, existe un desconocimiento sobre la caracterización de estas maquinarias celulares en los tumores cerebrales y las consecuencias específicas de esta desregulación en estas patologías.

Por lo tanto, en base a la información mencionada anteriormente, es necesario interrogar la alteración de los sistemas reguladores celulares que están implicados en los procesos de splicing y el control de calidad del ARNm en los tumores cerebrales malignos, lo que llevaría al descubrimiento de nuevos mecanismos moleculares para comprender mejor el comportamiento de estos tumores e identificar nuevos biomarcadores con potencial diagnóstico, pronóstico y terapéutico.

Asimismo, dado que la heterogeneidad tumoral es una de las principales razones por las que la mayoría de las terapias contra los tumores cerebrales malignos son ineficaces, sería muy importante tener en cuenta el entorno metabólico de los pacientes en la búsqueda de nuevas opciones terapéuticas en pacientes con tumores cerebrales malignos. En este contexto, una conocida estrategia para tratar enfermedades humanas se basa en el reposicionamiento de fármacos ya aprobados para tratar otras patologías, siendo más rápida y fácil su traslación a la práctica clínica que los nuevos fármacos. En este sentido, la metformina (un fármaco antidiabético oral de primera línea) y las estatinas (por ejemplo, la simvastatina, fármaco antihipercolesterolémico), comúnmente utilizados para tratar patologías relacionadas con el metabolismo, han sido probadas en distintos tipos de cáncer; sin embargo, algunas de las acciones antitumorales de estos fármacos, como el mecanismo molecular asociado, son todavía controvertidas y/o han sido poco exploradas. Por lo tanto, son necesarios más estudios para definir claramente si estos fármacos de carácter metabólico por sí solos, pero especialmente en combinación, ejercen acciones antitumorales en diferentes tipos de cáncer, incluidos los tumores cerebrales.

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Teniendo en cuenta toda esta información, el OBJETIVO GENERAL de esta Tesis Doctoral ha sido explorar la presencia, desregulación potencial y/o posible papel funcional de los componentes de las maquinarias celulares clave implicadas en los procesos reguladores críticos [i..e., el splicing y el metabolismo/control de calidad del ARNm, así como sus marcadores subsidiarios (por ej., variantes de splicing alternativo)], así como de moduladores metabólicos clave (elementos endógenos y tratamientos exógenos) que podrían estar asociados con el desarrollo, progresión y agresividad de los tumores cerebrales malignos, con el objetivo final de descubrir nuevos biomarcadores y herramientas terapéuticas para mejorar el diagnóstico, pronóstico, tratamiento y, por tanto, manejo de los pacientes con tumores cerebrales malignos. Para conseguir este objetivo principal, hemos llevado a cabo diferentes objetivos específicos cuyos resultados se han recogido en cuatro apartados, correspondientes a cinco artículos científicos derivados directamente de esta Tesis Doctoral (3 artículos aceptados en revistas de primer nivel del ámbito de la "Neurociencia", "Neurología Clínica",

"Oncología" y "Bioquímica y Biología Molecular", y 2 manuscritos que se están preparando para su publicación o ya están en revisión en revistas especializadas).

La primera sección de esta Tesis Doctoral tenía como objetivo caracterizar el patrón de expresión de los elementos relacionados con el proceso de splicing (componentes del spliceosoma y SFs) en una batería representativa de tumores cerebrales malignos clínicamente bien caracterizados en comparación con las muestras control (no tumorales), y explorar su asociación con parámetros clínicos clave, con el fin de determinar la utilidad de algunos de estos elementos clave relacionados con la desregulación del spliceosoma como posibles biomarcadores de diagnóstico y pronóstico.

Además, esta sección se centró en analizar el papel funcional y los mecanismos moleculares asociados de los elementos clave relacionados con la desregulación del spliceosoma en modelos de tumores cerebrales malignos in vitro e in vivo. En resumen, los resultados de esta sección desvelaron nuevas vías conceptuales y funcionales en GBM, con

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7 potenciales implicaciones terapéuticas, al demostrar por primera vez una drástica desregulación de la maquinaria de splicing (núcleo del spliceosoma y SFs; especialmente, SRSF3/RBM22/PTPB1/RBM3) en distintos modelos (humanos/murinos) de astrocitomas de grado III y GBM. Estos resultados podrían ser clínicamente relevantes, ya que esta desregulación se asocia directamente a las características de desarrollo y agresividad de los GBM. Además, se demuestra el papel clave de SRSF3 en procesos fisiopatológicos cruciales de los GBM, como la proliferación celular, migración, apoptosis, secreción de VEGF y formación de tumorosferas, lo que explicaría la asociación relevante y directa de los niveles reducidos de SRSF3 con la reducción de la progresión tumoral y una mejora de la tasa de supervivencia observada en diferentes cohortes humanas y modelos animales de GBM. Estas acciones están probablemente mediadas a través de la modulación de vías de señalización clave (PDGFRB y PI3K) involucrando una alteración del splicing de factores de transcripción específicos que a su vez controlan la expresión de PDGFRB. Por lo tanto, nuestro estudio proporciona pruebas sólidas y convincentes que demuestran que SRSF3 tiene un papel funcional en la fisiopatología de los GBMs e invita a sugerir que el desarrollo y el uso de fármacos dirigidos a SRSF3 podría convertirse en una opción terapéutica prometedora para tratar a los pacientes con esta devastadora patología.

