Chronic Myeloid Leukemia (CML) is caused by the BCR/ABL fusion gene. If untreated CML progresses within 3 years from a mild and easy to control form, called chronic phase (CP), into an aggressive and deadly acute leukemia called blast crisis (BC). Despite the aggressiveness of BC and the poor overall survival of BC patients, little is known about the molecular mechanisms responsible for the progression of the disease. Therefore to gain insight into the molecular lesions responsible for BC, I used a two prong approach. First, I performed whole-exome SEQ analysis of paired CP/BC CML samples from patients that underwent progression to BC after standard therapy. By comparing exome-sequences of 11 paired CP (used as a control) and BC samples we found a total of 38 single nucleotide mutations occurring in BC and that were absent in the corresponding CP sample. By using this approach we found recurrent somatic single nucleotide mutations in RUNX1 and UBE2A in 2 out of 11 BC samples. UBE2A is here associated with CML progression for the first time. In addition, Copy Number Alteration analysis of 9 matched BC/CP exomes reveals the presence of large chromosomal alterations acquired during BC transformation, among which the bulky alteration of chromosome 7 in 3/9 BC samples. In conclusion, despite some heterogeneity in the genetic alterations identified in BC samples, we were able to find 2 recurrently mutated genes associated with blastic transformation RUNX1 and UBE2A, with the last one never been detected in CML samples. Ongoing analysis on additional BC/CP samples and in vitro experiments will help to clarify the role of UBE2A mutations in CML progression. The second approach involves the study of the BCR promoter. After the oncogenic translocation, the BCRABL gene is transcriptionally controlled by the BCR promoter. In spite of all the research performed in the field, little is known in the regulation of BCR promoter. We thus need to understand what are the transcription factors or the mechanisms that regulate BCRABL expression. By in-silico analysis and in-vitro Chromatin Immunoprecipitation experiments we found that COUP-TF1, CTCF, MYC and MAX proteins bind at BCR promoter at specific sites. As many studies have indicated the involvement of MYC in CML progression, we here focused the study on MYC and its co-factor MAX for our further analysis. In the present study we demonstrate that MYC_MAX heterocomplex binds to the BCR promoter at four different loci, leading to upregulation of BCR and BCRABL at both transcriptional and protein level. In contrast, silencing of MYC expression in various BCRABL positive cell lines causes significant downregulation of BCR and BCRABL, which consequently leads to decreased proliferation and induction of apoptosis. Taken together all these results demonstrate that MYC_MAX heterocomplex regulates BCR promoter basal activity and can contribute to BCRABL overexpression and blast aggressiveness of CML advanced phase.

La Leucemia Mieloide Cronica(CML) è caratterizzata dalla presenza del gene di fusione BCRABL, prodotto dalla traslocazione tra i cromosomi 9 e 22. Se non trattata questa patologia progredisce entro 3 anni da una forma cronica(CP) a una forma acuta, la crisi blastica(BC). I meccanismi molecolari alla base della progressione della malattia non sono ancora completamente chiariti. Per raggiungere questo scopo ho quindi utilizzato un duplice approccio: 1)Grazie alla disponibilità di campioni CP/BC derivanti dagli stessi pazienti progrediti in BC dopo terapia standard, abbiamo sequenziato l’intero esoma(Whole Exome Sequencing-WES) e analizzato i dati utilizzando il campione CP come controllo. In questo modo l’analisi ha permesso di evidenziare solamente le alterazioni genetiche acquisite dopo progressione in BC. Ho quindi individuato la presenza di 2 mutazioni ricorrenti a carico dei geni RUNX1 e UBE2A, con quest’ultimo associato per la prima volta all’evoluzione in leucemia acuta e trovato mutato in circa l’11per cento dei campioni BC. Analisi successive invitro e invivo permetteranno di chiarire in particolare il ruolo delle mutazioni a carico di UBE2A nella progressione di CML. 2)Il secondo approccio si basa sullo studio del promotore del gene BCR. Dopo la traslocazione il gene di fusione BCRABL è sotto il controllo del promotore di BCR. Attualmente i meccanismi di regolazione di questo promotore sono poco chiari. Nel nostro laboratorio è stata precedentemente identificata una deregolazione trascrizonale di entrambi i geni BCR e BCRABL durante la progressione in BC. Ho quindi messo a punto un’analisi insilico per identificare i fattori di trascrizione che possano avere un ruolo nella regolazione del promotore BCR. Utilizzando tecniche di immunoprecipitazione di cromatina ho confermato in-vitro il legame dei fattori di trascrizione MYC e MAX a siti di legame specifici sul promotore di BCR(PBS1-4). Ho quindi osservato come l’overespressione di MYC e MAX sia in grado di indurre un aumento dell’espressione di BCR e di BCRABL, e che questo aumento risulta più evidente quando entrambi i fattori di trascrizione sono overespressi. Il silenziamento specifico di MYC nelle stesse linee cellulari ha dimostrato ulteriormente l’effetto regolatorio su entrambi i promotori. Per confermare questi risultati ho messo a punto un saggio di luciferasi inserendo il promotore di BCR nel plasmide pGL3. Cellule silenziate o meno per il gene MYC sono state poi trasfettate con i plasmidi di interesse. I risultati di questi esperimenti dimostrano l’importanza di MYC nella regolazione del promotore BCR. Ho inoltre osservato come il silenziamento di MYC in cellule BCRABL+ induca anche una alterazione del potenziale proliferativo e un aumento del tasso apoptotico. Questi dati descrivono quindi per la prima volta un meccanismo di regolazione del promotore BCR basato sul legame specifico di MYC e MAX e indicano una diretta associazione tra i livelli di espressione di MYC e quelli di BCRABL, entrambi upregolati durante l’evoluzione della malattia. E’ quindi suggerito un meccanismo molecolare alla base dell’aumento dell’espressione di BCRABL e dell’evoluzione in BC.

