Dissertations / Theses on the topic 'B-cell malignancie'

Non-Hodgkin’s lymphoma (NHL) is a group of B-cell malignancies that is the 5th most common cancer in males and the 4th most common cancer in females. Three of the four most common histological subtypes of NHL are Diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL) and B-cell Chronic Lymphocytic Leukemia (B-CLL), which together make up over 60% of NHL cases. These diseases vary in both aetiology and aggressiveness, with patient prognosis predicted using indices that rely on biological surrogates in order to predict disease behavior. This results in a large degree of heterogeneity within prognostic groups, with some high-risk patients demonstrating long survival and low-risk patients undergoing rapid transformation and succumbing to an early death. The use of molecular profiling allows interrogation of genetic and transcriptional features of tumor samples that are directly indicative of cellular mechanisms of disease pathogenesis. This is therefore an attractive option for more accurately predicting patient prognosis and thereby allowing design of risk-adapted therapeutic regimes in order to increase survival rates. In this work whole-genome gene expression and single nucleotide polymorphism (SNP) microarrays have been employed in order to interrogate the transcriptome and genome of NHL tumor samples. In order to allow informed analysis of genomic data-sets within the context of gene networks, novel bioinformatic methods were required. This work therefore included the development of novel methods for informed analysis of genome-wide SNP microarray data, visualization of the subsequent gene-set enrichment analysis (GSEA) results, as well as bioinformatic methods for mapping loss-of-heterozygosity without the need for patient-matched control samples. Following development, the utility of these genome-wide approaches for the elucidation of pathogenic mechanisms with the use of small sample sizes was investigated by the interrogation of a novel immunodeficiency disorder. This resulted in our characterization of the first described case of immunodeficiency linked with a chromosomal duplication of the IL25 locus at 14q11.2 and an aberrant Th2-switching mechanism. It also provided proof that whole-genome analyses could be used to determine pathogenic mechanisms of disease.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Medical Science<br>Griffith Health<br>Full Text

La importància de la funció de MYC en el càncer (i l’origen del nom de la oncoproteïna) es va descobrir a finals dels 70, amb la identificació de la seqüència del retrovirus aviar causant de la leucèmia mielocítica. Durant més de 40 anys d’investigació, s’ha subratllat la rellevància d’aquesta proteïna en la divisió cel·lular normal i la seva implicació en la transformació tumoral. De fet, una de les primeres connexions entre la sobreexpressió de proto-oncògens (com MYC), reordenaments gènics i el càncer es va fer en el limfoma de Burkitt, la leucèmia mieloide crònica i els plasmacitomes de ratolí. Tenint en compte el paper de MYC en els càncers, sembla òbvia la necessitat de desenvolupar estratègies terapèutiques contra aquesta proteïna. No obstant, dirigir teràpies contra MYC era i segueix sent, un repte, donades les seves propietats úniques: no disposa d’estructura tridimensional, localització nuclear i absència d’un domini enzimàtic. Malgrat aixó, molts estudis han demostrat l’impacte terapèutic potencial de la inhibició de MYC, ja sigui directa o indirectament. En aquesta tesi, descrivim el potencial de la inhibició directa de MYC en el limfoma de Burkitt (BL) i el mieloma múltiple (MM), fent ús de dues estratègies diferents: petites molècules peptidomimètiques i disruptors del complex MYC / MAX / ADN (la mini-proteïna Omomyc i una variant derivada, anomenada variant 26 o V26): - La validació del potencial terapèutic dels peptidomimètics es va fer en col·laboració amb una startup biotecnològica. Tot i la prometedora eficàcia demostrada in vitro, un cop administrats in vivo, els compostos van provocar toxicitat severa local acompanyada de canvis en el comportament dels animals, fet que ens va portar a suspendre aquesta línia d’investigació. - Pel que fa a la validació de la mini-proteïna Omomyc com a estratègia farmacològica per tractar el BL i el MM, aquí vam mostrar la seva eficàcia in vitro i resultats preliminars en un model de peix zebra, on el pretractament amb Omomyc evita la colonització de la medul·la òssia. D’aquesta manera, s’evidencia per primera vegada l’ús potencial d’aquest “candidat a fàrmac” per al tractament de tumors líquids. Els nostres resultats in vivo en ratolins mostren que, tot i que l’administració d’Omomyc com a monoteràpia té una eficàcia limitada (probablement a causa d’un lliurament insuficient de pèptid a les cèl·lules de BL i MM), la seva combinació amb un inhibidor del proteasoma (teràpia estàndard del MM) resulta en efectes sinèrgics tant in vitro com in vivo en models de MM. A més, hem demostrat que l’administració intravenosa de Omomyc encapsulada en liposomes és segura i que aquesta formulació liposomal allarga la vida mitjana d’Omomyc en el sèrum, encara que no aconseguim augmentar l’entrada a les cèl·lules diana. - Per últim, hem caracteritzat la V26, un derivat de Omomyc, dissenyada per millorar la localització nuclear i la capacitat d’escapar dels endosomes. Aquí mostrem que la V26 pot homodimerizar i heterodimerizar amb MAX, a més d’unir-se a l’ADN en qualsevol de les dues formes dimèriques. Tal i com esperàvem, la V26 va mostrar ser més nuclear i va induir mort cellular in vitro. No obstant, també va resultar ser menys soluble que Omomyc, de manera que caldria fer millores en la seva formulació per poder ser utilitzada in vivo. En conjunt, els resultats suggereixen que la mini-proteïna Omomyc, o altres derivats com la V26, poden servir com a base pel disseny de nous fàrmacs anti-MYC pel tractament del BL i del MM. Així mateix, la combinació d’aquests amb les teràpies estàndard podrien constituir una estratègia prometedora, mentre que l’encapsulació en liposomes podria ajudar a resoldre aquells possibles problemes de biodisponibilitat derivats del seu ús in vivo.<br>La importancia de la función de MYC en el cáncer (y el origen del nombre de la oncoproteína) se descubrió a finales de los años 70, con la identificación de la secuencia del retrovirus aviar causante de la leucemia mielocítica. Durante más de 40 años de investigación, se ha subrayado la relevancia de esta proteína en la división celular normal y su implicación en la transformación tumoral. De hecho, una de las primeras conexiones entre la sobreexpresión de proto-oncogenes (como MYC), reordenamientos génicos y el cáncer se hizo en el linfoma de Burkitt, la leucemia mieloide crónica y los plasmacitomas en ratón. Teniendo en cuenta el papel que desempeña MYC en los cánceres, parece obvia la necesidad de desarrollar estrategias terapéuticas contra esta proteína. Sin embargo, dirigir terapias contra MYC era y sigue siendo, un reto, dadas las propiedades únicas que lo caracterizan: su carencia de estructura tridimensional, localización nuclear y ausencia de un "bolsillo" enzimático. A pesar de estas particularidades, muchos estudios han demostrado el impacto terapéutico potencial que tendría la inhibición de MYC, ya sea directa o indirecta. En esta tesis, describimos el potencial de la inhibición directa de MYC en el linfoma de Burkitt (BL) y el mieloma múltiple (MM), usando dos estrategias distintas: pequeñas moléculas peptidomiméticas y disruptores del complejo MYC/MAX/ADN (la mini-proteína Omomyc y una variante derivada, llamada variante 26 o V26): - La validación del potencial terapéutico de los peptidomiméticos se hizo en colaboración con una "startup" biotecnológica. A pesar de la prometedora eficacia demostrada in vitro, los compuestos provocaron toxicidad severa local in vivo, además de cambios en el comportamiento de los animales, que nos llevaron a discontinuar la investigación. - Respecto a la validación de la mini-proteína Omomyc como estrategia farmacológica para tratar el BL y el MM, aquí mostramos su eficacia in vitro y resultados preliminares en un modelo de pez cebra, donde el pretratamiento con Omomyc previene la colonización de la médula ósea. De esta forma, se evidencia por primera vez el uso potencial de este "candidato a fármaco" para el tratamiento de tumores líquidos. Nuestros resultados in vivo en ratones muestran que, aunque la administración de Omomyc como monoterapia tiene una eficacia limitada (probablemente debido a la insuficiente llegada de péptido a las células de BL y MM), su combinación con un inhibidor del proteasoma (terapia estándar del mieloma) resulta en efectos sinérgicos tanto in vitro como in vivo en modelos de mieloma. Además, hemos mostrado que la administración intravenosa de Omomyc encapsulada en liposomas es segura y que dicha formulación liposomal prolonga la vida media de Omomyc en el suero, aunque no conseguimos aumentar la entrada en las células diana. - Por último, hemos caracterizado la V26, un derivado de la mini-proteína Omomyc, diseñada con el objetivo de mejorar la localización nuclear y el escape de los endosomas. Aquí mostramos que, como Omomyc, la V26 puede homodimerizar y heterodimerizar con MAX, además de unirse al ADN en cualquiera de las dos formas diméricas. Como esperábamos, la V26 se mostró más nuclear e indujo muerte celular in vitro. Sin embargo, también resultó ser menos soluble que Omomyc, de forma que sería necesario hacer mejoras en su formulación para poder usarla in vivo. En conjunto, nuestros resultados sugieren que la mini-proteína Omomyc, u otros derivados, como la V26, pueden servir como base para el diseño de nuevos fármacos anti-MYC para el tratamiento del BL y MM. Además, la combinación de éstos con las terapias estándar podrían constituir una estrategia prometedora, mientras que la encapsulación en liposomas podría ayudar a resolver aquellos posibles problemas de biodisponibilidad derivados de su uso in vivo.<br>The importance of MYC function in cancer (and the origin of the oncoprotein's name) was discovered in the late '70s when the sequence of the avian retrovirus that causes myelocytic leukaemia was identified. Since then, over 40 years of unceasing research have highlighted the significance of this protein in regular cell division, and importantly, its involvement in malignant transformation. Indeed, some of the earliest connections between the higher expression of proto-oncogenes (such as MYC), genetic rearrangements and their relation to cancer development were made in Burkitt lymphoma, chronic myeloid leukaemia and mouse plasmacytomas. Given the role of MYC in cancer, the need for the design of therapeutic strategies against it seems obvious. However, targeting MYC was - and somehow, still is - challenging due to its unique properties: lack of defined three-dimensional, structure nuclear localisation and absence of enzymatic pocket. Despite these difficulties, many studies have shown the potential therapeutic impact of direct or indirect MYC inhibition. In this thesis, we outline the potential of direct MYC inhibition in Burkitt lymphoma (BL) and multiple myeloma (MM) making use of 3 different strategies: small molecule peptidomimetics and disruptors of the MYC/MAX/DNA complex (Omomyc mini-protein and the derivative variant 26 or V26): - In the first case, the validation of the peptidomimetics therapeutic potential was done in collaboration with a start-up biotech company. Despite evidencing some promising efficacy in vitro, the compounds displayed severe local toxicity in vivo, accompanied by changes in animal behaviour that prompted us to discontinue the investigation. - Regarding the validation of Omomyc mini-protein as a pharmacological approach in the treatment of BL and MM, we demonstrated in vitro efficacy and showed preliminary results in the prevention of bone marrow homing upon Omomyc pre-treatment in a zebrafish model, indicating, for the first time, the potential use of this drug candidate to treat liquid tumours. Our in vivo data in mice show that, even if the administration of Omomyc as monotherapy has limited efficacy (probably due to insufficient delivery of peptide to BL and MM target cells), the combination with a proteasome inhibitor (the standard of care for myeloma) both in vitro and in vivo displays synergic effects in myeloma models. In addition, we were able to show that intravenous administration of the Omomyc mini-protein encapsulated in liposomes was safe and the liposomal formulation prolonged the serum half-life of the mini-protein, although it did not promote increased penetrance in MM target cells. - Lastly, we characterised V26, a rationally designed derivative of the Omomyc mini-protein, meant to display improved nuclear localisation and endosomal escape. Here we evidenced that, like Omomyc, V26 can homodimerise and heterodimerise with MAX, as well as bind DNA in both dimeric forms. As expected, V26 displayed better nuclear localisation and induced cell death in vitro. However, it also turned out to be less soluble than Omomyc, indicating that it would require further formulation efforts to be used in vivo. Altogether, our results suggest that Omomyc mini-protein itself or other Omomyc-derivatives, like V26, can serve as the backbone for the design of new anti-MYC agents to treat BL and MM. In this context, combination therapy with the standard of care seems to be a promising strategy, while encapsulation in liposomes might help to address potential bioavailability issues that might arise from its use in vivo.