Además, en esta sección también investigamos la presencia de una variante de splicing específica, el receptor de somatostatina 5 truncado (sst5TMD4), en los tumores cerebrales malignos, así como su papel fisiopatológico utilizando diferentes enfoques in vitro.

Los resultados de este estudio han revelado nuevas vías conceptuales y funcionales en GBM con potenciales implicaciones clínicas, al demostrar que sst5TMD4 se sobreexpresa en GBM y se asocia con la supervivencia/progresión de GBM y con procesos fisiopatológicos clave en la biología de los GBM (i.e., la capacidad de proliferación y migración), probablemente mediante la modulación de diferentes vías de señalización oncogénica (AKT/JAK-STAT/NF- κB/TGF-β). Asimismo, nuestro estudio demostró que la modulación de los niveles de expresión de sst5TMD4 podría ser una posible vía terapéutica que debería explorarse en el

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futuro en GBM, ya que su silenciamiento disminuyó las tasas de proliferación y migración en las células de GBM y sensibilizó a estas células al efecto antitumoral del pasireótido (un análogo de la somatostatina ampliamente utilizado para tratar algunas patologías tumorales).

En conjunto, estos resultados señalan a sst5TMD4 como un biomarcador útil para el diagnóstico y el pronóstico, así como una diana potencial en el futuro desarrollo de enfoques terapéuticos en pacientes con GBM, ofreciendo otra oportunidad clínicamente relevante que también debería probarse para su uso en humanos.

La segunda sección de esta Tesis Doctoral se centró en abordar un componente clave del spliceosoma así como el ambiente metabólico como nuevas vías terapéuticas en los tumores cerebrales malignos. En primer lugar, nos propusimos caracterizar la implicación oncogénica de SF3B1 (un componente del spliceosoma esencial y susceptible de ser inhibido con fármacos), sus mutaciones somáticas, su perfil de expresión y su asociación con características moleculares y parámetros clínicos en tumores cerebrales malignos, así como evaluar el potencial terapéutico de pladienolide B (un inhibidor de SF3B1) en modelos de tumores cerebrales malignos en comparación con la condición control (no tumoral) in vitro e in vivo. Nuestros resultados demuestran que SF3B1 es una atractiva diana terapéutica en GBM, ya que su inhibición altera procesos fisiopatológicos clave en la biología del GBM (i.e., proliferación, migración, formación de tumorosferas, apoptosis, etc.) probablemente mediante la modulación de diferentes vías de señalización oncogénica (AKT-mTOR y ß-catenina) asociadas a la supervivencia/iniciación/progresión del GBM, y con la alteración en el splicing del transcrito del gen BCL2L1. Además, descubrimos que la sobreexpresión de SF3B1 en GBM se asocia con características moleculares y clínicas clave, como la supervivencia, el mal pronóstico y la resistencia a los fármacos. Por lo tanto, estos resultados señalan a SF3B1 como un potencial biomarcador de diagnóstico/pronóstico y una prometedora diana farmacológica para tratar a los pacientes con GBM.

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9 En segundo lugar, evaluamos las posibles asociaciones entre los pacientes con tumores cerebrales malignos tratados con metformina y/o simvastatina y los parámetros de agresividad tumoral que los acompañan, explorando a su vez los efectos funcionales y las vías moleculares asociadas a los tratamientos con metformina, simvastatina y, especialmente, su combinación en los tumores cerebrales malignos. En concreto, nuestros resultados apoyan y amplían los datos anteriores que indican que las acciones de la metformina, la simvastatina, y especialmente su combinación, en las células cancerosas no se limitan a la reducción de las características de agresividad (i.e., proliferación, migración, formación de colonias y tumorosferas, secreción de VEGF e inducción de la apoptosis), sino también a la regulación de la expresión de genes clave implicados en el fenotipo secretor asociado a la senescencia (SASP, de sus siglas en inglés senescence-associated secretory phenotype), el complejo shelterina/telomerasa y la maquinaria de splicing, que, en conjunto, podrían promover un estado de senescencia y un alargamiento de los telómeros en las células del GBM. Por lo tanto, estos hallazgos sugieren una posible oportunidad para estos fármacos metabólicos, especialmente su combinación, como una nueva herramienta terapéutica para el manejo de los GBM en humanos.

