Studying the Transcriptional and Epigenetic Perturbations of Haematopathologies Expressing NPM1-ALK

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Ducray, Stephen Paul 

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase involved in the development of the gut and nervous system, initially cloned from cases of T-cell lymphoma. ALK has been frequently implicated in diverse cancers through oncogenic translocations, mutations, chromosomal inversion, or amplification events. Such translocations or inversions have involved different fusion partners, which create novel fusion proteins that facilitate autophosphorylation of ALK, resulting in a constitutively active tyrosine kinase with oncogenic potential. ALK fusion proteins enable transcriptional programmes that drive the pathogenesis of a range of ALK-related malignancies including anaplastic large cell lymphoma (ALCL), diffuse large B-cell lymphoma (DLBCL), and non-small cell lung cancer (NSCLC). In addition to oncogenic fusion proteins, ALK is activated as a consequence of amplification and mutation in neuroblastoma (NB); a malignancy of the peripheral nervous system typically affecting children, and most commonly arising in the adrenal glands. Irrespective of the mechanism, hyperactivated ALK proteins are involved in diverse cellular signalling pathways such as Ras/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/Akt, and Janus protein tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT). Furthermore, ALK is implicated in epigenetic regulation including DNA methylation and miRNA expression as well as the activation and transcription of effector transcription factors. ALK interactions with nuclear proteins have also been described. Given ALK’s oncogenic activities and central role in many malignancies, ALK has presented itself as an amenable therapeutic target. Hence, directed therapeutics (ALK-tyrosine kinase inhibitors; TKI) have been used in the treatment of ALK-driven cancers. Unfortunately, clinical and research studies have suggested that resistance to ALK inhibitors can develop through mutation or activation of ALK signalling bypass tracks. As such, a need for the development of novel front-line, dual-combination, and second-line therapies has been identified.

Given the above, this thesis has focussed on two topics. Firstly, an investigation of the transcriptional roles the NPM1-ALK fusion protein plays in lymphoma, specifically ALCL, including its interaction with Brahma-related gene-1 (BRG1, encoded by the SMARCA4 gene, the catalytic subunit of the hSWI/SNF chromatin remodelling complex). I have demonstrated that an NPM1-ALK-BRG1 axis exists whereby loss of NPM1-ALK catalytic activity results in the proteasomal degradation of BRG1. As such, the transcriptional activity of BRG1 was investigated. These data suggest BRG1 holds roles in the transcriptional potentiation of cell division in ALCL and indicate that the protein is a vital effector in maintaining NPM1-ALK+ ALCL cell survival. Additionally, the cell-type dependent and independent transcriptional roles of aberrantly expressed ALK (e.g., through the activation of STAT3) were investigated and contrasted across ALK-driven malignancies (such as NSCLC and neuroblastoma). This showed that ALK holds mutually inclusive transcriptional roles in both cytokine-cytokine receptor interaction and further altering the transcriptional landscape (via expression of transcription factors and non-coding RNA).

The second focus of investigation was on the targeting of ALK-orchestrated epigenetic and transcriptional mediators (and their effects) for the treatment of NPM1-ALK+ ALCL and the plethora of other aberrant ALK-driven malignancies. As ALK was shown to be indispensable in mediating transcription in NPM1-ALK-driven ALCL, EML4-ALK-driven NSCLC, and ALK-driven NB, a large-scale drug library comprising of approximately 300 clinically approved drugs and novel agents targeting transcriptional and epigenetic mediators was applied to several in vitro models. Such models included cell lines representing ALK-driven malignancies (ALCL, DLBCL, NSCLC, and NB) as well as ex vivo primary patient-derived lines (notably an ALK-TKI sensitive primary patient-derived ALCL model, a second ALK-TKI resistant relapse patient-derived ALCL model, and a primary patient-derived neuroblastoma model). Several of the identified drugs have been previously clinically trialled and/or used for the treatment of various cancers (e.g., aurora kinase, topoisomerase, and HDAC inhibitors) and were employed as internal controls for the drug screens. Additionally, an array of novel drugs, not previously described in the context of the treatment of ALK-driven cancers, was also identified. Notably, one such drug was nanaomycin A, a selective inhibitor of DNA methyltransferase 3B (DNMT3B), which reportedly inhibits DNA methylation and thus reactivates the expression of silenced genes. Nanaomycin A was one of the most potent drugs in 13/14 ALK-driven lines tested and may thus serve as a novel front-line therapy. I also demonstrated that nanaomycin A works synergistically when used with ALK-TKIs and remains efficacious against ALK-TKI resistant cell lines. Given these promising results, a larger library consisting of approximately 1400 U.S. Food and Drug Administration (FDA)-approved drugs was subsequently used to determine novel therapies for NPM1-ALK+ ALCL cell lines in addition to the two novel ex vivo patient-derived xenograft (PDX) models. Many compounds targeting transcriptional means of regulation were identified and validated as potent therapies, several of which supported the previous epigenetics screen findings (e.g., kinase and HDAC inhibitors). Drugs targeting kinase effectors were of particular interest to this study given NPM1-ALK’s role in mediating transcriptional regulation through tyrosine phosphorylation cascades, especially the STAT3 inhibitor napabucasin. Experimentally, napabucasin was found to be efficacious across all systemic ALCL cell lines treated; regardless of ALK status. Importantly, the compound was also potent in both neuroblastoma and NSCLC, and had validated efficacy in cell lines demonstrating ALK-TKI resistance. Subsequently, a novel STAT3 inhibitor, DR-1-55, was also explored and found to be efficacious across the cohort of ALK-STAT3-driven malignancies, highlighting the promise STAT3 holds as a therapeutic target in this context.

In conclusion, ALK was demonstrated to be important in manipulating the transcriptional underpinning of ALK-driven malignancies such as ALCL, NB, and NSCLC. Through understanding this pathophysiology, several therapeutics were identified that may function as alternatives for the harsh conventional chemotherapeutics currently employed.

Turner, Suzanne D
ALK, ALCL, NPM-ALK, EML4-ALK, NSCLC, ALK-translocation proteins, epigenetics, Neuroblastoma, pediatric cancer, tyrosine kinase inhibitor (TKI), STAT3
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (675712)
This project has received funding from the European Union’s Horizon 2020 Marie Skłodowska-Curie Innovative Training Networks (ITN-ETN) under grant agreement No.: 675712 (ALKATRAS).