El microambient immune tumoral juga un paper fonamental en les etapes inicials de la formació dels tumors i en la progressió d’aquests. Teràpies dirigides a aquest microambient ofereixen noves opcions terapèutiques i també serveixen per a millorar les teràpies actuals enfront de molts càncers, incloent els que afecten les cèl·lules B. No obstant això, són necessàries més recerques per a entendre en major profunditat els mecanismes d’evasió del sistema immune que afavoreixen la progressió dels tumors i dissenyar immunoteràpies més precises. Els nostres principals objectius són aportar noves evidències sobre mecanismes immunes associats a la progressió tumoral i les bases pre-clíniques per al desenvolupament de noves estratègies terapèutiques amb potencial immunomodulador. Per a això, ens centrem en la leucèmia limfàtica crònica (LLC) i en el limfoma cerebral primari (LCP). Els mecanismes de progressió en LLC des d’estadis inicials no són coneguts íntegrament. Encara que l’adquisició d’alteracions moleculars és escassa suggerint que la LLC no progressa exclusivament per mecanismes d’evolució clonal, encara no s’ha dut a terme una anàlisi exhaustiva del microambient immune que demostri que la progressió sí que pugui deure’s a canvis immunes. Per això, hem realitzat un estudi longitudinal abastant tant els escenaris genètics com immunològics en pacients de LLC sense tractar que han progressat clínicament i en pacients asimptomàtics durant un llarg període de temps. Els nostres resultats mostren que els pacients que progressen experimenten un increment de cèl·lules T CD8+ efectores de memòria i terminalment exhaustes T-betmid/-*EomeshiPDhi a la progressió. Aquest increment no s’observa en els pacients de LLC que no han progressat. A més, les cèl·lules T a la progressió adquireixen un perfil transcripcional diferent. Això va acompanyat d’un augment en les propietats immunosupressores de les cèl·lules leucèmiques a la progressió. Vam demostrar que les cèl·lules de LLC en el moment de la progressió tenen major capacitat d’induir exhaustió tant en cèl·lules T CD8+ de LLC com aquelles procedents d’individus sans, i que ho fan mitjançant un mecanisme dependent de factors solubles que inclou IL-10. Els escassos canvis genètics que trobem després de seqüenciar el exoma dels nostres pacients ens permeten concloure que les variacions immunes que hem identificat són fonamentals per a la progressió de la LLC. El desenllaç dels pacients diagnosticats amb LCP és normalment desfavorable a causa de l’escassetat d’opcions terapèutiques efectives. Les cèl·lules malignes de LCP presenten amb freqüència una desregulació de la via del receptor de la cèl·lula B (de l’anglès, BCR), però la seva inhibició mitjançant ibrutinib mostra respostes molt breus en pacients. No obstant això, la via del BCR també pot bloquejar-se mitjançant la inhibició de la exportina nuclear XPO1 amb selinexor. Selinexor travessa la barrera hemato-encefàlica i ha mostrat activitat en un pacient diagnosticat amb limfoma difús de cèl·lules grans B amb recaiguda en el sistema nerviós central. Per consegüent, decidim avaluar els efectes de selinexor en monoteràpia i combinat amb ibrutinib en models preclínics murinos de LCP. La nostra anàlisi mostra que selinexor bloqueja el creixement tumoral i prolonga la supervivència en un model de ratolí bioluminiscent i la combinació amb ibrutinib prolonga encara més la supervivència. Vam demostrar que els limfomes cerebrals en ratolí estan infiltrats amb macròfags pro-tumorals M2 que expressen PD-1 i SIRPα. A més, el tractament amb selinexor i ibrutinib afavoreix la resposta immune anti-tumoral induint un canvi en la polarització dels macròfags cap a un perfil pro-inflamatori i reduint l’expressió de PD-1 i SIRPα en els macròfags M2 associats al tumor.<br>El microambiente inmune tumoral juega un papel fundamental en las etapas tempranas de la formación de los tumores y en la progresión de éstos. Terapias dirigidas a este microambiente ofrecen nuevas opciones terapéuticas y también sirven para mejorar las terapias actuales frente a muchos cánceres, incluyendo los que afectan a las células B. Sin embargo, son necesarias más investigaciones para entender en mayor profundidad los mecanismos de evasión del sistema inmune que favorecen la progresión de los tumores y diseñar inmunoterapias más precisas. Nuestros principales objetivos son aportar nuevas evidencias sobre mecanismos inmunes asociados a la progresión tumoral y las bases pre-clínicas para el desarrollo de nuevas estrategias terapéuticas con potencial inmuno-modulador. Para ello, nos centramos en la leucemia linfática crónica (LLC) y en el linfoma cerebral primario (LCP). Los mecanismos de progresión en LLC desde estadios tempranos no son conocidos en su totalidad. Aunque la adquisición de alteraciones moleculares es escasa sugiriendo que la LLC no progresa exclusivamente por mecanismos de evolución clonal, todavía no se ha llevado a cabo un análisis exhaustivo del microambiente inmune que demuestre que la progresión sí pueda deberse a cambios inmunes. Por ello, hemos realizado un estudio longitudinal abarcando tanto los escenarios genéticos como inmunológicos en pacientes de LLC sin tratar que han progresado clínicamente y en pacientes asintomáticos durante un largo periodo de tiempo. Nuestros resultados muestran que los pacientes que progresan experimentan un incremento de células T CD8+ efectoras de memoria y terminalmente exhaustas T-betmid/-EomeshiPDhi a la progresión. Este incremento no se observa en los pacientes de LLC que no han progresado. Además, las células T a la progresión adquieren un perfil transcripcional diferente. Esto va acompañado de un aumento en las propiedades inmunosupresoras de las células leucémicas a la progresión. Demostramos que las células de LLC en el momento de la progresión tienen mayor capacidad de inducir exhaustión tanto en células T CD8+ de LLC como aquellas procedentes de individuos sanos, y que lo hacen mediante un mecanismo dependiente de factores solubles que incluye IL-10. Los escasos cambios genéticos que encontramos tras secuenciar el exoma de nuestros pacientes nos permiten concluir que las variaciones inmunes que hemos identificado son fundamentales para la progresión de la LLC. El desenlace de los pacientes diagnosticados con LCP es normalmente desfavorable debido a la escasez de opciones terapéuticas efectivas. Las células malignas de LCP presentan con frecuencia una desregulación de la vía del receptor de la célula B (del inglés, BCR), pero su inhibición mediante ibrutinib muestra respuestas muy breves en pacientes. Sin embargo, la vía del BCR también puede bloquearse mediante la inhibición de la exportina nuclear XPO1 con selinexor. Selinexor atraviesa la barrera hemato-encefálica y ha mostrado actividad en un paciente diagnosticado con linfoma difuso de células grandes B con recaída en el sistema nervioso central. Por consiguiente, decidimos evaluar los efectos de selinexor en monoterapia y combinado con ibrutinib en modelos pre-clínicos murinos de LCP. Nuestro análisis muestra que selinexor bloquea el crecimiento tumoral y prolonga la supervivencia en un modelo de ratón bioluminiscente y la combinación con ibrutinib prolonga aún más la supervivencia. Demostramos que los linfomas cerebrales en ratón están infiltrados con macrófagos pro-tumorales M2 que expresan PD-1 y SIRPα. Además, el tratamiento con selinexor e ibrutinib favorece la respuesta inmune anti-tumoral induciendo un cambio en la polarización de los macrófagos hacia un perfil pro-inflamatorio y reduciendo la expresión de PD-1 y SIRPα en los macrófagos M2 asociados al tumor.<br>The tumor immune microenvironment (TIME) plays a critical role in the early formation of tumors and their progression. Targeting the TIME has offered new therapeutic approaches and improved current ones in several cancers, including B-cell malignancies. Nonetheless, further investigation is needed in order to more deeply understand immune evasion mechanisms that lead to tumor progression and to design therapies that modulate the immune system more precisely. Here, our main objectives are to provide new insights into immune mechanisms that favor tumor progression and a pre-clinical rationale for the design of new therapeutic strategies with immunomodulatory potential. To accomplish these goals our study will focus on chronic lymphocytic leukemia (CLL) and primary central nervous system lymphoma (PCNSL). Mechanisms driving the progression of CLL from its early stages are not fully understood. This hampers detecting progression in advance and developing therapies that could intervene in the early stages. Although the limited acquisition of molecular changes suggests that CLL progression is not mainly driven by clonal evolution, a deeper analysis of the immune microenvironment that demonstrates immune variations over time that contribute to progression has not been performed. Hence, we longitudinally studied the immune and genetic landscapes of untreated progressing and non-progressing patients. Our results show that progressed CLL patients experience an increase in effector memory and terminally exhausted T-betmid/-EomeshiPDhi CD8+ T cells over time, not observed in non-progressing patients. In addition, T cells at progression acquire a distinct transcriptional profile. This is accompanied by enhanced immunosuppressive properties in leukemic cells at progression. We prove that progressed CLL cells are intrinsically more capable of inducing CD8+ T-cell exhaustion in T cells affected by CLL and healthy T cells by a mechanism dependent on soluble factors including IL-10. In addition, the reduced genetic changes we found by whole-exome sequencing in our cohort indicate these immune variations are fundamental for progression in CLL. Patients diagnosed with PCNSL often face dismal outcomes due to the limited availability of therapeutic options. PCNSL cells frequently have deregulated B-cell receptor (BCR) signaling, but its inhibition using ibrutinib only offers a brief effective response in PCNSL patients. Nonetheless, the BCR pathway can also be blocked by inhibiting the nuclear exportin XPO1 using selinexor. Selinexor is able to cross the blood–brain barrier and has shown positive clinical activity in a patient with refractory diffuse large B-cell lymphoma in the CNS. Accordingly, we evaluated the effects of selinexor alone and also combined it with ibrutinib in pre-clinical mouse models of PCNSL. Our analysis shows that selinexor blocks tumor growth and prolongs survival in a bioluminescent mouse model and its combination with ibrutinib further increases survival. We demonstrate that CNS lymphomas in mice are infiltrated by tumor-promoting M2-like macrophages expressing PD-1 and SIRPα. Moreover, the treatment with selinexor and ibrutinib favors an anti-tumoral immune response by shifting macrophage polarization toward an inflammatory phenotype and diminishing the expression of PD-1 and SIRPα in M2 tumor-associated macrophages.