La tercera y última sección de esta Tesis Doctoral se centró en la caracterización del patrón de expresión de los componentes de la maquinaria celular del ARN-exosoma en una batería representativa de tumores cerebrales malignos clínicamente bien caracterizados en comparación con muestras control (no tumorales), en explorar su asociación con parámetros clínicos clave, y en analizar el papel funcional de los componentes clave identificados de esta maquinaria celular, todo ello con el fin de determinar su utilidad como posibles biomarcadores de diagnóstico/pronóstico y dianas terapéuticas. Nuestros resultados demostraron por primera vez una clara desregulación global del perfil de expresión de los componentes del complejo ARN-exosoma en los tumores cerebrales malignos en comparación con los tejidos cerebrales control. Además, demostramos que un componente catalítico/exorribonucleasa clave de esta

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maquinaria celular podría representar un biomarcador novedoso y útil, así como una herramienta terapéutica para abordar el GBM.

El conjunto de los resultados de esta Tesis Doctoral demuestra la existencia nuevas vías conceptuales y funcionales en GBM con potenciales implicaciones clínicas, al demostrar que la desregulación de ciertos componentes de las maquinarias de splicing y ARN-exosoma junto con la alteración del proceso de splicing podrían contribuir fuertemente al desarrollo, progresión y agresividad del GBM, representando una fuente de nuevas dianas diagnósticas, pronósticas y terapéuticas que podrían utilizarse para mejorar el diagnóstico, manejo y supervivencia de los pacientes con GBM. Asimismo, los resultados de esta tesis demuestran la eficacia de la metformina, la simvastatina y, en particular, su combinación, como potencial herramienta terapéutica útil para el manejo del GBM humano.

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13

Summary

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15 Primary brain tumors represent a highly variable and heterogeneous group of neoplasms (~150 different types) that includes non-malignant and malignant tumors. The most frequent malignant brain tumors are gliomas (representing the 80.8 %), which also comprise a diverse group of heterogeneous pathologies that can be classified according to three criteria:

1) cell type of origin, 2) tumor grade, and 3) molecular genetic features.

Specifically, according to the cell type of origin gliomas are classified into: i) ependymomas, from ependymal cells in the brain and spinal cord; ii) Oligodendrogliomas, derived from oligodendrocytes; and, iii) astrocytomas, originated from astrocytes. Regarding the second criterion, they are stratified in grade I and grade II (low-grade gliomas; sometimes with a tendency to progression in a few years) and grade III and grade IV (high-grade gliomas).

The third criterion [the latest added in the World Health Organization (WHO) classification]

constitutes a series of molecular genetic features that improve the classification of these tumors. For instance, the identification of 1p/19q co-deletion status to identify oligodendrogliomas and the presence of IDH1 mutation (R132H) which has been associated with better prognosis and low-grade tumors. Among them, astrocytomas represent the majority of gliomas which also involved the most aggressive and prevalent malignant brain tumor (~50

%), known as glioblastoma (GBM; astrocytoma grade IV), the subtype with the lowest survival rate.

This Doctoral Thesis has been focused on the study of malignant brain tumors from glial origin, i.e., astrocytoma grade III (known as anaplastic astrocytoma) and, especially, in astrocytoma grade IV or GBM, both known as high-grade astrocytomas, particularly due to the high prevalence, sample availability, and lower survival rate. Indeed, one of the main problems with these tumor types are the inefficacy of the current treatments. Specifically, the standard of care in adult brain tumors with malignant behavior often consists of surgery for the tumor resection followed by chemotherapy and/or radiotherapy; however, the survival rate of a patient who is diagnosed with a malignant brain tumor remains devastating [i.e., slight

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improvement in the overall survival (OS) by 1 % in the last 30 years, which is very low in comparison with other cancer types such as bowel (19 %) and breast cancer (18 %)]. For all these reasons, it is urgently necessary to identify new biomarkers and molecular targets that allow a better understanding of malignant brain tumor behavior, their appropriate classification and stratification, and the development of new therapeutic strategies for the treatment and control of these devastating neoplasms.

In this context, a common hallmark of cancer, including brain tumors, is the atypical presence of splicing variants belonging to certain pathways that are important in the control of the normal physiology of the cells. Indeed, a substantial amount of evidence supports the idea of an association between the aberrant presence of alternative mRNA splicing variants and the development and progression of different cancer types, including malignant brain tumors.

Thus, it has been suggested that a dysregulation in the spliceosome, the macromolecular machinery driving the splicing process, might be associated with the presence of these splicing variants in cancer. In fact, it has been demonstrated the existence of described different alterations (i.e., mutation, loss, overexpression…) and a potential oncogenic role of some spliceosome components and splicing factors (SFs) in numerous tumor pathologies [i.e., PTBP1, SF3B1, ESRP1/2 RNA-binding motifs (RBM) proteins, and hnRNPs, among others].

