ERBB2 as a driver of an invasive phenotype of cells grown in 3D culture and an important regulator of oncogenic miRNAs' expression in breast cancer

Breast cancer is often associated with deregulated activity of two receptor tyrosine kinases (RTKs) from the ERBB family: EGFR and ERBB2 (HER2/Neu). Whereas the EGF receptor is often mutated in breast cancer, ERBB2 is up-regulated in up to 20% of invasive breast tumors and plays a triggering role in cell proliferation, invasion and metastasis. Patients overexpressing ERBB2 are treated with targeted therapies and in particular with the monoclonal antibody trastuzumab, which binds an extracellular domain of the receptor and abrogates receptor-kinase activation. However, ERBB2-positive breast cancer patients overexpress ERBB2 to different extent. The expression range is very wide, raising the question whether the tumors expressing moderate, high and very high ERBB2 levels have different outcome and patients carrying them can be treated as one entity or not. To better understand the impact of different ERBB2 levels I developed stable cell line pools overexpressing ERBB2 at different levels. I observed that in 3D culture as well as in matrigel-based invasion assays, cells expressing very high ERBB2 levels showed more invasive properties than those expressing moderate ERBB2 levels, even in the absence of EGF. This was accompanied by disruption of cell polarity in 3D-grown spheroids and anchorage-independent growth which occured only when ERBB2 expression was very high. These phenotypes could be at least partially explained by ERBB2-dose dependent epithelial-mesenchymal transition (EMT), an increase in HB-EGF transcription and constitutive ERK and AKT signaling pathway activation at the very high ERBB2 levels. As recently several miRNAs have been reported to regulate EMT-related processes and because their expression is frequently deregulated in cancer, I also hypothesized that their expression would change with the ERBB2 expression level in 3D culture. To verify my hypothesis, I performed small RNA sequencing from stable cell line pools grown in matrigel. This identified several miRNAs which are ERBB2-level dependent. I focused then on the functional characterization of miR-301b and miR-130b whose expressions were ERBB2-dependent in 3D, but not in 2D culture. Overexpression of these miRNAs phenocopied ERBB2 effects on cell invasion and proliferation as well as induction of EMT, as did overexpression of the related miR-301a

The central role of the transcriptional regulator IκBζ in psoriasis

IκBζ belongs to the group of atypical NF-κB inhibitors (IκBs). In contrast to classical IκBs, IκBζ is only inducibly expressed in the cell nucleus, where it can inhibit, but more importantly, also activate the expression of a particular subset of target genes. NFKBIZ, the gene encoding IκBζ, has been identified as new risk gene in psoriasis. Moreover, IκBζ knockout mice are protected in certain models of experimental psoriasis. The aim of this thesis was to examine the global role of IκBζ in psoriasis in order to find a new therapy approach. It could be shown that IκBζ is a regulator not only of IL-17 but also of IL-36 signaling, which plays a major role in certain forms of psoriasis. The IL-36-mediated induction of IκBζ was driven by NF-κB and STAT3, and led to the expression of pro-inflammatory genes that initiate the development of psoriasis. Accordingly, IκBζ knockout mice were completely protected from IL-36- mediated experimental psoriasis. Thus, IκBζ represents a central regulator in psoriasis, which promotes inflammation regardless of the type of stimulus. Based on this finding, screenings for small-molecule inhibitors were performed that are able to repress the induction of IκBζ. Thereby, I could identify a new pro-inflammatory signal pathway in keratinocytes. In this pathway, CDK4/6 phosphorylated the methyltransferase EZH2, which in turn methylated and activated STAT3, which transcriptionally induced IκBζ. The pharmacological inhibition of CDK4/6 or EZH2 inhibited the pathogenesis of psoriasis in vitro and in vivo. In conclusion, this thesis not only validates IκBζ as an essential mediator of psoriasis, but also identifies the CDK4/6-EZH2 axis as a novel mechanism whose inhibition could provide a potential therapeutic option for the treatment of psoriasis