This study consists of two pieces of work investigating haematological malignancies; Acute Lymphoblastic Leukaemia (ALL) and Diffuse Large B Cell Lymphoma (DLBCL). Firstly, Pre-B ALL represents the most common paediatric malignancy and despite increasingly improved outcomes for patients, ~ 20% of all patients diagnosed with ALL relapse. Activating mutations in the RAS pathway are common (~50%) and result in hyperactivation of the MAPK pathway. I identified Erk negative feedback control via DUSP6 to be crucial for NRASG12D-mediated pre-B cell transformation and investigated its potential as a therapeutic target. I showed that a small molecule inhibitor of DUSP6 (BCI) selectively induced cell death in patient-derived pre-B ALL cells; with a higher sensitivity observed in relapse pre-B ALL. I also discovered that a high level of Erk activity is required for proliferation of normal pre-B cells, but dispensable in leukemic pre-B ALL cells. In addition, I found that human B cell malignancies can be grouped into three categories that fundamentally differ in their ability to control Erk signalling strength. Secondly, DLBCL is the most common haematological malignancy and although potentially curable with chemotherapy, 40% of patients still succumb from their disease. Recent exome sequencing studies have identified hundreds of genetic alterations but, for most, their contribution to disease, or their importance as therapeutic targets, remains uncertain. I optimised a novel approach to screen the functional importance of these mutations. This was achieved by reconstituting non-malignant, primary, human germinal centre B cells (GC B cells) with combinations of wildtype and mutant genes to recapitulate the genetic events of DLBCL. When injected into immunodeficient mice, these oncogene-transduced GC B cells gave rise to tumours that closely resemble human DLBCL, reinforcing the biological relevance of this system. To screen potential tumour suppressor mutations in this system in a high throughput fashion, I developed a lymphoma-focused CRISPR library of 692 genes recurrently altered in B cell lymphomas. These experiments identified GNA13 as an unexpectedly potent tumour suppressor in human GC B cells and provided new understanding to its mechanism of action. These findings provide novel understanding of the complexity of oncogenic mechanisms in human B cell malignancies.

Splenic marginal zone lymphoma (SMZL) and chronic lymphocytic leukaemia (CLL) are B cell malignancies, predominately affecting the elderly. The disease course of both SMZL and CLL is highly variable, with some patients dying rapidly within a month whilst other remain stable and live a normal lifespan. Biomarkers are used to help to distinguish those patients who may progress. Genomic abnormalities such as copy number alterations and mutation of genes may have prognostic value in CLL and SMZL. Several technologies were used to assess the genomic landscape in B cell malignancies; low resolution technology such as multiplex ligation-dependant probe amplification (MLPA) and Sanger sequencing as well as higher resolution methods such as SNP 6.0 arrays and next generation sequencing technologies; whole exome sequencing, TruSeq and Nextera XT. MLPA was used to assess both the copy number alterations (CNA) and mutational abnormalities in the well-defined CLL4 clinical trial cohort. Though the technology is restrictive in terms of probe location and sensitivity, there was statistical concordance with Sanger sequencing and fluorescence in situ hybridisation (FISH). Novel CNA, independently associated with survival were uncovered; 1) a very indolent disease course in patients carrying a biallelic 13q deletion with IGHV mutated genes and 2) a very poor prognosis in patients with a 9p deletion. Exome sequencing of CLL (n=6) and SMZL (n=7) patients uncovered novel variants via a bioinformatical pipeline; this pipeline was validated using more traditional sequencing methods. The candidate genes; U2AF1, BIRC3, POT1 and MYD88 are implicated in CLL and were screened in a larger cohorts of patients. Analysis of the 11q loci in relation to ATM and BIRC3 gene mutations, identified alterations of ATM that impact most significantly on survival. Nextera technology was optimised to screen ibrutinib treated CLL patients for BTK and PLCG2 mutations. DNAH9 and SPEN were also sequenced in a small cohort of patients. A breakpoint deletion was identified in the DNAH9 gene, suggesting this gene may be relevant in CLL. Further studies are needed to examine these newly defined high and low risk groups, and other genetic abnormalities in another well-defined CLL cohort, to ascertain their pathogenic and biological significance.

The ZFP36 proteins are a family of post-transcriptional regulator proteins that bind to adenine uridine rich elements (AREs) in 3’ untranslated (3’UTR) regions of mRNAs. The members of the human family, ZFP36L1, ZFP36L2 and ZFP36 are able to degrade mRNAs of important cell regulators that include cytokines, cell signalling proteins and transcriptional factors. This project investigated two proposed targets for the protein family that have important roles in B cell biology, BCL2 and CD38 mRNAs. BCL2 is an anti-apoptotic protein with key roles in cell survival and carcinogenesis; CD38 is a membrane protein differentially expressed in B cells and with a prognostic value in B chronic lymphocytic leukaemia (B-CLL), patients positive for CD38 are considered to have a poor prognosis. This project provides evidence of a functional interaction between the three ZFP36 proteins and the 3’UTR AREs of BCL2 and CD38 mRNAs. 3’UTR dual luciferase reporter assay results showed that the three ZFP36 proteins bound the 3’UTR ARE of BCL2 mRNA and CD38 mRNA. Zinc finger mutant versions of ZFP36L1 failed to bind the 3’ UTR AREs for each target, proving that intact zinc finger domains are the functional binding domains of the protein and are required for interaction with AREs. A complete ARE sequence is also needed and when mutated BCL2 3’UTR ARE was tested, lacking the adenine uridine rich core element, the BCL2 transcript was not bound by ZFP36L1 protein. For CD38 further experiments have demonstrated that down regulation of ZFP36L1 by siRNAs in HeLa cells resulted in an increase in CD38 expression as measured by immunofluorescence and flow cytometry and by Western blot analysis. These results provide further evidence that ZFP36L1 negatively regulates CD38 mRNA. Analysis of BCL2, CD38 and ZFP36L1 protein expression in primary B-CLL cells by Western blot analysis did not show an inverse relationship between the proposed targets and ZFP36L1. Protein expression analysis in B-CLL for the whole family of ZFP36 proteins showed that ZFP36L1 was heterogeneously expressed; ZFP36L2 was detected at very low levels or was undetectable and ZFP36 was low and homogeneously expressed. In cell lines representing different B cell stages, but mainly representing mature and plasma cell stages, ZFP36L2 was detected at relatively high levels but also heterogeneously and there was very low or undetectable expression of ZFP36L1 in all cells. Immunohistochemistry analysis of ZFP36L1, BCL2 and CD38 in normal lymphoid tissue and FL indicated that areas of normal lymphoid tissues associated with highest levels of BCL2 and CD38 were associated with low or undetectable levels of ZFP36L1. In FL (FL) ZFP36L1 was detected in follicular centre cells, where BCL2 is also reported to be highly expressed due to a translocation that leads to over expression of BCL2. CD38 expression was also detected within FL follicle centres with some cells showing a high level of expression within the neoplastic follicle and amongst scattered cells outside of it. Overall, the results support the hypothesis that ZFP36L1 (and also ZFP36 and ZFP36L2) negatively regulates BCL2 and CD38 mRNAs. In a wider context, the results of this project support the view that ZFP36L1, and perhaps other ZFP36 family proteins, play important roles in controlling mature B cell survival and differentiation by targeting important regulatory mRNAs in these cells.

Immunotherapy for cancer is a young research field progressing at high speed. The first chimera of an antibody and a signaling chain was designed by Zelig Eshhar and was later further developed to enhance existing T cell therapy by combining a single-chain fragment of an antibody with the CD3 zeta chain of the TCR complex. T cells expressing these chimeric antigen receptors (CARs) could recognize and specifically kill tumor cells. However the T cells, lacked in persistence and tumor rejection did not occur. Thus, the CAR constructs have been improved by providing the T cell with costimulatory signals promoting activation. The focus of this thesis has been to evaluate second and third generation αCD19-CAR T cells for the treatment of B cell leukemia and lymphoma. B cell tumors commonly upregulate anti-apoptotic proteins such as Bcl-2, which generates therapy resistance. In the first paper a second generation (2G) αCD19-CD28-CAR T cell was combined with the Bcl-2 family inhibitor ABT-737. ABT-737 sensitized tumor cells to CAR T cell therapy and may be an interesting clinical combination treatment. In paper II, the phenotype and function of a third generation (3G) αCD19-CD28-4-1BB-CAR T cell were evaluated. B cell-stimulated CAR T cells showed increased proliferation and an antigen-driven accumulation of CAR+ T cells. 3G CAR T cells had equal cytotoxic capacity, similar lineage, memory and exhaustion profile phenotype compared to 2G CARs. However, 3G CAR T cells proliferated better and had increased activation of intracellular signaling pathways compared to 2G CAR T cells. In paper III, αCD19-CD28-4-1BB-CAR T cells were used to stimulate immature dendritic cells leading to an upregulation of maturation markers on co-cultured dendritic cells. Hence, CAR T cells may not only directly kill the tumor cells, but may induce bystander immunity that indirectly aids tumor control. This thesis also include supplementary information about the development and implementation of protocols for GMP production of CAR T cell batches for a phase I/IIa clinical trial currently ongoing for patients with refractory B cell leukemia and lymphoma. So far, two patients have safely been treated on the lowest dose.

Chimeric antigen receptor (CAR) T-cells are genetically engineered to express a synthetic receptor which redirects their specificity and effector functions to a tumour associated antigen. They have induced unsurpassed clinical responses in patients with relapsed and refractory CD19-positive haematological malignancies who hitherto had limited treatment options. Currently, all commercially available CAR T-cell products are manufactured using viral vectors for transduction, an efficient but highly costly and complex process. Thus, access to CAR T-cell therapy is inequitable. Historically, the use of lower-cost non-viral plasmid-based transduction methods have been limited by the high toxicity and low transfection efficiencies of the electroporation process required for gene transfer and integration. This thesis furthers previous work using the super piggyBac plasmid-based system to generate CAR T-cells. Chapter 3 outlines the optimisation of an electroporation system with available current Good Manufacturing Practice reagents. It describes the effect of altering pre-electroporation, electroporation and post-electroporation variables on transfection efficiency and evaluates the feasibility of CAR T-cell production using the methodology described. Multiple myeloma is an incurable B-cell haematological malignancy without uniform CD19 expression. Its considerable heterogeneity poses both opportunities and challenges for CAR T-cell immunotherapy. In Chapter 4, design modifications to a novel CAR construct created with the piggyBac transposon/transposase system and directed against kappa myeloma antigen (KMA) to improve its clinical applicability for multiple myeloma are described. The non-viral superPiggyBac plasmid-based system is a feasible, cost-effective alternative for the production of CAR T-cells. It is envisaged that its use will improve access to CAR T-cells for patients with CD19-positive and CD19-negative haematological malignancies.