Similar to the splicing process, the surveillance of mRNA quality and the presence of aberrant transcripts are highly supervised in the cells under normal conditions. This control of the mRNA metabolism is tightly supervised by other cellular machineries/ biological complexes (i.e., nonsense-mediated decay, and RNA-exosome). Consequently, their dysregulation might also contribute to the development, progression, and/or aggressiveness of various human pathologies, including different tumor types. However, although there is plenty of evidence indicating that the dysregulation of the cellular machineries controlling the splicing and surveillance of mRNA quality processes could be important in cancer development, progression and/or aggressiveness, there is a lack of knowledge about the characterization of

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17 these cellular machineries in brain tumors and the specific consequences of this dysregulation in these pathologies.

Therefore, based on the information mentioned above, it is necessary to interrogate the alteration of critical cellular regulatory systems involved in the splicing and surveillance of mRNA quality processes in malignant brain tumors, which would lead to: 1) the discovery of novel molecular mechanisms to better understand the behavior of these tumors, and 2) uncover new biomarkers with diagnostic, prognostic, and therapeutic potential.

Likewise, since heterogeneity is one of the main reasons why most therapies against malignant brain tumors are ineffective, it would be very important to consider the metabolic environment of the patients when searching for new therapeutic options in patients with malignant brain tumors. In this context, a well-known strategy to treat human diseases is based on the repositioning of drugs already approved to treat other pathologies since they are faster and easier to translate to clinical practice than new drugs. In this scenario, metformin (a first- line oral antidiabetic drug) and statins (e.g., simvastatin, anti-hypercholesterolemic drug), commonly used to treat metabolism-related pathologies, have been tested in some cancer types, showing promising results; however, some of the antitumor actions of these drugs, as well as the molecular mechanism associated, are still controversial and poorly understood.

Therefore, further studies are necessary to clearly define if these metabolic drugs alone, but especially in combination, exert antitumor actions in different cancer types, including brain tumors.

Taken all this information together, the GENERAL AIM of this Doctoral Thesis was to explore the presence, potential dysregulation and/or functional role of components of key cellular machineries involved in critical regulatory processes [i.e., splicing and mRNA metabolism and surveillance, and their subsidiary markers (e.g., alternative splicing variants)], as well as of key metabolic modulators (endogenous elements and exogenous treatments) that could be associated with the development, progression and aggressiveness of malignant

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brain tumors, with the ultimate goal of discovering novel biomarkers and therapeutic tools to improve the diagnosis, prognosis, treatment and, therefore, the management of malignant brain tumors patients. To achieve this main aim, we have carried out different specific objectives whose results have been included in four sections, corresponding to five scientific manuscripts directly derived from this Doctoral Thesis (3 accepted manuscripts in top ranking journals of the “Neuroscience”, “Clinical Neurology”, “Oncology” and “Biochemistry & Molecular Biology” fields, and 2 manuscripts that are being prepared for submission or already under review in specialized journals).

The first section of this Doctoral Thesis was aimed to characterize the expression pattern of splicing-related elements (spliceosome components and SFs) in a representative battery of clinically well-characterized malignant brain tumors in comparison with control (non- tumor) samples, and to explore their association with key clinical parameters, in order to determine the utility of key dysregulated spliceosome-related elements as putative diagnostic and prognostic biomarkers. Moreover, this section was focused on analyzing the potential functional role and associated molecular mechanisms of key spliceosome-related elements in malignant brain tumor models in vitro and in vivo. Briefly, the results of this section unveiled new conceptual and functional avenues in GBM, with potential therapeutic implications, by demonstrating for the first time a drastic dysregulation of the splicing machinery (spliceosome core and SFs; especially, SRSF3/RBM22/PTPB1/RBM3) in grade III astrocytomas/GBM of different species. This might be clinically relevant, because this dysregulation directly associates to development and aggressiveness features of GBMs. Moreover, we unveil a role of SRSF3 in crucial pathophysiological processes of GBMs, such as cell proliferation, migration, apoptosis, VEGF secretion and tumorsphere formation, which would underlie the relevant direct association of lower SRSF3 levels with reduced tumor progression and enhanced survival rate observed in different GBM human cohorts and animal models. These actions are likely mediated through the modulation of key signaling pathways (PDGFRB and PI3K) and may involve the distinct alteration of alternative splicing event of specific

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19 transcription factors controlling PDGFRB expression. Therefore, our study provides solid, convincing evidence demonstrating that SRSF3 has a functional role in the pathophysiology of GBMs and invites to suggest that the development and use of SRSF3-targeting drugs could become a promising option to treat patients with this devastating pathology, offering a potential clinically relevant opportunity that should be soon tested for their use in humans.