Analysis of the c-MYC and AP4 axis in breast cancer

The deregulated expression of the c-MYC oncogene activates p53 which is presumably mediated by ARF/INK4, as well as replication-stress-induced DNA damage. Here, we aimed to determine whether the c-MYC-inducible AP4 transcription factor plays a role in this context using a genetic approach. We used a CRISPR/Cas9 approach to generate AP4- and/or p53-deficient derivatives of MCF-7 breast cancer cells harboring an ectopic, inducible c-MYC allele. Cell proliferation, senescence, DNA damage, and comprehensive RNA expression profiles were determined after activation of c-MYC. In addition, we analyzed the expression data from primary breast cancer samples. Loss of AP4 resulted in elevated levels of both spontaneous and c-MYC-induced DNA damage, senescence, and diminished cell proliferation. Deletion of p53 in AP4-deficient cells reverted senescence and proliferation defects without affecting DNA damage levels. RNA-Seq analyses showed that loss of AP4 enhanced repression of DREAM and E2F target genes after p53 activation by c-MYC. Depletion of p21 or the DREAM complex component LIN37 abrogated this effect. These p53-dependent effects were conserved on the level of clinical and gene expression associations found in primary breast cancer tumors. Our results established AP4 as a pivotal factor at the crossroads of c-MYC, E2F, and p53 target gene regulation.Die deregulierte Expression des c-MYC-Onkogens aktiviert p53, was vermutlich durch ARF/INK4 sowie durch Replikationsstress-induzierte DNA-Schäden vermittelt wird. Ob der c-MYC-induzierbare Transkriptionsfaktor AP4 in diesem Zusammenhang eine Rolle spielt, wollten wir hier mit einem genetischen Ansatz klären. Wir verwendeten einen CRISPR/Cas9-Ansatz, um AP4- und/oder p53- defiziente Derivate von MCF-7-Brustkrebszellen zu erzeugen, die ein ektopisches, induzierbares c-MYC-Allel beinhalten. Zellproliferation, Seneszenz, DNA-Schäden und umfassende RNA-Expressionsprofile wurden nach Aktivierung von c-MYC bestimmt. Darüber hinaus analysierten wir die Expressionsdaten von primären Brustkrebsproben. Der Verlust von AP4 führte zu Erhöhung von spontaner als auch c-MYC-induzierter DNA-Schädigung, Seneszenz und verminderter Zellproliferation. Die Deletion von p53 in AP4-defizienten Zellen hob die Seneszenz und Proliferationsdefekte auf, ohne das Ausmaß der DNA-Schädigung zu beeinflussen. RNA-Seq-Analysen zeigten, dass der Verlust von AP4 die Repression von DREAM- und E2F-Zielgenen nach p53-Aktivierung durch c-MYC verstärkt. Die Depletion von p21 oder der DREAM-Komplex-Komponente LIN37 hob diesen Effekt auf. Diese p53- abhängigen Effekte blieben auf der Ebene von klinischen und Genexpressions Assoziationen erhalten, die in primären Brustkrebstumoren gefunden wurden. Unsere Ergebnisse etablierten AP4 als zentralen Faktor an der Schnittstelle der c-MYC, E2F und p53-vermittelten Genregulation

Globale Expressionsprofile Pneumokokken-infizierter Bronchialepithelzellen - Einfluss der miRNA-3135b und des Nicotinamidstoffwechselweges auf die bakterielle Replikation

Streptococcus pneumoniae (S. pneumoniae), auch als Pneumococcus bezeichnet, ist ein grampositives Bakterium, welches gewöhnlich als Kommensale asymptomatisch den humanen Nasopharynx besiedelt, jedoch auch schwere Erkrankungen bis hin zur Sepsis oder Meningitis auslösen kann. Pneumokokken sind Hauptverursacher der Pneumonie beim Menschen und fordern jährlich mehrere Millionen Opfer weltweit. Weiterhin können Koinfektionen mit Influenza A Viren die Erkrankung verschlimmern. Die Epithelzellen des humanen Respirationstraktes bilden die erste Verteidigungsbarriere gegen die Infektion. Es sind jedoch viele Aspekte der Interaktion zwischen Epithelzellen und S. pneumoniae noch nicht umfassend geklärt. Um diese Interaktion detailliert zu analysieren, wurde ein Expressionsprofil aus mRNAs, Proteinen und miRNAs von infizierten Bronchialepithelzellen erstellt. Zusätzlich wurde ein Koinfektionsmodell in humanem ex vivo Lungengewebe zu Vergleichszwecken untersucht. Signalweg‐Analysen der infizierten Epithelzellen ergaben eine verstärkte Regulation des Zellzyklus zum späten Zeitpunkt der Infektion (16 h). Eine Vernetzung der Daten mit dem miRNA‐Profil offenbarte wenige, bereits bekannte Verknüpfungen. Dennoch konnten mit Hilfe der miRNA‐Untersuchungen behandlungsabhängige Expressionsmuster detektierten werden, welche S. pneumoniae‐spezifische miRNAs, wie die induzierte miRNA‐3135b, aufzeigten. Bei dieser hypothetischen miRNA könnte es sich tatsächlich um ein t‐RNA‐deriviertes Fragment (tRF) handeln. Eine Überexpression der miRNA‐3135b resultierte in einer signifikanten Reduktion der Pneumokokken‐Last, was auf einen Abwehrmechanismus der Epithelzellen hindeutet. Zudem zeigte die RNA‐Sequenzierung nach miRNA‐3135b‐Überexpression verschiedene putative Ziel‐mRNAs, deren Funktionen bisher nur eingeschränkt bekannt sind. Des Weiteren weisen funktionelle Analysen der mRNAs und Proteine auf eine Regulation des Nicotinamidmetabolismus hin. Die in den Epithelzellen durchgeführte Depletion von NAMPT, dem Schlüsselenzym dieses Stoffwechselweges, führte zu einer verminderten Replikation von S. pneumoniae. Weiterhin bewirkte die Zugabe von Nicotinamid‐Mononukleotid (NMN) eine gesteigerte Replikationsrate der Bakterien. Dies deutet auf NMN als wichtige Nährstoffquelle von Pneumokokken hin. Die Daten dieser Arbeit erweitern die Kenntnisse zur Interaktion von humanen Epithelzellen und Pneumokokken und könnten zur Identifizierung alternativer und neuer Therapiestrategien genutzt werden