Despite significant advances, the prognosis in relapsed/refractory (R/R) B-cell malignancies remains poor. In the Phase I study of the selective Bruton’s Tyrosine Kinase inhibitor (BTKi) tirabrutinib in R/R B-cell malignancies, I showed that targeting BTK demonstrated remarkable clinical responses and tolerability in Chronic Lymphocytic Leukaemia and Mantle Cell Lymphoma. Targeted DNA sequencing demonstrated that no mutations were associated with a lack of response in CLL. However, in activated B-cell like diffuse large B-cell lymphoma (ABC DLBCL), only 35% of patients responded to treatment and median duration of response was 12 weeks. This prompted my laboratory studies to explore mechanisms of resistance to BTKi in ABC DLBCL. Using BTKi resistant cell lines TMD8 RO and TMD8 RI, I undertook biological and genetic studies. BTK expression and subcellular localisation was not altered in the resistant cell lines. Apoptosis induced by the BTKi ibrutinib and tirabrutinib occurred 24 hours following drug exposure. A decrease in the expression of the anti-apoptotic proteins MCL1, BCLxL and BCL2A1 was observed in TMD8 but not TMD8 RO following treatment with tirabrutinib, consistent with modulation of the BCR pathway. No significant change was identified in apoptotic gene expression. Study of the BCR signalling pathway showed an increase in cell surface expression of sIgM and CD20 in the resistant cell lines. No change in IgM RNA levels nor CD20 were observed. However, gene expression of IGJ was downregulated in the resistant cell lines. Both TMD8 RO and TMD8 RI showed increased basal levels of phosphor-tyrosine phosphorylation and amplified BCR signalling following BCR ligation. Collectively, these studies indicate hyperactivation of the BCR signalling pathway in the development of resistance to BTKi, with changes occurring upstream of BTK. Further studies to characterise changes at the cell surface are required to identify novel therapeutic approaches to the development of BTKi resistance.

Despite several recent advancements in the treatment of B lymphoproliferative disorders, still a considerable number of lymphoma cases either cannot be cured or become incurable when relapsing. This issue reflects the need for better and more effective therapies that can be designed once novel pathogenic mechanisms have been designed and suitable preclinical models have been established. Therefore, aim of this thesis was to understand the molecular mechanisms leading to mature B lymphoid malignancies by using different mouse models, in order to identify novel potential therapeutic targets and obtain useful models for preclinical studies. In chapter 2 we show the establishment of a new xenograft model obtained by injecting the CLL cell line MEC1 into immunodeficient Rag2‒/‒gammac‒/‒ mice. This model resembles the aggressive form of human CLL and is conceivably useful to test the efficacy of new therapeutic agents. In chapter 3 we show that HS1 is involved in the trafficking and homing of leukemic B cells and that its deficiency is responsible for an earlier onset of the disease and a reduced survival in the Emu-TCL1 mouse model of CLL. The same animal model was used to investigate the role of TLR pathway in CLL. In chapter 4 we report that the absence of TIR8 accelerates the appearance of the disease and favors the progression into an aggressive form characterized by the accumulation of “prolymphocytoid” cells. Finally in chapter 5 we demonstrate that mice lacking the negative regulator SIGLEC-G are susceptible to B cell lymphoma development with age indicating that the downregulation of SIGLEC10 may be involved in the malignant transformation of human B lymphocytes.

This thesis contains two distinct parts: Current models of Chronic Lymphocytic Leukaemia (CLL) pathogenesis invoke specialised anatomical microenvironments that harbour proliferating cells. Such proliferating CLL cells are more resistant to current immuno-chemotherapeutic regimens than cells in the peripheral blood and are thought to be the cause of disease relapse. Using a system to recapitulate CLL proliferation centres in vitro, I have observed that CLL cells undergo proliferation. Unexpectedly, under these conditions an induction of wild-type TP53 protein was also observed in all cases of CLL analysed. The results reported here were undertaken to understand how CLL cells upregulate TP53 protein and proliferate. For reasons that remain unclear, TP53 is unable to transactivate its classic target genes to induce cell-cycle arrest or apoptosis. However, it remains able to trigger a full apoptotic response after further DNA damage and a higher threshold of protein levels is reached. We propose a model whereby oxidative stress induced by proliferation in CLL triggers TP53 protein expression. Hairy Cell Leukaemia (HCL) represents approximately 2% of all leukaemias, follows an indolent course and remains an incurable disease. Recently, virtually all HCL patients shown to carry the BRAFV600E mutation, thought to be a disease-defining event. The BRAF V600E mutation results in constitutive activation of the MEK-ERK pathway resulting in aberrant proliferation, and targeted inhibitors have shown efficacy in BRAFV600E positive tumours. We wanted to test whether this efficacy can be extrapolated to HCL. Here we report in vitro studies using PLX4720 and in vivo trial of Vemurafenib in a patient with refractory HCL. While BRAF inhibition showed no effect on HCL survival in vitro, it resulted in rapid loss of viability of hairy cells in vivo. The results obtained show that efficacy of BRAF inhibition achieved did not occur via the expected inhibition of MEK-ERK activation.

This thesis presents a light scattering-based method for biomarker detection, which could potentially be used for the quantification of multiple biomarkers specific to B-cell malignancies. This method uses fabricated gold nanoparticle probes to amplify inelastic light scattering in a process referred to as surface-enhanced Raman scattering. These gold nanoparticle probes were conjugated to antibodies for specific and targeted molecular binding. The spectrum of the amplified inelastic light scattering was detected using a spectrometer and a detector. To detect the light scattering signal from the gold nanoparticle probes, several commercial Raman spectrometer instruments were evaluated. Initial results from these evaluations are presented in this thesis. After system evaluation, a custom Raman microscope system was designed, built, and tested. This system was used for the development of a surface-enhanced Raman spectroscopy-based immunoassay. The development of this assay confirms the successful design of gold nanoparticle probes for the specific targeting and detection of immunoglobulins. The immunoassay also shows promise for the simultaneous detection of multiple biomarkers specific to B-cell malignancies.

CD19-specific chimeric antigen receptor (CAR19) T cells are effective against relapsed/refractory B cell malignancy; but, most are individualised autologous products that have been generated using viral vectors. The complexity and cost of this approach is a barrier to widespread use. The plasmid-based piggyBac transposon system is a simple and economical alternative to viral vectors for transgene delivery. However, CAR19 T cells previously generated with piggyBac lacked in vivo efficacy, likely due to deleterious interactions of the CAR with other immune cells. This thesis describes the clinical translation of efficacious piggyBac CAR19 T cells. Pre-clinical evaluation of piggyBac CAR19 T cells expressing re-engineered CARs demonstrated potent efficacy and persistence in vivo, providing the rationale for clinical translation. The influence of CAR design on CAR19 T cell phenotype and function was also demonstrated in vitro. A first-in-human phase I clinical trial of escalating doses of HLA-matched sibling donor-derived piggyBac CAR19 T cells for the treatment of relapsed/refractory B cell malignancies post allogeneic haematopoietic stem cell transplant was initiated. Early results show that donor-derived piggyBac CAR19 T cells effectively induce disease remission, and suggest that expansion, persistence, acute toxicity and relapse profiles may be similar to autologous CAR19 T cells produced with viral vectors. But, development of a CAR19 T cell malignancy in a single patient is a serious safety issue under investigation. CAR19 T cell production with piggyBac was refined by replacing plasmid components with doggybones. These are minimal DNA vectors that can be inexpensively produced enzymatically, and lack bacterial DNA sequences and antibiotic resistance genes that are problematic for clinical use. If serious safety issues can be resolved, simple and affordable CAR T cell production with piggyBac shows promise for accelerating research and improving clinical access.

2013/2014<br>B-cell malignancies are a heterogeneous group of clinical conditions including indolent diseases such as Chronic Lymphocytic Leukemia (CLL) and highly aggressive lymphoproliferative disorders such as Burkitt’s lymphoma. B-cell malignancies treatments take advantage of dose-intensive chemotherapeutic regimens and immunotherapy via monoclonal antibodies. Unfortunately, they may lead to insufficient tumor distribution of therapeutic agents and cause several adverse effects. Thus, we propose a novel therapeutic approach for the treatment of CLL and Burkitt’s lymphoma in which high-doses of the association of hydroxychloroquine and chlorambucil (HCQ/CLB) or fludarabine were loaded inside biodegradable nanoparticles (BNPs) coated with an anti-CD20 antibody. First of all, a Burkitt’s lymphoma cell line (BJAB), two CLL cell lines (MEC1 and EHEB) and cells purified from patients’ blood samples were used to confirm CD20 expression and to assess BNPs binding and internalization. These studies demonstrated BNPs ability to bind malignant B cells and to enter inside cells in a process different from endocytosis. Then, BNPs therapeutic effect was evaluated by MTT test, AnnV/PI assay and western blot to put in evidence apoptosis induction and autophagy inhibition. These experiments demonstrated drugs-loaded BNPs ability to kill malignant B cells with comparable effects than those obtained with free drugs whereas empty BNPs were practically ineffective. In vivo BNPs characterization included the evaluation of their toxicity, biodistribution and therapeutic effect. C57/BL mice were used to evaluate BNPs toxicity which was studied considering survival, loss of body weight and several tissue markers in the blood. Mice receiving 8 injections of free HCQ+CLB died in this experiment whereas animals challenged with the same amount of drugs encapsulated inside BNPs did not show toxic effects suggesting BNPs safety. The importance of antiCD20 antibody in the homing of BNPs was confirmed by in vivo Time-Domain Optical Imaging performed in localized B-cell malignancy-bearing mice. This analysis suggested the ability of antiCD20-conjugated BNPs to specifically target tumor B-cells, with a pick after 24-48 hours. On the contrary, untargeted BNPs localization inside tumor was significantly decreased. In this analysis it was also evident that the liver is the main site of BNPs’ elimination while in the other organs the presence of fluorescent BNPs was very low. Finally, BNPs ability to treat a new xenograft human/SCID leukemia and Burkitt’s lymphoma mouse model was studied. Drugs-loaded BNPs were able to improve HCQ/CLB efficacy in vivo allowing the cure of treated all Burkitt’s lymphoma-bearing mice and 3 out of 7 leukemia-bearing animals. All these data together put the basis for the potential use of BNPs in the treatment of B-cell malignancies.<br>I tumori a cellule B sono un gruppo eterogeneo di patologie che comprendono sia malattie indolenti, come la leucemia linfatica cronica (LLC), sia aggressive, come il linfoma di Burkitt. Il trattamento delle patologie a cellule B prevede sia l’utilizzo di chemioterapici (agenti alchilanti e analoghi delle purine) che di anticorpi monoclonali. Nonostante la varietà di terapie esistenti, l’efficacia di questi farmaci è limitata dalla mancata specificità per le cellule tumorali e dall’induzione di gravi effetti collaterali. Per ovviare ai limiti delle terapie attuali, è stato quindi proposto l’utilizzo di nanoparticelle coniugate con un anticorpo antiCD20, specifico per le cellule B, e contenenti alte concentrazioni di chemioterapici (idrossiclorochina e clorambucile o fludarabina). Le nanoparticelle sono state caratterizzate in vitro e in vivo durante questo progetto di dottorato. Inizialmente sono stati effettuati studi in vitro al fine di valutare l’espressione del CD20 sulla superficie di una linea cellulare di linforma di Burkitt (BJAB), due linee di LLC (MEC1 e EHEB) e cellule purificate da campioni di sangue di pazienti affetti da LLC. In seguito, il legame e l’internalizzazione delle nanoparticelle a queste cellule sono stati dimostrati suggerendo anche come le nanoparticelle vengano internalizzate attraverso un meccanismo diverso dall’endocitosi. L’effetto terapeutico in vitro delle nanoparticelle è stato valutato con test MTT, AnnessinaV/PI e tramite western blot al fine di evidenziare l’induzione di apoptosi e l’inibizione dell’autofagia, meccanismi attraverso cui i farmaci utilizzati sono noti agire. Questi esperimenti hanno dimostrato che le nanoparticelle cariche di chemioterapici sono in grado di uccidere le cellule B tumorali con effetti paragonabili a quelli ottenuti da pari concentrazioni di farmaci liberi dimostrando come il processo di produzione delle nanoparticelle non influisca sull’efficacia dei chemioterapici. Al contrario, nanoparticelle vuote non sono in grado di uccidere le cellule dimostrando la mancata tossicità dei polimeri da cui sono costituite. Dopo aver confermato il legame e l’internalizzazione delle nanoparticelle che inducono la morte delle cellule B tumorali, sono stati effettuati esperimenti in vivo tra cui studi di tossicità al fine di valutare eventuali effetti collaterali indotti dal trattamento, studi di biodistribuzione e la valutazione degli effetti terapeutici. Gli studi di tossicità sono stati effettuati in topi sani valutando parametri quali la perdita di peso, la sopravvivenza e la tossicità sistemica. Nanoparticelle cariche di farmaci presentano un profilo tossicologico sicuro mentre pari dosi di farmaci liberi inducono la morte di tutti gli animali trattati. Questi esperimenti dimostrano quindi come l’inserimento di farmaci all’interno di nanoparticelle prevenga gli effetti collaterali normalmente indotti dai chemioterapici. Secondariamente, sono stati effettuati studi di biodistribuzione di nanoparticelle coniugate o meno con un anticorpo antiCD20. Questi studi effettuati tramite Optical Imaging dimostrano come nanoparticelle coniugate con l’anticorpo antiCD20 si localizzino preferenzialmente nella massa tumorale in 24-48 ore in quantità maggiore rispetto a nanoparticelle non coniugate con l’anticorpo. Inoltre, da queste analisi risulta evidente come il fegato sia il maggiore sito di eliminazione delle nanoparticelle mentre in altri organi la presenza di nanoparticelle è molto bassa. Infine, un modello disseminato di linfoma di Burkitt e un modello di LLC sono stati sviluppati in topi SCID (Severe Combined ImmunoDeficiency) iniettando rispettivamente cellule BJAB intraperitoneo e cellule MEC1 endovena. I modelli sono stati caratterizzati e utilizzati per valutare la potenziale applicazione delle nanoparticelle nel trattamento di queste patologie. Questi studi hanno dimostrato come le nanoparticelle siano in grado di aumentare l’efficacia dei chemioterapici e di curare tutti i topi affetti da linfoma di Burkitt e 3/7 topi affetti da leucemia. Concludendo, questi risultati suggeriscono la potenziale applicazione delle nanoparticelle cariche di chemioterapici nel trattamento di LLC e linfoma di Burkitt.<br>XXVII Ciclo<br>1986