Additionally, in this section we also investigated the presence of a specific splicing variant, the truncated somatostatin receptor subtype 5 (sst5TMD4), in malignant brain tumors as well as its pathophysiological role using different in vitro approaches. The results of this study unveiled new conceptual and functional avenues in GBM with potential clinical implications, by demonstrating that sst5TMD4 is overexpressed in GBM and associated with GBM survival/progression and key pathophysiological processes in GBM biology (i.e., proliferation and migration capacity), likely by modulating different oncogenic signaling pathways (AKT/JAK-STAT/NF-κB/TGF-β). Moreover, our study demonstrated that the modulation of sst5TMD4 expression levels could be a putative therapeutic avenue that should be explored in the future in GBM since its silencing decreased proliferation and migration rates in GBM cells and sensitized these cells to the antitumor effect of pasireotide (a somatostatin analogue widely used to treat some tumor pathologies). Altogether, these results underscore sst5TMD4 as a useful diagnostic and prognostic biomarker, and as a potential target in the future development of therapeutic approaches in GBM patients, offering another clinically relevant opportunity that should be also tested for use in humans.

The second section of this Doctoral Thesis was focused on targeting key spliceosome components and metabolic milieu as new therapeutic avenues in malignant brain tumors. First, we aimed to characterize the oncogenic implication of SF3B1 (an essential and druggable spliceosome component), its somatic mutations, expression profile and association with molecular features and clinical parameters in malignant brain tumors, as well as to assess the therapeutic potential of pladienolide B (a SF3B1 inhibitor) on malignant brain tumors in

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comparison with control (non-tumor) models in vitro and in vivo. Our results demonstrate that SF3B1 is an attractive therapeutic target in GBM since its inhibition impaired key pathophysiological processes in GBM biology (i.e., proliferation/migration/tumorspheres formation/apoptosis, etc.) likely by modulating different oncogenic signaling pathways (AKT- mTOR/ß-catenin) associated with GBM survival/initiation/progression, and an imbalance of BCL2L1 splicing. Moreover, we found that SF3B1 overexpression in GBM is associated with key molecular and clinical features including OS, poor prognosis, and drug resistance.

Therefore, these results point out SF3B1 as a potential diagnostic/prognostic biomarker and a promising pharmacological target to treat patients with GBM.

Secondly, we assessed the putative associations between patients with malignant brain tumors treated with metformin and/or statins and the accompanying oncological aggressiveness parameters, and also explored the functional effects and mechanistic underpinnings of the treatments with biguanides, statins and specially their combination in malignant brain tumors. Specifically, our results support and extend previous data indicating that the actions of metformin, simvastatin, and specially their combination, in cancer cells are not only confined to the reduction in aggressiveness features (i.e., proliferation/migration rates, tumorsphere/colony formation, VEGF secretion, and apoptosis induction) but also included the regulation of the expression of key genes involved in the Senescence-Associated Secretory Phenotype (SASP), the shelterin-complex/telomerase, and the splicing machinery, which, in conjunction, might promote a senescence-state and a telomere lengthening in GBM cells.

Therefore, these findings suggest a putative window of opportunity for these metabolic drugs, especially their combination, as a potential new therapeutic tool for the management of human GBM.

The third and final section of this Doctoral Thesis was focused on the characterization of the expression pattern of the components of the RNA-exosome cellular machinery in a representative battery of clinically well-characterized malignant brain tumors in comparison

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21 with control (non-tumor) samples, to explore their association with key clinical parameters, and to analyze the potential functional role of the key components identified in malignant brain tumor of this cellular machinery in order to determine the utility as putative diagnostic/prognostic biomarkers and therapeutic targets. Our results demonstrated for the first time a clear overall dysregulation of the expression profile of the components of the RNA-exosome complex in malignant brain tumors compared to the control brain tissues.

Moreover, we demonstrated that the catalytic/exoribonuclease component (DIS3) of this cellular machinery might represent a novel, useful tool as diagnostic and prognostic biomarker and a potential therapeutic target to tackle GBM.

Taken together all the results of this Doctoral Thesis unveiled new conceptual and functional avenues in GBM with potential clinical implications, by demonstrating that the dysregulation of certain components of the splicing and RNA-exosome machineries and the alteration of the splicing process could strongly contribute to the development, progression, and aggressiveness of GBM, representing a source of novel diagnostic, prognostic, and therapeutic targets that could be used to improve the diagnosis, management, and survival of GBM patients. Moreover, results of this thesis demonstrate the efficacy of metformin, simvastatin, and, particularly, their combination, as a potential and useful therapeutic tool for the management of human GBM.

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1. Introduction

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1.1. Brain Tumors

Primary brain tumors represent a highly variable and heterogeneous neoplasms group of ~150 different types divided into non-malignant and malignant tumors¹. The most frequent malignant brain tumors are gliomas (representing the 80.8 %)², which are originated from three different glial cell types: astrocytes, oligodendrocytes, and ependymal cells³. Likewise, the cell of origin is used to classify them in astrocytomas, oligodendrogliomas, and ependymomas, respectively³. Among them, astrocytomas represent the majority of gliomas which also involve the most aggressive and prevalent malignant brain tumor (~50 %), known as glioblastoma (GBM), the subtype with the lowest survival rate².