(2015). CHARACTERIZATION OF SOME MOLECULAR MECHANISMS ASSOCIATED TO CML PROGRESSION. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

CHARACTERIZATION OF SOME MOLECULAR MECHANISMS ASSOCIATED TO CML PROGRESSION

SHARMA, NITESH DEVINARAYAN
2015

Abstract

Chronic Myeloid Leukemia (CML) is caused by the BCR/ABL fusion gene. If untreated CML progresses within 3 years from a mild and easy to control form, called chronic phase (CP), into an aggressive and deadly acute leukemia called blast crisis (BC). Despite the aggressiveness of BC and the poor overall survival of BC patients, little is known about the molecular mechanisms responsible for the progression of the disease. Therefore to gain insight into the molecular lesions responsible for BC, I used a two prong approach. First, I performed whole-exome SEQ analysis of paired CP/BC CML samples from patients that underwent progression to BC after standard therapy. By comparing exome-sequences of 11 paired CP (used as a control) and BC samples we found a total of 38 single nucleotide mutations occurring in BC and that were absent in the corresponding CP sample. By using this approach we found recurrent somatic single nucleotide mutations in RUNX1 and UBE2A in 2 out of 11 BC samples. UBE2A is here associated with CML progression for the first time. In addition, Copy Number Alteration analysis of 9 matched BC/CP exomes reveals the presence of large chromosomal alterations acquired during BC transformation, among which the bulky alteration of chromosome 7 in 3/9 BC samples. In conclusion, despite some heterogeneity in the genetic alterations identified in BC samples, we were able to find 2 recurrently mutated genes associated with blastic transformation RUNX1 and UBE2A, with the last one never been detected in CML samples. Ongoing analysis on additional BC/CP samples and in vitro experiments will help to clarify the role of UBE2A mutations in CML progression. The second approach involves the study of the BCR promoter. After the oncogenic translocation, the BCRABL gene is transcriptionally controlled by the BCR promoter. In spite of all the research performed in the field, little is known in the regulation of BCR promoter. We thus need to understand what are the transcription factors or the mechanisms that regulate BCRABL expression. By in-silico analysis and in-vitro Chromatin Immunoprecipitation experiments we found that COUP-TF1, CTCF, MYC and MAX proteins bind at BCR promoter at specific sites. As many studies have indicated the involvement of MYC in CML progression, we here focused the study on MYC and its co-factor MAX for our further analysis. In the present study we demonstrate that MYC_MAX heterocomplex binds to the BCR promoter at four different loci, leading to upregulation of BCR and BCRABL at both transcriptional and protein level. In contrast, silencing of MYC expression in various BCRABL positive cell lines causes significant downregulation of BCR and BCRABL, which consequently leads to decreased proliferation and induction of apoptosis. Taken together all these results demonstrate that MYC_MAX heterocomplex regulates BCR promoter basal activity and can contribute to BCRABL overexpression and blast aggressiveness of CML advanced phase.
GAMBACORTI PASSERINI, CARLO
Chronic Myeloid Leukemia, UBE2A , BCR promoter, MYC_MAX
MED/15 - MALATTIE DEL SANGUE
English
28-gen-2015
Scuola di Dottorato in Scienze Mediche Sperimentali e Cliniche
EMATOLOGIA SPERIMENTALE - 05R
26
2013/2014
open
(2015). CHARACTERIZATION OF SOME MOLECULAR MECHANISMS ASSOCIATED TO CML PROGRESSION. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/63684
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