The proper differentiation and survival of human peripheral immature B cells relies on two critical signaling pathways: B cell receptor (BCR) signaling and the B cell activating factor (BAFF)/BAFF-receptor (BAFF-R) signaling axis. The quality of the BCR signal is regulated in a developmental manner. Self-reactive early immature B cells are eliminated in response to strong BCR-induced signals, while late immature B cells require BCR-induced signals for survival and further differentiation. Although components and events downstream of the BCR are well known, the mechanisms of BCR signaling and its role in the regulation of BAFF signaling are still poorly understood. Through the use of transgenic and knockout murine models, the effects of BAFF on murine B cell maturation and survival are well characterized. There is a crucial need to better understand the functions of BAFF in humans. High levels of soluble BAFF, reduced expression of BAFF-R and BCR signaling abnormalities in B cells have been identified in a large group of clinically heterogeneous diseases including autoimmune and inflammatory conditions, allergy, viral infections and lymphoid cancers. In order to better understand BCR signaling mechanisms, functional properties of human BAFF and factors regulating BAFF-R expression, this thesis describes: 1) the phenotypic, molecular and functional characterization of rare unknown inherited monogenic immunodeficiencies involving defects in early B cell development and the BAFF/BAFF-R pathway and, 2) the functional characterization of dysregulated BAFF/BAFF-R signaling in B cell malignancy. This led to the discovery of two novel primary immunodeficiencies involving MALT1 deficiency and gain-of-function PLCγ2 mutation. Our results indicate that MALT1 is essential for antigen-receptor mediated NF-κB activation and plays a role in the surface expression of BAFF-R and proper development of human B cells. The PLCγ2 mutation led to hyper-reactive BCR signaling and increased apoptosis of transitional B cells. Work-up of this patient also allowed us to investigate how soluble BAFF down-modulates surface expression of it’s principal receptor, BAFF-R, through receptor internalization, in normal B cells. Further analysis of the BAFF/BAFF-R pathway in pre-B acute lymphoblastic leukemia provides evidence of different structural and functional BAFF isoforms.<br>Medicine, Faculty of<br>Pathology and Laboratory Medicine, Department of<br>Graduate

B-lymphocytes rearrange their cytoskeleton and undergo dramatic morphological changes when searching for antigens and when forming immune synapses upon contacting cells that display antigens on their surface. Although these morphological changes are essential to B cell function, the signaling pathways underlying these processes are not fully understood. The aim of this thesis is to investigate how B cell receptor (BCR) and integrin signaling regulate B cell morphological changes. The Rap GTPases (Rap) are molecular switches that regulate integrin activation, adhesion, migration in B cells and other cell types. I hypothesize that activation of the Rap GTPases is important for regulating changes in B cell morphology. Indeed, in this thesis I showed that activation of Rap is essential for B cell cytoskeletal rearrangements. I found that Rap activation is important for BCR- and lymphocyte function-associated antigen-1 (LFA-1)-induced spreading, for BCR-induced immune synapse formation, and for particulate BCR ligands to induce localized F-actin assembly and membrane process extension. Rap activation and F-actin assembly were also required for optimal BCR signaling in response to particulate antigens leading to B cell activation. Consistent with Rap activation being important for B cell adhesion and migration, I showed that Rap activation is important for the dissemination of B cell lymphomas in vivo. B cell lymphomas are common malignancies in which transformed B cells enter the circulation, extravasate into tissues, and form tumors in multiple organs. Lymphoma cells are thought to exit the vasculature and enter tissues via the same chemokine- and adhesion molecule-dependent mechanisms as normal B cells. Using A20 murine B lymphoma cells, I showed that Rap activation is important for circulating lymphoma cells to invade tissue and form tumors in vivo in syngeneic mice. Moreover, using in vitro models I showed that Rap activation is required for these cells to extend membrane processes between vascular endothelial cells and undergo transendothelial migration. Thus, by controlling B cell morphology and cytoskeletal organization, the Rap GTPases play a key role in both malignant and normal B cell functions, and may be a potential therapeutic target for treatment of B cell-related diseases.

Chronic Lymphocytic Leukaemia (CLL) remains the most abundant leukaemia in those aged over 65 years. It is characterised by the expansion of malignant monoclonal B-lymphocytes that were originally described as being functionally incompetent. Identified by immunophenotypic expression of monoclonal light chain restriction, it falls into the classification of chronic B-cell lymphoproliferative disorders (B-LPD). This thesis aims to demonstrate that CD160, an activating NK cell receptor, is aberrantly expressed in B-LPD and can function as a tumour specific antigen, which has clear translation roles within the clinical environment, aiding in the diagnosis of CLL and monitoring of minimal residual disease (MRD). More so, this study aims to provide an insight into the potential biological roles of CD160 within chronic B-cell malignancies. CD160 is an activating NK cell receptor whose major form is a glycosylphosphatidylinositol (GPI)-anchored cell surface molecule with a single immunoglobulin domain. In-vitro studies on a large cohort of B-LPD patients demonstrated that CD160 was primarily restricted to cases of CLL (98%) and Hairy Cell Leukaemia (HCL, 100%) with only a minor population of other B-LPDs expressing the antigen. More so, within the B-cell lineage, CD160 can be considered a tumour specific antigen (TSA) in that when looking for both transcript and protein, they were absent throughout the normal B-cell hierarchy. Many clinical studies base their entry criteria on clinical and biological prognostication, as this provides insights into the biology of CLL and its response to therapy. Disease eradication has been shown to be prognostic. This study demonstrates the feasibility and clinical importance of MRD detection utilising CD160 as novel marker of residual disease. Subsequently, CD160 analysis by flow cytometry (CD160FCA) demonstrated to be as sensitive and specific as other methodologies, and independent of the type of therapy. Further to this the early detection of MRD was correlated with known biological prognostic risk groups. Patients in CR had significantly different EFS based on their MRD status following treatment using the CD160FCA. For those patients with adverse prognostic markers (including CD38, ZAP-70 and M), the time to detection of MRD or relapsing disease ß2using CD160FCA, was significantly shorter than those with a normal or good prognosis. Within normal NK and T lymphocytes, CD160 has a multifunctional role that upon triggering results in a unique profile of cytokine production via the recruitment of Phosphatidylinositol 3-kinase (PI3K). In CLL cells, CD160 stimulation resulted in the recapitulation of these observations including cell survival, an increase in Bcl-2 family antiapoptotic proteins, and cell cycle progression. This thesis has demonstrated that CD160 is aberrantly expressed in malignant B-cells, it has a clear clinical translation role in terms of diagnosis and MRD monitoring, and multiple biological functions which recapitulate those observed in NK-cells.

Cyclin D1 expression is deregulated by chromosome translocation in mantle cell lymphoma and a subset of multiple myeloma. The epigenetic mechanisms involved in this long-distance gene deregulation were studied. The cyclin D1 promoter was hypomethylated and hyperacetylated in expressing cell lines and patient samples. Domains of hyperacetylated histones and hypomethylated DNA extended over 120 kilobases upstream of the cyclin D1 gene. Interestingly, hypomethylated DNA and hyperacetylated histones were also located at the cyclin D1 promoter but not upstream regions in cyclin D1 nonexpressing, nontumorigenic B and T cells. The findings of complete cyclin D1 promoter demethylation were surprising given the presence of both the translocated or inserted and the normal chromosome 11. The cyclin D1 locus and sequences 90 kilobases upstream were methylated in genetic variants that had lost the translocated or inserted but retained one or more copies of normal chromosome 11. These observations indicate a trans demethylating effect of the translocated chromosome containing the immunoglobulin heavy chain (IgH) locus that resembles transvection in Drosophila and paramutation in maize. RNA polymerase II (Pol II) binding was demonstrated at the cyclin D1 promoter, upstream sequences, and 3' IgH regulatory regions only in malignant B cell lines with deregulated expression of the cyclin D1 gene. These results suggest Pol II bound at IgH regulatory sequences can activate the cyclin D1 promoter by either a long-range polymerase transfer or tracking mechanism. The observation that the DNA binding protein CTCF concurrently binds to the cyclin D1 promoter region and IgH regulatory elements only in the cyclin D1(+) MCL cell lines but not in the cyclin D1(-) EBV-transformed B lymphocytes suggests that CTCF may be crucial in the long distance interaction between IgH regulatory elements and the cyclin D1 gene locus. I propose that, in cyclin D1 expressing B cell malignancies, after chromosomal translocation, concurrent binding of Pol II, CTCF, and other transcription factors at the IgH regulatory regions and cyclin D1 gene promoter juxtaposes these two loci, affecting cis and trans DNA hypomethylation and histone hyperacetylation, and initiating deregulated expression of cyclin D1.