More specifically, astrocytomas arise from astrocytes or neural precursor cells located in the Central Nervous System (CNS) and are originated when they accumulate different molecular alterations. In this context, several genes [e.g., Cyclin-dependent kinases (CDKs), Tumor protein P53 (TP53), Phosphatase and tensin homolog (PTEN), Epidermal growth factor receptor (EGFR), Platelet derived growth factor subunit A (PDGFA), B-Raf proto-oncogene serine/threonine kinase (BRAF), Vascular endothelial growth factor receptor (VEGFR), Telomerase reverse transcriptase (TERT), Alpha thalassemia/mental retardation syndrome X - (ATRX), and Isocitrate dehydrogenase (NADP(+)) 1 (IDH1)] suffer mutations, copy number variations (CNVs), and dysregulations in their expression which translate into the alteration of their original function^1,3–10. In addition, chromosome deletions and epigenetic alterations are also common (e.g., 1p19q and O-6-Methylguanine-DNA Methyltransferase (MGMT) promoter methylation status)^1,3,11. All these genes are involved in crucial biological processes such as cell cycle control, cell migration, angiogenesis, and genomic stability which directly result in an oncogenic transformation of glial cells. In fact, these molecular changes are usually associated with the prognosis of the tumor, and they also help to stratify the brain tumors from low to high aggressiveness following the World Health Organization (WHO) criteria ³.

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In spite of these molecular abnormalities, additional key causes underlying the development of these tumors are still unknown and the tools available for their diagnosis, predicting their prognosis, and, especially, for their treatment, are very limited, inefficient, or controversial.

1.1.1. Classification and Epidemiology

First, it should be mentioned that the classification used in this Doctoral Thesis corresponds to the report published in 2016 by the WHO³. Epidemiology data mentioned below is based on the Central Brain Tumor Registry of the United States [CBTRUS; data from 2013- 2017; the largest population-based registry focused exclusively on primary brain and other CNS tumors²].

Brain tumors, including other CNS tumors, are one the most important form of solid cancer, having, whether malignant or not, an incidence of 23.79 per 100 000 population, being higher in females in comparison with males (26.31 vs. 21.09 per 100 000 population, respectively). Across all the brain tumors, 29.7 % of all primary brain tumors are malignant vs.

70.3 % of non-malignant. Regarding mortality, this type of tumors has a mortality rate of 4.42 per 100 000 population being 62.5 % of deaths from all malignant primary brain tumors. The five-year survival rate from diagnosis depends on the type of tumor and the age, being the mean of malignant tumor 75.4 % in persons aged 0-14 years, 72.5 % in 15-39 years or 21.5 % in persons > 40 years (Figure 1, Right panel). These data are higher in the case of non- malignant tumors: 97.3 % in persons aged 0-14 years, 98.2 % in 15-39 years, or 90.2 % in persons > 40 years (Figure 1, Right panel). It should be pointed out that those malignant primary brain tumors diagnosed in patients aged > 40 years, commonly known as adult brain tumors, are the 8^th most common cancer type with an incidence of 42.85 per 100 000 population^1,2 (Figure 1, Left panel).

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Figure 1: Summary of brain tumors statistics from CBTRUS Statistical Report 2020². Left Panel: Incidence of the eight most common tumors in patients aged > 40 years. Right Panel: Distribution of primary brain and other CNS Tumors by behavior including the 5 year-survival in patients aged > 40 years.

In this context, the most frequent adult malignant brain tumors are gliomas [81.14 % of all malignant gliomas (24.1 % out of 29.7 % of gliomas); Figure 1, Right panel); which encompass a highly diverse group of pathologies, having a classification based on three criteria: 1) cell type of origin, 2) tumor grade, and 3) molecular genetic features³ (Figure 2).

1.1.1.1. Classification by Cell Type of Origin

Gliomas can be classified, fundamentally, according to the cell type of origin into: i) ependymomas (6.6 % of all malignant brain tumors), complex tumors that can spread throughout the CNS from ependymal cells in the brain and spinal cord, with a high prevalence in childhood²; ii) Oligodendrogliomas (5.3 % of all malignant brain tumors), which are derived from oligodendrocytes being usually benign, with a higher prevalence in people over 40 years of age and, finally, iii) astrocytomas (76.6 % of all malignant brain tumors, the most common glioma type), which are originated from astrocytes and have different ages of prevalence² (Figure 2).

1.1.1.2. Classification by Tumor Grade

The tumor grade refers to the rate of division and the aspect of the cells determined by histologic criteria. Then, they are stratified in grade I and grade II (low-grade gliomas;

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sometimes with a tendency to progression in a few years) and grade III and grade IV (high- grade gliomas)³ (Figure 2).

