RNA interference of gene expression (siRNA/shRNA) is a well-established technique used for loss-of-function studies and is particularly useful for identifying and validating drug discovery-relevant targets. However, for cell lines which grow in suspension (e.g. lines derived from leukemias or hematological malignancies) reliable and reproducible siRNA-mediated gene knockdown with standard liposomic reagents has to date proven difficult to achieve, due to low efficiency of oligonucleotide delivery and high sensitivity of such cells to transfection conditions. We developed a novel approach for large-scale transfection of siRNA oligonucleotide libraries and image-based phenotypic screening which overcomes some of the limitations related to the use of non-adherent cell lines. Suitable experimental conditions were identified for efficient and nontoxic transfection of siRNA oligonucleotides in the myelogenous leukemia cell line K562. The approach was validated through screening of the K562 cells against a subset of a commercial siRNA library, targeting a subset of the druggable genome (about 6000 siRNA oligonucleotides). Results of the screen confirmed robustness of assay conditions, with hits including oligonucleotides targeting genes known to be required for K562 cell viability, such as the kinase ABL1, which is a driver oncogene in this line, as wells as additional genes whose products are more generally required for cell viability and proliferation (e.g., PLK1 and WEE1 kinases, proteasome subunits, DNA and RNA polymerases). Among non-canonical hits, not expected a priori to inhibit cell growth, 14 genes were confirmed as novel potential molecular targets in K562, with a role in promoting proliferation/viability or, notably, in preventing terminal differentiation. In order to further study the role of a selection of the screening hits, we established a high throughput high-content screening platform based on multiplexed imaging on a K562 cell line variant (β-K562), which expresses significant levels of β-globin in addition to the α- and γ-globins which are normally expressed in parental K-562 cells. Setup of a method for the simultaneous analysis of Hoechst DNA staining, together with immunocytochemical staining for detection of adult hemoglobin (HbA, composed of an α2βb2 globin heterotetramer) and fetal hemoglobin (HbF, composed of an α2γ2 globin heterotetamer), resulted in a robust and sensitive assay able to detect variations in γ-globin/β-globin ratio in response to drug treatments, as validated by treatment with known γ-globin inducers (hydroxyurea, butyric acid, hemin). The availability of this single cell high-content analysis assay permits investigation of the degree of population heterogeneity in response to drug treatment and to discriminate between drugs acting specifically on either γ- and β-globin expression and those acting indiscriminately on the expression of both genes. By applying this assay to β-K562 cells transfected with siRNA oligos selected among the screening hits described above, several genes were found to play a role in cell differentiation and hemoglobinization. Most notably amongst these, HMOX-2 (Heme oxygenase-2) silencing was shown to greatly increase γ-globin expression. We observed that also enzymatic HMOX-2 inhibition by Tin protoporphyrin-IX resulted in increased γ-globin expression, confirming Heme oxygenases as potential targets for the pharmacological γ-globin reactivation. This is of particular interest because several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other HbF inducers) to improve the treatment of β-hemoglobinopathies

La tecnica dell'RNA interference (siRNA/shRNA) rappresenta un approccio ormai consolidato per studiare il fenotipo cellulare derivante dalla perdita di funzione di un dato gene, e si presta particolarmente all'identificazione e validazione di nuovi potenziali bersagli molecolari per terapie farmacologiche mirate. Tuttavia, per linee cellulari che crescono in sospensione (per esempio, linee leucemiche), gli approcci di siRNA basati sull'uso di reagenti di trasfezione liposomiali si sono mostrati fin'ora di difficile applicazione, a causa della bassa efficienza di trasferimento degli oligonucleotidi e dell'alta sensibilità di tali modelli cellulari alle condizioni di trasfezione. E' stato perciò sviluppato un nuovo approccio metodologico di trasfezione su larga scala di librerie di oligonucleotidi siRNA, abbinato allo screening fenotipico di analisi di immagine, che permette di superare alcune delle limitazioni derivanti dall'uso di cellule che crescono in sospensione. Abbiamo identificato condizioni sperimentali ottimali per ottenere una significativa efficienza di trasfezione, a fronte di una bassa tossicità, nella linea cellulare di leucemia mieloide cronica K562. L'approccio è stato validato tramite lo screening di una collezione di circa 6000 oligonucleotidi siRNA disegnati per silenziare circa 2000 geni i cui prodotti rappresentano putativi bersagli farmacologici. I risultati di questo screening hanno confermato la robustezza delle condizioni sperimentali e hanno permesso di identificare un numero di geni la cui funzione è richiesta per la vitalità o la proliferazione delle K562. Tra questi, è emersa la chinasi ABL, che è un oncogene essenziale in questa linea, insieme ad altri geni coinvolti in processi cellulari fondamentali (per esempio, le chinasi PLK1 e WEE1, alcune subunità del proteasoma, DNA e RNA polimerasi). Tra i geni emersi dallo screening in maniera inattesa, 14 sono stati confermati come nuovi potenziali bersagli molecolari in K562, con un ruolo nel promuovere la vitalità o la proliferazione, oppure nel prevenire il differenziamento terminale. Allo scopo di approfondire lo studio di alcuni di questi geni, è stata implementata una piattaforma di screening fenotipico ad alto contenuto (“high-content screening”) basata sull’analisi di immagine di una variante clonale della linea cellulare K562 che esprime livelli significativi di β-globina, oltre alle globine α e γ normalmente espresse nella linea parentale, che abbiamo nominato “β-K562”. Utilizzando questa linea cellulare, abbiamo sviluppato un saggio multiparametrico che abbina l’analisi del contenuto di DNA e della morfologia nucleare (tramite colorazione con Hoechst 33342) con l’immunofluorescenza della γ-globina (componente dell’emoglobina fetale, HbF) e della β-globina (componente dell’emoglobina adulta, HbA). Questo saggio si è dimostrato sufficientemente robusto e sensibile da permettere l’analisi di variazioni nell’espressione delle globine γ e β in un unico esperimento, ed è stato validato tramite trattamento con noti induttori della γ-globina (idrossiurea, acido butirrico o hemin). L’applicazione di questo saggio alla trasfezione della linea β-K562 con oligonucleotidi siRNA emersi dallo screening ha permesso di identificare diversi geni coinvolti nel differenziamento eritrocitario e nella emoglobinizzazione. Tra di essi, è risultato particolarmente interessante HMOX-2 (Heme oxygenase-2), il cui silenziamento induce un marcato aumento dei livelli di γ-globina. Ulteriori esperimenti hanno dimostrato che l’inibizione enzimatica di HMOX-2, per mezzo della stagno protoporfirina IX, determina un aumento dei livelli di γ-globina, confermando questo enzima come un potenziale bersaglio molecolare per la riattivazione farmacologica dell’emoglobina fetale.