Cytokine-induced killer (CIK) cells are a heterogeneous population of lymphocytes obtained in vitro within 21 days from mononuclear cells under the influence of cytokines. CIK cells show potent MHC-unrestricted cytotoxicity against a variety of tumor cells, in particular hematological malignancies, and minimal tendency to induce graft-versus-host disease. The expanded bulk CIK culture consists of over 90% CD3+ cells, of which the majority coexpress CD56 and the remaining cells are CD56-. CD3+CD56+ “true” CIK cells are terminally differentiated non dividing lymphocytes which could deliver potent MHC-unrestricted cytotoxicity for the immediate destruction of tumor cells. The other less cytotoxic CD3+CD56- cell subset represents a progenitor reservoir that could proliferate and differentiate into CD3+CD56+ CIK cells. CD3+CD56+ CIK cells express activating NK receptors including NKG2D, DNAM-1 and low levels of NKp30. Cell signalling not only through TCR/CD3, but also through NKG2D, DNAM-1 and NKp30, leads to CIK cell activation resulting in granule exocytosis and cytotoxicity. Antibody blocking experiments revealed that NKG2D, DNAM-1 and NKp30 are actually involved in tumor cell recognition and killing. Anti-CMV specific CIK cells could be expanded in standard CIK conditions and mediate both specific, MHC-restricted recognition of a CMV-pulsed autologous target and NK-like non specific cytolytic activity against leukemic cell targets. Antibody blocking of NKG2D and NKp30 only inhibited NK-like cytotoxicity. Their dual effector function suggests that CIK cells, when used in a clinical setting, may control both neoplastic relapses and viral infections, two frequently associated complications in transplanted patients. B-cell non-Hodgkin lymphoma is only partially susceptible to CIK-mediated lysis. The addition of anti-CD20 monoclonal antibodies GA101 or rituximab increased cytotoxicity mediated by CIK cell cultures by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum inhibited NK-cell activation induced by rituximab, but not activation induced by GA101. Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 mcg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. The combined use of CIK cells with anti-CD20 mAbs could represent a novel immunotherapy protocol for the treatment of B lymphoma patients with resistant disease.

Oncolytic measles virus (MV) is being tested in several ongoing clinical trials with encouraging results. There is a demonstrable need to explore the role of the immune system in addition to the direct oncolytic effect of MV. My laboratory has previously shown that neutrophils are involved in MV-mediated tumour regressions, becoming activated, upon MV infection. This thesis further explores the role of neutrophils, one of the key players of the innate immune system in MV oncolysis. First, I showed that acute lymphoblastic leukaemia (ALL) shows marked sensitivity to MV oncolysis (Patel, Dey et al., 2011). I attempted to enhance neutrophil function at tumour sites by generating a novel strain of MV expressing the human granulocyte colony-stimulating factor (GCSF), a known neutrophil survival factor and enhancer of antibody dependent cellular cytotoxicity (ADCC). Evaluating the effects in two different models of B-cell malignancy, I showed that neutrophil depletion abrogated the MV therapeutic effect in an in-vivo Raji - but not Nalm-6 - tumour model. MVhGCSF enhanced the oncolytic capacity of MV in the Raji model in-vivo, whereas in the Nalm-6 model, the opposite was unexpectedly the case. MVhGCSF replicated within an MV-infectable CD46 transgenic mouse model with detectable serum levels of hGCSF but no toxicity. My data suggest that a "one-size-fits-all" model of immune response to viral oncolysis is not appropriate, and each tumour target will need full characterisation for the potential of MV to generate benefit (Dey et al., 2016). 5 Next, I showed that ADCC was NOT a mechanism by which neutrophils kill MV-infected cells. Finally, I showed that MV infection of target cells can stimulate neutrophils to develop a cytotoxic effector phenotype, all aspects of which are blocked by fusion inhibition. Hence, I suggest a new mechanism for MV-mediated oncolysis; fusion between infected target cells and neutrophils.

Les lymphocytes B mémoires circulants incluant ceux infectés par EBV de façon latente retournent périodiquement vers les territoires lymphoïdes secondaires où ils subissent une différenciation en cellules productrices d'immunoglobulines permettant au virus d'initier la réplication virale. Cependant, le suivi et la gestion de la réactivation de EBV et son association avec des néoplasies lymphoïdes chez les sujets infectés par le VIH restent un sujet de controverse et nécessite une meilleure compréhension des mécanismes impliqués. Dans cette étude, nous avons proposé de nouveaux outils biologiques pour la quantification de l'ADN EBV permettant la discrimination entre le réservoir latent et le cycle lytique du virus. Nous avons montré que la taille de ces réservoirs est étroitement associée à une activation polyclonale plus ou moins importante des cellules B. Nous avons également observé une association entre les marqueurs d'activation immunitaire et de réactivation de EBV avec la survenue de lymphome B. En outre, nous avons décrit l'évolution de gammapathies monoclonales chez des sujets infectés par le VIH sous traitement antirétroviral, et la persistante du pic monoclonal d'immunoglobulines était associée à des charges virales EBV plus élevés. Par conséquent, l'activation des lymphocytes B et subséquemment la réactivation EBV joueraient vraisemblablement les rôles principaux dans la survenue de tumeurs bénignes ou malignes des lymphocytes B au cours de l'infection VIH<br>It is assumed that circulating memory B cells including those latently infected by EBV return periodically to lymphoid nodes where they are stimulates and undergo differentiation into immunoglobulin-producing cells allowing the virus to initiate viral replication. However, the monitoring and the management of EBV reactivation and it association with lymphoid malignancies in HIV-infected patients are still being controversies and need a better understanding of the probable mechanisms involved. In this study, we proposed novel biological tools for EBV DNA quantification allowing discriminating latent and lytic reservoir. We showed that the EBV reservoir levels are closely associated with the polyclonal B-cell activation. We also observed an association between immune activation and EBV reactivation markers with the occurrence of B-cell lymphoma. Moreover, we described a long term evolution of monoclonal gammapathies in HIV-infected subjects and the persistence of the immunoglobulis monoclonal pike was found to be associated with higher EBV reservoir levels. Therefore, the B-cell activation and subsequently EBV reactivation likely play the main roles on the occurrence of B lymphocytes malignancies during HIV infection

Maintenance of telomere length, required for the unlimited cell proliferation displayed by cancer cells, is provided by telomerase, a ribonucleoprotein complex containing a specialized reverse transcriptase, encoded by TERT gene, that uses an internal RNA template to maintain telomeres length, thus playing a critical role in tumor formation and progression. TERT is usually repressed in normal somatic cells, but is detectable in the vast majority of tumors. Recent studies have suggested that TERT, besides maintaining telomere, is involved in other cellular functions, and it may contribute to carcinogenesis also via telomere length-independent mechanisms; therefore its inhibition could represent a promising strategy to improve cancer treatment, regardless of telomere length. The possible therapeutic effects of BIBR1532 (BIBR), a specific TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein-Barr virus (EBV)-driven and virus-unrelated B-cell malignancies. The aim of this study was to characterize the biological effects of short-term TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt’s lymphoma (BL) cell lines; in addition, we investigated the effects of short-term BIBR treatment in vivo in wild type zebrafish embryos. We found that short-term inhibition of TERT by BIBR, in in vitro models of B-cell malignancies, led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately increased apoptosis. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone. In agreement with in vitro results, short-term inhibition of Tert by BIBR in wild type zebrafish embryos reduced cell proliferation, induced an accumulation of cells in S-phase, increased apoptosis, and triggered the activation of DDR. These effects were telomere length-unrelated, since the range of telomere length was not affected by the short-term BIBR treatment and the DNA damage foci were distributed randomly, rather than specifically located at telomeres. All these effects were specifically related to Tert inhibition since BIBR treatment showed no effect in Tert-negative zebrafish embryos. Taken together these data demonstrate that TERT inhibition impairs cell proliferation and induces pro-apoptotic effects unrelated to telomere dysfunction, enforcing the concept that TERT per se exerts telomere length-independent tumor-promoting effects, and thus supporting the introduction of TERT inhibitors to complement current anticancer treatment modalities.<br>Il mantenimento dei telomeri, necessario per la proliferazione illimitata delle cellule tumorali, è esercitato dalla telomerasi, un complesso ribonucleoproteico contenente una trascrittasi inversa specializzata, codificata dal gene TERT, che utilizza un templato ad RNA per sintetizzare nuove sequenze telomeriche, svolgendo quindi un ruolo critico nella formazione e nella progressione dei tumori. TERT viene infatti solitamente represso in normali cellule somatiche, mentre è rilevabile nella maggior parte dei tumori. Studi recenti hanno suggerito che TERT è coinvolto in altre funzioni cellulari e può contribuire alla carcinogenesi anche attraverso meccanismi indipendenti dal mantenimento dei telomeri, quindi la sua inibizione potrebbe rappresentare una strategia promettente per migliorare il trattamento antitumorale, al di là dell’effetto sui telomeri. I possibili effetti terapeutici di BIBR1532 (BIBR), un inibitore specifico del TERT, sono stati valutati in diversi contesti cellulari, ma non sono attualmente disponibili dati ottenuti su modelli di neoplasie delle cellule B sia associate al virus di Epstein-Barr (EBV) che virus-indipendenti. Lo scopo di questo studio era di caratterizzare gli effetti biologici dell'inibizione di TERT a breve termine da parte del BIBR su linee cellulari linfoblastoidi immortalizzate da EBV (LCL) e su modelli in vitro di linfoma di Burkitt (BL); inoltre, sono stati studiati gli effetti del trattamento con BIBR a breve termine in vivo negli embrioni di zebrafish. I risultati ottenuti hanno dimostrato che l'inibizione a breve termine di TERT da parte di BIBR, in modelli in vitro di tumori delle cellule B, ha portato a una diminuzione della proliferazione cellulare, all'accumulo di cellule nella fase S e infine all'aumento dell'apoptosi. L'arresto del ciclo cellulare e l'apoptosi, conseguenti all'inibizione di TERT a breve termine, erano associati e probabilmente dipendenti dall'attivazione della risposta al danno del DNA, come evidenziato dall’aumento dei livelli di γH2AX e dall'attivazione dei pathway di ATM e ATR. L’analisi della media e del range di lunghezza dei telomeri e dei foci di danno al DNA ha indicato che la risposta al danno attivata in seguito all’inibizione TERT a breve termine non era legata a disfunzioni telomeriche, suggerendo quindi che TERT, oltre a stabilizzare il telomero, può proteggere il DNA tramite meccanismi telomero-indipendenti. In particolare, LCL-TERT positive trattate con BIBR in combinazione con fludarabina o ciclofosfamide hanno mostrato un aumento significativo del numero di cellule apoptotiche rispetto a quelle trattate con agenti chemioterapici da soli. In accordo con i risultati in vitro, l'inibizione a breve termine di Tert da parte del BIBR in embrioni di zebrafish ha ridotto la proliferazione cellulare, indotto un accumulo di cellule nella fase S, aumentato il tasso di apoptosi e innescato l'attivazione della risposta al danno al DNA. Questi effetti non erano legati a disfunzioni telomeriche, poiché il range di lunghezza dei telomeri non era influenzato dal trattamento a breve termine con BIBR e i foci di danno al DNA erano distribuiti casualmente, piuttosto che localizzati in modo specifico sui telomeri. Tutti questi effetti erano specificamente associati all'inibizione di Tert poiché il trattamento con BIBR non mostrava alcun effetto negli embrioni di zebrafish Tert-negativi. Nel complesso questi dati dimostrano che l'inibizione del TERT compromette la proliferazione cellulare e induce effetti pro-apoptotici non associati a disfunzioni telomeriche, rafforzando il concetto che TERT esercita di per sé funzioni pro-tumorali indipendenti dalla lunghezza del telomero e quindi supportando l'introduzione di inibitori di TERT per integrare le attuali modalità di trattamento antitumorale.