Accordingly, oligodendroglioma can be grade II or III (Anaplastic Oligodendroglioma), and ependymomas can be grade II or III (Anaplastic Ependymoma), having both subtypes a low prevalence (4.5 % and 3.2 % of all malignant brain tumors, respectively)³.

On the other hand, in the case of astrocytomas (64.6 % of all malignant brain tumors), they are graded from II to IV regarding the adult brain tumors³. Astrocytoma grade II is named as diffuse astrocytoma, astrocytoma grade III as anaplastic astrocytoma, and astrocytoma grade IV as glioblastoma (GBM)³. Particularly, GBM is the most aggressive and common malignant cancer in the brain and CNS (48.6 % of all malignant tumors)³.

Figure 2: Criteria of 2016 WHO Glioma Classification³. Classification of gliomas based on cell type of origin, tumor grade, and molecular genetic features. Molecular genetic features in brackets are required for diagnosis.

1.1.1.3. Classification by Molecular Genetic Features

The third criterion and the latest added in the WHO classification of gliomas constitutes a series of molecular genetic features that improve the classification of these tumors. For

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29 instance, the identification of 1p/19q co-deletion status is now indisputably associated with the classification of a brain tumor as oligodendroglioma¹². Furthermore, the presence of IDH1 mutation (Arginine132Histidine; R132H) has been associated with better prognosis and low- grade tumors; however, the grade IV astrocytomas with IDH mutated preserved its denomination as GBM^1,3, but not in the recent published 2021 WHO classification where have not been considered as GBM because of its better prognosis⁹(Figure 2).

Due to the high prevalence, sample availability, and lower survival rate, this Doctoral Thesis is focused on malignant brain tumors (i.e., astrocytoma grade III or anaplastic astrocytoma and, especially, in astrocytoma grade IV or GBM, both known as high-grade astrocytomas).

1.1.2. Management of Malignant Brain Tumors in Adults

Malignant brain tumors management in adults is continuously being challenged because of the correct decision making¹. Although, after brain tumor diagnosis, the first-line therapy is widely established, there are several factors (e.g., tumor type, tumor grade, patient age, location, and size) that could influence this decision making¹.

1.1.2.1. Diagnosis

The brain tumor diagnosis starts after the identification of common generalized symptoms of brain tumor (i.e., abnormal headaches, balance alteration, and loss of arm/leg movement, among others). The first step is to perform a magnetic resonance imaging (MRI with gadolinium) after suspicion of brain tumor which would confirm the presence of an unusual mass in the brain. Additionally, multimodal MRI such as diffusion weighted imaging as well as diffusion tensor imaging, magnetic resonance (MR) spectroscopy, and MR perfusion are used to better characterize the tumor in terms of cellularity, vascularity, and metabolism, respectively¹³.

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Nonetheless, to corroborate the malignancy of and to classify the tumor, it is necessary to obtain a piece of the tumor from stereotactic biopsy or surgery. Thus, an accurate diagnosis of the tumor type needs a histological analysis together with certain molecular determinations.

Specifically, the most common molecular determinations are the 1p/19q co-deletion to diagnose tumors as oligodendrogliomas, and the presence of IDH1mut as a marker of favorable prognosis^14,15. EGFR amplification, BRAF fusion, MGMT methylation status, and mutations in TERT promoter, ATRX, and TP53 are also useful to grade the tumors^14,15.

Therefore, the robustness and accuracy of diagnosis depend on the molecular diagnostic tools available in the hospital. Nowadays, following the WHO guidelines, it is crucial for the correct diagnosis a thoughtful molecular analysis at genomic, epigenomic, and transcriptomic levels which will be reflected in a correct decision making for treatment.

1.1.2.2. First-line therapy

The Standard of care in adult brain tumors with malignant behavior often consists of surgery for the tumor resection followed by chemotherapy and/or radiotherapy¹. In fact, the complete or near-complete surgical resection of the tumor mass is beneficial for patient outcomes. Sometimes this consists of a challenge for neurosurgeons if the tumor location is close to an eloquent area since they must avoid injuring brain tissue which is important to the patient's neurological function¹³.

Afterward, radiotherapy in combination with concurrent and adjuvant chemotherapy (temozolomide; TMZ) has been demonstrated to improve overall survival (OS) in a subset of patients with malignant brain tumors. However, the intensity and duration of chemo/radiotherapy depend on some clinical and molecular parameters^1,13,15. For instance, for an elderly patient diagnosed with GBM with MGMT promoter methylation, TMZ without radiation could be a promising option for treatment. MGMT is crucial in DNA repair being one of the most causes of TMZ resistance. Thus, if its expression is silenced by the promoter

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31 methylation, the cell cannot repair its DNA after TMZ treatment resulting in a better effect of this chemotherapeutic drug¹³.