(2015). Identification and functional analysis of genes with a role in proliferation and erythroid differentiation of a chronic myelogenous leukemia cell line. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

Identification and functional analysis of genes with a role in proliferation and erythroid differentiation of a chronic myelogenous leukemia cell line

DURLAK, MARTA
2015

Abstract

RNA interference of gene expression (siRNA/shRNA) is a well-established technique used for loss-of-function studies and is particularly useful for identifying and validating drug discovery-relevant targets. However, for cell lines which grow in suspension (e.g. lines derived from leukemias or hematological malignancies) reliable and reproducible siRNA-mediated gene knockdown with standard liposomic reagents has to date proven difficult to achieve, due to low efficiency of oligonucleotide delivery and high sensitivity of such cells to transfection conditions. We developed a novel approach for large-scale transfection of siRNA oligonucleotide libraries and image-based phenotypic screening which overcomes some of the limitations related to the use of non-adherent cell lines. Suitable experimental conditions were identified for efficient and nontoxic transfection of siRNA oligonucleotides in the myelogenous leukemia cell line K562. The approach was validated through screening of the K562 cells against a subset of a commercial siRNA library, targeting a subset of the druggable genome (about 6000 siRNA oligonucleotides). Results of the screen confirmed robustness of assay conditions, with hits including oligonucleotides targeting genes known to be required for K562 cell viability, such as the kinase ABL1, which is a driver oncogene in this line, as wells as additional genes whose products are more generally required for cell viability and proliferation (e.g., PLK1 and WEE1 kinases, proteasome subunits, DNA and RNA polymerases). Among non-canonical hits, not expected a priori to inhibit cell growth, 14 genes were confirmed as novel potential molecular targets in K562, with a role in promoting proliferation/viability or, notably, in preventing terminal differentiation. In order to further study the role of a selection of the screening hits, we established a high throughput high-content screening platform based on multiplexed imaging on a K562 cell line variant (β-K562), which expresses significant levels of β-globin in addition to the α- and γ-globins which are normally expressed in parental K-562 cells. Setup of a method for the simultaneous analysis of Hoechst DNA staining, together with immunocytochemical staining for detection of adult hemoglobin (HbA, composed of an α2βb2 globin heterotetramer) and fetal hemoglobin (HbF, composed of an α2γ2 globin heterotetamer), resulted in a robust and sensitive assay able to detect variations in γ-globin/β-globin ratio in response to drug treatments, as validated by treatment with known γ-globin inducers (hydroxyurea, butyric acid, hemin). The availability of this single cell high-content analysis assay permits investigation of the degree of population heterogeneity in response to drug treatment and to discriminate between drugs acting specifically on either γ- and β-globin expression and those acting indiscriminately on the expression of both genes. By applying this assay to β-K562 cells transfected with siRNA oligos selected among the screening hits described above, several genes were found to play a role in cell differentiation and hemoglobinization. Most notably amongst these, HMOX-2 (Heme oxygenase-2) silencing was shown to greatly increase γ-globin expression. We observed that also enzymatic HMOX-2 inhibition by Tin protoporphyrin-IX resulted in increased γ-globin expression, confirming Heme oxygenases as potential targets for the pharmacological γ-globin reactivation. This is of particular interest because several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other HbF inducers) to improve the treatment of β-hemoglobinopathies
RONCHI, ANTONELLA ELLENA
siRNA; K562; βK562; immunostaining; interference
BIO/18 - GENETICA
English
25-giu-2015
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
27
2013/2014
open
(2015). Identification and functional analysis of genes with a role in proliferation and erythroid differentiation of a chronic myelogenous leukemia cell line. (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/10281/83982
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