Global evaluation of transcripts associated to human epidermal aging with DNA microarrays

Orientador: Nilson Ivo Tonin ZanchinTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Com o aumento do tempo de vida da população humana muitas modalidades médicas, incluindo a dermatologia, deparam-se com uma revolução na forma de garantir saúde e qualidade de vida aos pacientes. Em contato com o ambiente externo, a pele representa um órgão no qual as mudanças com o envelhecimento causam danos funcionais, além de potencial impacto estético e psicossocial. A epiderme, camada mais externa da pele, constitui uma barreira seletiva com destacada capacidade de renovação e manutenção da homeostasia corporal. Entretanto, o entendimento de diversos mecanismos associados à fisiologia e envelhecimento da epiderme permanece como desafio para a comunidade científica. Com base nesse cenário, o objetivo do presente trabalho foi compreender o atual estado da arte no tema de envelhecimento da epiderme e realizar experimentos voltados para lacunas existentes, com foco na integração de aspectos clínicos, fisiológicos, morfológicos, celulares e moleculares. O capítulo de abertura descreve uma avaliação global de transcritos associados ao envelhecimento da epiderme humana, com a técnica de microarranjos de DNA e coleta não invasiva com fitas adesivas. O estudo indica características moleculares específicas do fotoenvelhecimento epidermal, com alterações relevantes e complementares a dados clínicos e morfológicos prévios, como modulação das vias de organização do citoesqueleto de actina e sinalização de cálcio, expressão gênica alterada de proteínas do envelope córneo, e avaliação de um painel segmentado por décadas de vida que sugere aspectos inéditos de regulação homeostática da epiderme, além de genes com modulação contínua ao longo das idades. O segundo capítulo compara o envelhecimento nas regiões folicular e interfolicular da epiderme. Como um sistema biológico de simples obtenção e fácil manuseio, os bulbos dos folículos pilosos representam uma fonte rica de material epidermal distinto, conforme evidencias na ampla modulação gênica diferenciada. O terceiro capítulo inclui uma avaliação in vitro do envelhecimento da epiderme, com queratinócitos de indivíduos de diferentes idades cultivados em monocamada e no modelo de pele equivalente. Os resultados evidenciam diferenças na expressão de marcadores moleculares de proliferação e diferenciação entre queratinócitos neonatais e adultos, mas não entre adultos de diferentes idades. Não houve diferença nas populações de células tronco, entretanto, observou-se aumento de células na fase proliferativa do ciclo celular em neonatos, assim como predominância de células na fase estacionária do ciclo celular em adultos mais velhos. Concluindo, os resultados obtidos no presente trabalho contribuem de forma significativa para o avanço do entendimento dos mecanismos moleculares afetados pelo avanço da idade da epiderme, possilitando a busca de novas alternativas no tratamento do envelhecimento cutâneoAbstract: With the increase in lifetime of the human population many medical disciplines, including dermatology, are facing a revolution in the approaches to ensure healthcare and quality of life for patients. In contact with the external environment, the skin is an organ in which the changes of aging cause functional damage, in addition to potential aesthetic and psychosocial impact. Epidermis, the outermost skin layer, is a selective barrier with outstanding capacity for renewal and maintenance of the body homeostasis. However, the understanding of several mechanisms associated with skin physiology and aging remains a challenge for the scientific community. Considering this scenario, the objective of this work was to evaluate the state of the art knowledge on epidermal aging and to conduct experimental approaches to cover gaps that still exist on that theme, focusing on the integration of clinical, physiological, morphological, cellular and molecular aspects of epidermis aging. The opening chapter describes a study based on global transcriptional evaluation associated with aging of the human epidermis, using DNA microarrays and noninvasive tape stripping. This study reveals molecular characteristics specific of epidermal photoaging, with relevant findings complementary to previous clinical and morphological data, such as modulation of the actin cytoskeleton and calcium signaling pathways; altered gene expression of proteins of the cornified envelope; and evaluation of a segmented panel structured by decades of life, which suggests new aspects of homeostatic regulation in the epidermis and unvails genes with continuous modulation throughout different ages. The second chapter compares the gene expression patterns of the follicular and interfollicular regions of epidermis undergoing aging. As a biological system easily sampled and handled, the bulbs of plucked hair follicles represent a rich source of distinct epidermal material, as evidenced by the wide differential gene modulation that was detected. The third chapter includes an experimental in vitro evaluation of skin aging using keratinocytes isolated from individuals of different ages and cultured in monolayer and in skin equivalent models. Differences in the expression of proliferation and differentiation molecular markers between neonatal and adult keratinocytes were observed. No differences were found regarding the stem cell populations, however, neonates showed an increased percentage of cells in the proliferative phase of cell cycle, while older adults presented a predominance of cells in the stationary phase of cell cycle. The results herein presented provide novel insights on the molecular mechanisms affected by epidermal aging, enabling the search of new alternatives in the treatment of aging skinDoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecula

Mechanisms of tissue compartmentalization in human T cells

Mechanisms for human memory T cell differentiation and maintenance have predominantly been inferred from studies of peripheral blood, though the majority of T cells reside in lymphoid and non-lymphoid sites. Studies in mice have shown that memory T cells in non-lymphoid sites provide superior protection to pathogens compared to those in blood, defining a subset known as tissue-resident memory T cells (TRM), with emerging roles in lymphoid sites. There are many key unknown aspects of TRM biology in human tissues including if TRM have superior functional abilities, the mechanisms for maintenance of TRM in lymphoid and non-lymphoid sites, and the relatedness of tissue and blood localized T cell subsets. Through a collaboration with the local organ procurement agency, we obtained samples from >15 tissue sites from healthy organ donors of all ages. We analyzed CD8+ T cells in diverse sites and found the majority of TRM cells in lymph nodes (LNs) display an increased proliferative capacity, increased expression of TCF-1, and decreased turnover compared to TRM and effector memory (TEM) cells in other sites including blood, bone marrow (BM), spleen and lung. Further, we identified that exposure to type 1 interferons results in increased downregulation of TCF-1 expression during cell divisions driven by T cell receptor (TCR) stimulation. We investigated the relatedness of CD4+ and CD8+ T cell subsets, including central memory (TCM), effector memory (TEM), TRM, and terminal effectors (TEMRA) by sequencing TCR rearrangements. From diversity analysis of TCR repertoires we found that effector and memory subsets are maintained in a hierarchy from most to least diverse (TCM > TEM and TRM > TEMRA) that is largely conserved across tissues and CD4+ and CD8+ T cell lineages. Overlap analysis revealed the low and high relatedness of TCM and TEMRA cells respectively and this was highly conserved across tissues; in contrast, we found the relatedness of TEM and TRM was more dynamic across tissues. Together, these findings have implications for immune monitoring and modulation, highlighting that lymph nodes may function as reservoirs for long-lived memory T cells with high functional capacity; additionally, we identify cell extrinsic signals that regulate tissue-specific maintenance of T cell memory in lymph node sites

Globale Expressionsprofile Pneumokokken-infizierter Bronchialepithelzellen - Einfluss der miRNA-3135b und des Nicotinamidstoffwechselweges auf die bakterielle Replikation