1.1.2.3. Treatments in Trial

Currently, there are several clinical trials focused on malignant brain tumor therapy (~600 studies; source: https://clinicaltrials.gov/). A brief description of the most used potential treatments under research is included below:

1.1.2.3.1. Chemotherapy

Alkylating chemotherapeutics (i.e., iomustine, carmustine, procarbazine, and vincristine) could be used as second-line treatment, but to date most of the studies have reported any clear benefits in survival of patients¹⁶. Also, new TMZ schedules have been investigated and they have found no differences in comparison with current treatment¹⁶.

1.1.2.3.2. Radiotherapy

Radiotherapy is concurrently used with chemotherapy, but it is associated to some important side effects (e.g., neurological impairments). However, new radiotherapy dose- escalation, original strategies to deliver radioactive compounds, and implementation of the safety proton beam therapy are being currently investigated with fewer side effects and a slight improvement of OS^13,16.

1.1.2.3.3. Monoclonal Antibodies

Another potential treatment is bevacizumab, a monoclonal antibody for Vascular endothelial growth factor (VEGF) approved by the U.S. Food and Drug Administration (FDA) but not by the European Medicines Agency (EMA). This treatment could be used alone^17,18 or in combination with other chemotherapeutics drugs when the effect alone is very low¹⁹. However, there are no strong evidence that bevacizumab has the capacity to clearly improve

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the patient's OS, being a controversial topic. Remarkably, bevacizumab improves the quality of life of patients as a substitute for the use of cortisol resulting in a promising option for symptomatic recurrent tumors^16,17. Other immunotherapies using nivolumab¹⁸, pembrolizumab²⁰, durvalumab²¹ or atezolizumab²² have been interrogated in recurrent and metastatic brain tumors, but results were worse than expected¹³.

1.1.2.3.4. Tumor-treating Fields

Tumor-treating field (TTF) is a locoregional treatment that is adjuvant to TMZ. TTF impairs the cell mitosis alternating electric fields delivered by arrays applied to the pericranium²³. This treatment has demonstrated that increases the median survival in primary²³ but not in recurrence tumors²⁴. The cost, the accuracy of clinical trials, and skin toxicity limited the treatment to be essential in the standard of care. Nonetheless, eligible patients could receive the treatment since it is approved by FDA¹³.

1.1.2.3.5. Laser Interstitial Therapy

Finally, laser interstitial therapy (LITT) is being implemented in patients who are not eligible for standard tumor resection, for example, by patient age and complex accessibility²⁵. An MRI-guided laser-tip probe is introduced into the tumor to deliver low-powered laser- induced thermotherapy which is safe and improves patient survival¹⁶. Hence, LITT with adjuvant chemotherapy or radiotherapy in combination with immunotherapy is currently being investigated in gliomas.

1.1.3 Survival

Despite the efforts to improve the efficacy of the treatments over the last years, the survival rate of a patient who is diagnosed with a malignant brain tumor remains devastating.

Although the symptoms and clinical consequences of harboring a malignant brain tumor are

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33 decreasing, as it has been mentioned above, five-year survival of patients aged > 40 years remains 21.5 % which is very low in comparison with other ages1,2,13,16,26 (Figure 3, Left Panel).

The classification of tumors in different grades acts as a prognostic marker since survival rate is positively associated with the grade of the tumor (Figure 3, Right Panel).

Specifically, oligodendrogliomas and ependymomas (grade II-III) are surrounding the 80 % of patient’s 5-year survival¹. In the case of astrocytomas, the survival is lower than oligodendrogliomas and ependymomas: diffuse astrocytoma (grade II) 5-years survival is 50

% while anaplastic astrocytoma is 30 %¹. Finally, GBM (astrocytoma grade IV) is the tumor type with the lowest median survival (~8 months)² and 5-year survival equal to 5 %¹ (Figure 3, Left Panel) .

Figure 3: Survival rates from CBTRUS Statistical Report 2020². Right Panel: Hazard ratios and 95 % confidence intervals for age of the three glioma types with the lowest survival rate. Left Panel: Kaplan-Meier Survival Curves for gliomas subtypes in patients aged > 40 years.

In addition to these terrible statistics, unfortunately, brain tumors have slightly improved the OS by 1 % in the last 30 years, which is very low in comparison with other cancer types such as bowel (19 %) and breast cancer (18 %) (data based on the Australian Institute of Health and Welfare statistics; https://www.aihw.gov.au). For this reason, it is urgently necessary to identify new biomarkers and molecular targets that allow a better

Identification of Novel Markers and Molecular Mechanisms with Diagnostic, Prognostic, and Therapeutic Potential in Brain Tumors

DEPARTAMENTO DE BIOLOGÍA CELULAR, FISIOLOGÍA E INMUNOLOGÍA