En France, les hémopathies lymphoïdes, se situant au sixième rang des cancers les plus fréquents, sontun problème majeur de santé publique. Ce travail a pour objectif d’étudier l’impact de la prise en charge thérapeutiquesur la survie et sur la qualité de vie (QdV) des patients atteints de ce type d’hémopathies. Le premierobjectif de ce travail est un état des lieux de l’épidémiologie des hémopathies lymphoïdes avec l’étudede l’évolution de l’incidence et de la survie nette en Côte d’Or entre 1980 et 2009. L’incidence, en nette augmentationdepuis 1980, semble se stabiliser depuis les années 2000 pour certaines entités, notamment pourles lymphomes folliculaires (LF) et les lymphomes B diffus à grandes cellules (LBDGC). Nous observons globalementune amélioration de la survie nette avec, toutefois, un pronostic à court et à long terme qui restedéfavorable pour certaines entités. Les LF et les LBDGC sont les premiers lymphomes à bénéficier de l’introductiondes anticorps monoclonaux dans leur prise en charge thérapeutique. Notre deuxième étude a pourobjectif demesurer l’impact du rituximab sur la survie globale des patients atteints d’un LF ou d’un LBDGC enCôte d’Or en utilisant une méthodologie basée sur le score de propension. Nos résultats confirment le bénéficesignificatif du rituximab sur la survie globale en population générale, sans critère de sélection. En vue de cesrésultats, nous avons étudié la QdV de ces patients pendant et à la suite de la prise en charge thérapeutique. LaQdV évolue différemment au cours du suivi en fonction du type de lymphome<br>In France, hematologic malignancies, which are the sixthmost common cancers, are amajor public healthproblem. This work aimed to study the impact of the therapeutic management on survival and healt-relatedquality of life (HRQoL) in patients with these hematologic malignancies. The first objective of this work is topresent an overview of the epidemiology of lymphoid malignancies with a study of changes in the incidenceand net survival in the Côte d’Or department between 1980 and 2009. The incidence, which has increased since1980, seems to have stabilized since the 2000s for some entities, including follicular lymphoma (FL) and diffuselarge B-cell lymphoma (DLBCL). Overall, we observed an improvement in net survival, with, however, a lessfavorable prognosis in the short and long-term for some entities. FL and DLBCL were the first lymphomas tobenefit from the introduction of monoclonal antibodies in their therapeutic management. Our second studyaimed to assess the impact of rituximab on overall survival in patients with FL or DLBCL in the Côte d’Or departmentusing a methodology based on the propensity score. Our results confirmed the significant benefit ofrituximab on overall survival in an unselected population of patients. In view of these results, we studied theHRQoL of these patients during and after treatment. HRQoL evolved differently during follow-up dependingon the type of lymphoma

Few cancers arising in fluid organ systems can be cured with localized therapeutic modalities, such as radiation or surgical organ removal. Chemotherapy and hematopoietic stem cell transplants have long been employed as the standard of care for patients diagnosed with leukemias and lymphomas. Though research continues to propose new, more potent chemotherapeutic agents, a new paradigm of treating cancerous malignancies with tumor-specific monoclonal antibodies, adoptively transferred tumor-fighting cells, and other exogenously administered immunomodulatory agents, has emerged over the past decade. These immunotherapies have dramatically improved the outcomes of patients diagnosed with cancers of B lymphocytes, referred to as B-cell malignancies. Though curative FDA-approved therapies for patients diagnosed with B-cell malignancies have yet to be established, recent research in the field of adoptive T-cell therapy has produced promising results. Tumor infiltrating lymphocyte therapy (TIL therapy), T-cell Receptor Therapy (TCR therapy) and Chimeric Antigen Receptor T-cell Therapy (CAR T-cell therapy) are the three most extensively studied adoptive T-cell immunotherapies in the context of B-cell malignancies. TIL and TCR therapies, in which patients are provided with either the patient’s own tumor-specific T-cells or T-cells expressing engineered, tumor-specific TCRs, respectively, enhance the patient’s immune system to mount a more potent, anti-tumor response. However, these adoptive T-cell therapies do not change the mechanisms of the immune response. Cancerous cells can evade immune attack and dampen immune responses to survive and thrive in the body. By down-regulating their expression of human major histocompatibility complex I (MHC I), for example, cancer cells escape T-cell recognition, which is dependent on MHC expression. A chimeric antigen receptor (CAR), is composed of an antibody-derived (B-cell derived) extracellular, antigen-recognition domain, and T-cell derived intracellular domains. CAR T-cells, therefore, exploit the cytotoxic nature of CD8+ T-cells, and the MHC independent recognition of B-cell receptors, to identify and destroy all cells expressing a specific target. Consequently, many of the cancer cell’s mechanisms of immune evasion are less effective in the presence of CAR T-cells. Progressive generations of CAR T-cell designs couple these receptors with costimulatory molecules to amplify the activation, efficacy, and potency of these cells in-vivo. Over the past five years, phase I and IIa clinical trials have produced remarkable results in the treatment of advanced stage, high-risk B-cell malignancies, namely Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), and Non-Hodgkin’s Lymphoma (NHL). However, the significant oncogenic risks and fatal adverse events associated with this therapy necessitate further research to improve safety and reliable clinical efficacy of CAR T-cell therapy. In spite of these risks, the adoptive transfer of CD19-targeting, CAR expressing, cytotoxic T-cells (anti-CD19 CAR-T-cells) has produced sustained, complete remissions in patients diagnosed with progressive, advanced-stage, B-cell malignancies, for whom alternative treatments were not available. The unprecedented results of early clinical trials, as well as ongoing preclinical studies aimed at improving the design and production of CAR T-cells suggest a promising future for CAR T-cell therapy as a cure for B-cell malignancies.

(English) Using flow cytometry we analyzed absolute and relative proportions of hematopoietic stem and progenitors cells (HSPC) populations including hematopoietic stem cells (HSC), multipotent progenitors (MPP), multilymphoid progenitors (MLP) and pro B cells from bone marrow of patients with mature B cell malignancies and in healthy controls. We found lower absolute and relative numbers of MLP and higher relative numbers of HSC were observed in patients when compared to age-matched controls irrespective of bone marrow (BM) involvement. On the other hand significantly decreased absolute numbers of MPP were observed only in patients who had their BM infiltrated by disease. We also confirmed published data showing increasing absolute and relative percentages of MLP with increasing age, decreasing relative percentages of HSC with increasing age, and decreasing absolute and relative pro B cell frequencies with increasing age in healthy subjects. While decreased absolute and relative pro B cell numbers were also found in patient samples as age increased, no significant correlations were detected in patients HSC, MPP or MLP populations. Age-related sub-analysis of PTs samples demonstrated that most of the disease associated changes in HSPC frequencies were observable more prominently in the elderly (>45...

Research Doctorate - Doctor of Philosophy (PhD)<br>Haematological malignancies are a heterogenous group of cancers that originate in the mature or immature cells of the haemopoietic system. Some originate in the bone marrow, where stem cells differentiate into many types of immature blood cells, while others may form in the peripheral blood once the blood cell has matured. Each type of haematological malignancy is usually characterised by a distinct set of genomic changes that, when identified, can be used to make a specific diagnosis, predict response to therapy, provide a risk for relapse or chance of survival, characterise the disease further or provide a therapeutic target. These genomic changes are often well defined and recurrent in each type of haematological malignancy. Recent advances in technologies have also enabled the rapid discovery of novel findings – some of which have not been detectable by current standard clinical investigations performed at diagnosis. There are several techniques used to investigate the genome of haematological malignancies including G-banded karyotype, fluorescent in situ hybridisation (FISH) and various sequencing technologies. Currently, the primary technique used for the clinical genomic investigation of haematological malignancies is the G-banded karyotype, whereby analysis of banding patterns on chromosomes, which are visible in the metaphase of a cell cycle, is performed using a light microscope. This method of analysis has been the cornerstone of haematological malignancy characterisation since the discovery of the Philadelphia (‘Ph’) chromosome in 1960. However, there are many known limitations of this technique with low resolution and the need for dividing malignant cells being the most obvious. New cytogenomic tools, such as comparative genomic hybridisation microarray (CGH-microarray) and single nucleotide polymorphism microarray (SNP-microarray), overcome both the limitation of low resolution and the need for dividing cells by enabling the investigation of the whole genome at high resolution using DNA. However, the uptake of these techniques into clinical practice has been slow for haematological malignancy investigations. In this research endeavour, I sought to evaluate the clinical use of microarray technology for the investigation of haematological malignancies by assessing the performance of both CGH and SNP microarrays in comparison to current techniques. I focused on the platform’s ability to detect genomic changes that are known to hold prognostic value, as well as their ability to improve the detection of novel changes and those which are not detectable by current methodologies. In the first part of this research project I undertook a detailed literature review. Subsequently, I investigated the clinical use of CGH-microarrays for the detection of copy number variants (CNV’s) in haematological malignancies. The findings were compared to those observed using the current standard clinical investigations followed by a discussion of its prognostic implications. In the next step I used SNP-microarrays to further characterise the CGH-microarray findings. I evaluated the SNP-microarray for the identification and classification of prognostically important complex genomic signatures, such as chromothripsis and chromoanasynthesis, which are not identifiable by current clinical investigative tools. Finally, the impact of the detection of new prognostically important CNV’s identified at diagnosis by SNP-microarray were explored in a specific disease cohort. The literature clearly supports the use of new genomic profiling techniques that are only now beginning to transition from the research setting into clinical practice. Experts in the field of haematological malignancies recognise the role of genomic profiling as important for the understanding of this disease subset. However, research is still required to determine how significant the findings are and how to personalise treatment for patients based on these findings. It is imperative that the clinical diagnostic and treatment regimens do not fall too far behind advances made in research, particularly when it can have an immediate and positive effect on the patient.