Streptococcus pneumoniae (S. pneumoniae), auch als Pneumococcus bezeichnet, ist ein grampositives Bakterium, welches gewöhnlich als Kommensale asymptomatisch den humanen Nasopharynx besiedelt, jedoch auch schwere Erkrankungen bis hin zur Sepsis oder Meningitis auslösen kann. Pneumokokken sind Hauptverursacher der Pneumonie beim Menschen und fordern jährlich mehrere Millionen Opfer weltweit. Weiterhin können Koinfektionen mit Influenza A Viren die Erkrankung verschlimmern. Die Epithelzellen des humanen Respirationstraktes bilden die erste Verteidigungsbarriere gegen die Infektion. Es sind jedoch viele Aspekte der Interaktion zwischen Epithelzellen und S. pneumoniae noch nicht umfassend geklärt. Um diese Interaktion detailliert zu analysieren, wurde ein Expressionsprofil aus mRNAs, Proteinen und miRNAs von infizierten Bronchialepithelzellen erstellt. Zusätzlich wurde ein Koinfektionsmodell in humanem ex vivo Lungengewebe zu Vergleichszwecken untersucht. Signalweg‐Analysen der infizierten Epithelzellen ergaben eine verstärkte Regulation des Zellzyklus zum späten Zeitpunkt der Infektion (16 h). Eine Vernetzung der Daten mit dem miRNA‐Profil offenbarte wenige, bereits bekannte Verknüpfungen. Dennoch konnten mit Hilfe der miRNA‐Untersuchungen behandlungsabhängige Expressionsmuster detektierten werden, welche S. pneumoniae‐spezifische miRNAs, wie die induzierte miRNA‐3135b, aufzeigten. Bei dieser hypothetischen miRNA könnte es sich tatsächlich um ein t‐RNA‐deriviertes Fragment (tRF) handeln. Eine Überexpression der miRNA‐3135b resultierte in einer signifikanten Reduktion der Pneumokokken‐Last, was auf einen Abwehrmechanismus der Epithelzellen hindeutet. Zudem zeigte die RNA‐Sequenzierung nach miRNA‐3135b‐Überexpression verschiedene putative Ziel‐mRNAs, deren Funktionen bisher nur eingeschränkt bekannt sind. Des Weiteren weisen funktionelle Analysen der mRNAs und Proteine auf eine Regulation des Nicotinamidmetabolismus hin. Die in den Epithelzellen durchgeführte Depletion von NAMPT, dem Schlüsselenzym dieses Stoffwechselweges, führte zu einer verminderten Replikation von S. pneumoniae. Weiterhin bewirkte die Zugabe von Nicotinamid‐Mononukleotid (NMN) eine gesteigerte Replikationsrate der Bakterien. Dies deutet auf NMN als wichtige Nährstoffquelle von Pneumokokken hin. Die Daten dieser Arbeit erweitern die Kenntnisse zur Interaktion von humanen Epithelzellen und Pneumokokken und könnten zur Identifizierung alternativer und neuer Therapiestrategien genutzt werden

An ex vivo Familial Genetic Strategy for Determining Mechanism of Action

One of the greatest challenges in anticancer drug development is the discovery of molecular targets and biochemical interactions required for drug action. Lapses in drug efficacy and unanticipated toxicity, the two biggest causes of drug failure in clinical trials, are often attributed to our limited understanding of drug mechanism and cost the pharmaceutical industry millions. Genomics is rapidly emerging as tool for mechanism elucidation. Our approach is one of the latest to link drugs to the genes which influence their activity. This ex vivo familial genetics strategy uses a collection of extensively genotyped, normal, healthy, human cell lines from multigenerational families. Cell lines are phenotyped for cytotoxic response to anticancer agents, heritability analysis gives a measure of the degree to which genetic influences response, and linkage analysis suggests regions of the genome which are associated with the observed variation in response. To evaluate this strategy as method for mechanism elucidation, we first asked whether the system could produce pharmacological and genomic profiles related to a shared mechanism for a class of structurally related compounds. The in vitro sensitivity of CEPH cell lines the camptothecin, Topoisomerase 1 inhibitors (Top1), was studied. Heritability analysis estimates that genetics accounts for as much 20% of the observed variation in cytotoxic response to these drugs. Linkage analysis revealed a pattern of six quantitative trait loci (QTLs) that were shared by all of the camptothecins and independently replicated with a second of camptothecin analogues. The pattern of QTLs observed with the camptothecins was compared to those of the indenisoquinolones, a structurally distinct class of Top1 inhibitors. The objective was to identify which if any QTLs are related to the general mechanism of Top1 inhibition or should be considered class-specific. Finally, the model was assessed for its ability to stratify compounds by mechanism based on their biological and genomic profiles. Cell lines were phenotyped for response to approximately 30 drugs belonging to 8 mechanistic classes. Intraclass biological and genomic profiles were more similar to each other than to compounds belonging to distinct mechanistic classes. This work could have a significant impact on drug discovery and development as it provides a strategy for not only making predictions about mechanism of action for novel therapies, but for identifying genes involved in variable response to chemotherapeutic agents as well

Analysis of the genome and viral transcriptome of Epstein-Barr virus associated paediatric B cell lymphomas

Paediatric Hodgkin lymphoma (HL) and endemic Burkitt lymphoma (eBL) are Epstein-Barr virus (EBV) associated childhood malignancies. In HL, malignant Hodgkin/Reed-Sternberg (HRS) cells, are characterised by aneuploidy, and comprise <1% of tumour which is challenging for global genetic studies. In chapter 3, I describe the mutational landscape of paediatric HL using in vitro and in silico methods I established, including tumour DNA amplification from low numbers of (~150) microdissected cells. I found that protein-altering mutations converge on mitotic spindle functions and that EBV contributes to transcriptionally downregulate mitotic spindle genes. The contribution of the EBV to eBL pathogenesis remains poorly understood. In chapter 4, I show that the virus G protein-coupled receptor, BILF1 is apparently a latent gene expressed by tumour cells in a subset of BL. BILF1expression induced a transcriptional profile in primary human germinal centre (GC) B cells that partially recapitulates the transcriptional programme of EBV-positive eBL, and genes up-regulated by BILF1 have functions in oxidative phosphorylation and are also targets for MYC. In summary, my data identify novel pathogenic pathways contributing to the pathogenesis of two common paediatric B cell malignancies that are likely to have clinical, including therapeutic, relevance

Exploring BCR-ABL-independent mechanisms of TKI-resistance in chronic myeloid leukaemia

As the prevalence of Chronic Myeloid Leukaemia (CML) grows, due to the therapeutic success of tyrosine kinase inhibitors (TKI), we are witnessing increased incidences of drug resistance. Some of these patients have failed all currently licensed TKIs and have no mutational changes in the kinase domain that may explain the cause of TKI resistance. This poses a major clinical challenge as there are currently no other drug treatment options available for these patients. Therefore, our aim was to identify and target alternative survival pathways against BCR-ABL in order to eradicate TKI-resistant cells. To investigate alternative survival mechanisms in TKI-resistant CML cells, ponatinib-resistant cell line models were generated, which show resistance to all current TKIs, despite complete inhibition of BCR-ABL activity. Additionally, DNA sequencing revealed no mutational changes within the BCR-ABL kinase domain, which may explain TKI resistance and RNA-sequencing showed an impaired transcriptional response following ponatinib treatment when compared with parental TKI-sensitive cells. Using these models, we demonstrated that the TKI-resistant cells acquired alternative activation of mTOR. Using clinically relevant dual PI3K and mTOR inhibitors; NVP-Bez235, VS-5584, apitolisib and gedatolisib, we validated the PI3K-AKT-mTOR pathway as a therapeutic target in vitro in TKI-resistant CML cell lines and more importantly in bone marrow derived mononuclear cells from CML patients resistant to TKIs and with no known kinase domain mutational changes. We demonstrated in vitro that TKI-resistant cell lines are highly sensitive to PI3K and mTOR inhibitors, with EC50 values less than 30 nM compared to ponatinib, 647.3 nM. These inhibitors reduced cell viability by causing a significant induction of apoptosis and significant decrease in the clonogenic growth of primary TKI-resistant CML patient samples. Furthermore, we showed that NVP-Bez235 induced autophagy as a protective mechanism following PI3K/mTOR inhibition. The combination of NVP-Bez235 with pharmacological (Hydroxychloroquine (HCQ)) or specific autophagy inhibition, via ATG7 knockdown, the efficacy of NVP-Bez235 was enhanced shown by the dramatic reduction in clonogenic growth of TKI-resistant CML patient cells. In addition, we validated this in vivo using a murine model by transplanting luciferase tagged TKI-resistant cells, treated with NVP-Bez235 in combination with HCQ, which significantly reduced tumour burden and increased survival rates compared to controls. These data suggested that the PI3K-AKT-mTOR pathway may be a key player responsible for TKI-resistance and that pharmacological inhibition of this pathway, with the additional inhibition of autophagy, may represent a potential new treatment option for TKI-resistant CML patients, when resistance is driven by a BCR-ABL-independent mechanism