博士<br>中國醫藥大學<br>臨床醫學研究所博士班<br>99<br>Hematological malignancies can be grouped as either myeloid or lymphoid disorders. The standard treatment strategy for lymphoid malignancies is chemotherapeutic agents, usually in combination with monoclonal antibodies which have introduced great impact on the clinical situation. Although certain subtypes of lymphoid malignancies, e.g. diffuse large B cell lymphoma, have good response to initial treatment, some of patients will succumb to the disease because of development of drug resistance. Still others are primarily refractory to initial therapy. The prognosis for these patients is dismal. This highlights the necessity of developing approaches to improve the therapeutic efficacy for lymphoid malignancies. One approach is to develop compounds that have anti-tumor effects with mechanism different from conventional chemotherapeutic agents. Another strategy to improve the therapeutic efficacy is to sensitize cancer cells to cytotoxic effect of other agents. We hypothesize that agents targeting alternative pathways are effective in treatment of lymphoid malignancies. These agents can exhibit anti-tumor efficacy either by themselves or by sensitizing cancer cells to other cytotoxic agents. For this sack, we validate our hypothesis through three approaches. The first part demonstrates that OSU-DY7 induces cytotoxicity in chronic lymphocytic leukemia and Burkitt lymphoma through activating p38 mitogen-activated protein kinase pathway. The second part illustrates the effectiveness of a histone deacetylase inhibitor (S)-HDAC42 in multiple myeloma cells U266. The last part shows that OSU-03012, an Akt pathway inhibitor, sensitizes myeloma cells to imatinib mesylate via AMP-activated protein kinase and STAT3 pathways. Our study successfully proves the hypothesis that agents targeting p38 MAPK, Akt pathways or histone deacetylase are effective in treatment of lymphoid malignancies. Further studies are needed to shed more light on the road.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016<br>Lymphoma is the third most common neoplasia in the world. Within lymphomas, non-Hodgkin lymphoma (NHL) is the most common affecting mainly B cells. For more than three decades, chemotherapy and radiotherapy have been the only treatments available, but since the discovery of Rituximab, a new era of lymphoma therapy was inaugurated, once it was the first monoclonal antibody approved against the CD20 receptor. The CD20 receptor is an antigen expressed on the B-cells surface, and it is present nearly in all of its maturational development, being absent only in the stages of the pro-B lymphocyte and plasma cells. Furthermore, the CD20 receptor is also present on >90% of the B-cell NHL. These characteristics make this receptor an ideal target for immunotherapy. Besides the success of Rituximab, various mechanisms of resistance have been developed by the tumoral cells against this antibody, such as the decrease of the CD20 expression on B-cells, immunogenicity, structural changes affecting the binding region of the CD20 antibody or alterations in the cell membrane. New anti-CD20 antibodies have been developed to overcome the disadvantages of Rituximab, such as Ofatumumab and Obinutuzumab, presenting a different immunogenicity and binding to the different epitopes on the target with a different affinity. Apart from the progress in the lymphoma therapy, a significant population of patients still succumbs to this disease, as tumoral cells are always changing, making the search for better antibodies a never-ending process. Thus, the aim of this project consisted in the development and characterization of a new antibody against the CD20 receptor. To achieve this goal, the potential of rabbit derived single-domain antibodies (sdAbs) as therapeutic molecules were explored. For that, one rabbit was immunized with the CD20 receptor and an immune VH and VL sdAb library was generated. Then, a subtractive cell phage display screening was used for antibody selection. This approach allowed a specific selection of one sdAb in the VL format against the CD20 receptor in cells. In summary, the strategy explored in the present project resulted in an antibody that, according to its characteristics, could be a promising candidate in the treatment of NHL and other B-cell malignancies.<br>Os linfomas são a terceira neoplasia mais comum no mundo, sendo os linfomas não-Hodgkin os mais frequentes. Dentro dos linfomas não-Hodgkin, cerca de 85-90% são de células B. Os linfomas desenvolvem-se maioritariamente a partir dos nódulos linfáticos, no entanto podem formar-se a partir de qualquer outro tecido. Durante mais de três décadas, os únicos tratamentos disponíveis para esta doença eram a quimioterapia e a radioterapia. No entanto, com a descoberta do Rituximab, um anticorpo específico para o recetor CD20 presente nos linfomas de células B, começou uma nova era no campo das imunoterapias. O anticorpo Rituximab, uma vez que foi o primeiro a ser descoberto, faz parte da primeira geração de anticorpos anti-CD20. Este é um anticorpo quimérico, uma vez que é constituído por um IgG1 glicosilado composto por uma região constante kappa humana e regiões variáveis leves e pesadas murinas. Este anticorpo atua essencialmente através de três principais mecanismos de ação: citotoxicidade mediada por anticorpos (ADCC); citotoxicidade dependente do complemento (CDC) e indução da apoptose. O recetor CD20 é uma molécula expressa na superfície das células B e está presente na maioria das fases de maturação destas células, com exceção da fase de linfócitos pró-B e nas células plasmáticas. Para além disso, está presente em mais de 90% dos linfomas não-Hodgkin de células B. No entanto, apesar da sua função ainda não ser totalmente conhecida, pensa-se que possa estar envolvido no fluxo de cálcio. Este recetor é uma fosfoproteína não glicosilada com 4 domínios membranares: os domínios N e C terminal que são intracitoplasmáticos e 2 loops adicionais que são extracelulares. Devido a estas suas características, o recetor CD20 é considerado um bom alvo para imunoterapias, nomeadamente terapias para linfomas não-Hodgkin de células B. Apesar do sucesso do Rituximab, têm vindo a ser descritos vários mecanismos de resistência das células tumorais contra este anticorpo, nomeadamente a diminuição da expressão de CD20, mudanças estruturais que afetam o local de ligação do anticorpo à molécula ou alterações na membrana da célula, o que diminuiu a sua eficácia. Este aspeto fez com que fosse necessária a contínua procura por novos anticorpos para colmatar as lacunas do Rituximab. Assim, ao longo dos anos, foram descobertos novos anticorpos específicos para CD20, como o Ofatumumab e o Obinutuzumab, que têm uma diferente imunogenicidade e reconhecem diferentes epítopos na molécula CD20 aos quais se ligam com diferentes afinidades, atuando assim com diferentes mecanismos de ação. Ainda assim, este tipo de doença continua a afetar imensas pessoas, muitas vezes não existindo um tratamento eficaz devido às múltiplas resistências das células tumorais. Tendo em conta este contexto e uma vez que os anticorpos disponíveis no mercado não conseguem resolver estes problemas, a procura por novos anticorpos mais vantajosos continua. Atendendo a esta necessidade, o objetivo deste projeto é o desenvolvimento e caracterização de um anticorpo de pequeno domínio específico para CD20. Os anticorpos de pequenos domínios têm várias vantagens em relação aos IgGs completos, principalmente devido ao seu reduzido tamanho, que faz com que estes anticorpos tenham melhor acesso ao alvo na superfície da célula, para além disso podem ainda ser facilmente expressos em bactérias como proteína. Têm ainda a vantagem, como moléculas terapêuticas, de serem mais estáveis em circulação que os anticorpos completos. Estas características permitem uma administração de maiores quantidades por grama de produto, o que leva a um aumento significativo da potência por dose e na redução do custo de produção. Para o desenvolvimento do projeto, inicialmente foi imunizado um coelho com o péptido CD20 e com células HEK 293T transfetadas com o recetor CD20. Antes de cada imunização, o soro era recolhido para ser avaliado através de ELISA. Ao dia 89, quando foi atingido um elevado título de anticorpos, procedeu-se à recolha do soro final e à eutanásia do coelho. Os ensaios de ELISA permitiram confirmar que o soro extraído após as imunizações estava a reconhecer não só as células Raji, que contêm à sua superfície CD20, mas também o péptido CD20, por outro lado o soro recolhido antes das imunizações não reconhecia nem as células Raji, nem o péptido. Após a eutanásia, foram recolhidos o baço e a medula que são os órgãos onde se encontra um maior número de células plasmáticas que produzem anticorpos. A partir destes órgãos, procedeu-se à extração de ARN usando o reagente Tri. A partir do ARN extraído foi possível sintetizar a primeira cadeia de ADN. O cADN sintetizado foi utilizado para a construção de bibliotecas imunes de pequenos domínios de anticorpos específicos para o recetor CD20, através da amplificação por PCR das famílias de cadeias variáveis leves e pesadas. Esses produtos resultantes da amplificação foram clonados no vetor pComb3x, que é necessário na técnica de phage display, resultando numa biblioteca imune com uma grande diversidade, ideal para este tipo de seleção. Em seguida, as bibliotecas resultantes foram selecionadas através da tecnologia de phage display que permite a seleção de anticorpos específicos para CD20 em células que contenham esse recetor. Esta tecnologia baseia-se na engenharia genética de bacteriófagos e em várias rondas de seleção contra o antigénio e propagação dos fagos. Resumidamente, os fagos vão conter à sua superfície o fenótipo correspondente ao genótipo encapsulado no seu interior que contem a sequência correspondente aos fragmentos de ADN obtidos através da construção das bibliotecas de anticorpos. Seguem-se várias rondas de seleção onde vão sendo eliminados os anticorpos-fagos que não se ligam ou que possuem uma fraca ligação às células que contêm o antigénio, neste caso o recetor CD20, através de várias lavagens. Estas rondas vão sendo repetidas até obtermos uma população de anticorpos específica para o alvo. Para este phage display, foram utilizados 3 tipos de células: células Raji, que está descrito que expressam o CD20, pois são células de linfoma B; células HEK 293T que não expressam CD20 e por isso foram utilizadas para eliminar os anticorpos não específicos para este recetor; células Jurkat, que tal como as anteriores não possuem CD20, pois tratam-se de células T de leucemia aguda e que por isso foram também utilizadas para eliminar anticorpos não específicos. Esta seleção resultou num conjunto de anticorpos-fagos específicos para o recetor CD20, resultado que foi confirmado através de Western Blot. Após a seleção por phage display, foi necessário fazer uma seleção em larga escala para avaliar os anticorpos que melhor eram expressos e que melhor se ligavam ao alvo. Para esta avaliação da expressão foi necessário clonar o ADN resultante dos fagos selecionados por phage display, num outro vetor que permitisse a expressão de proteína, neste caso o vetor pT7-PL. Esta avaliação da ligação e expressão foi feita através de ensaios de ELISA com extrato proteico de células Raji e células Raji. Para além disso, após várias seleções os melhores 10 clones foram sequenciados para avaliar a homologia entre si e verificar os CDRs. Através do alinhamento da sequência de aminoácidos obtida a partir da sequenciação, verificou-se que 9 dos clones eram iguais, restando assim apenas 2 clones. A especificidade dos dois clones selecionados para o recetor CD20 foi avaliada através de Western Blot, sendo que um deles (anticorpo no formato VL) era específico para o recetor pretendido. Estas metodologias levaram à seleção de um anticorpo de pequeno domínio específico para CD20, que devido às suas características únicas e diferentes dos que já existem no mercado poderá ser um promissor candidato para ser usado como agente terapêutico para linfomas de células B que expressem o recetor CD20.