General introduction 1.

University of Groningen

Replication-stress induced mitotic aberrancies in cancer biology Schoonen, Pepijn Matthijs

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2019

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Schoonen, P. M. (2019). Replication-stress induced mitotic aberrancies in cancer biology. Rijksuniversiteit Groningen.

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General introduction 1.

10 Chapter 1

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GENERAL INTRODUCTION

C

Due to these mutations, cells acquire the capacity to escape cellular mechanisms that prevent unscheduled cell growth, causing rapid and uncontrolled cell division.⁽¹⁾ When cancer cells are not eradicated timely, some tumors can eventually grow out and acquire the ability to metastasize throughout the body. If no successful therapeutic intervention is provided, the disease will ultimately result in organ failure and death. Indeed, cancer is the responsible cause of death for approximately 8,7 million people worldwide in 2015 alone.

(2)

If cancer remains locally confined, surgical removal is possible and can lead to curation, but for treatment of metastasized disease, chemotherapy and/or radiotherapy might be required.

However, both these treatment options are harmful for normal cells. Damage to healthy tissues strongly limits the chemo and radiotherapy dose that can be tolerated, and thereby limits the cancer killing efficacy of cancer treatments.

(3) Furthermore, while patients often show remission following chemo and radiotherapy many tumors are not completely eradicated, resulting in patient relapse.

To resolve these issues, a more selective or ‘targeted’ approach is required, which seizes upon processes essential for cancer cell survival, but dispensable for non-transformed ‘healthy’ cells. An important example hereof is ‘oncogene addiction’, in which oncogenic growth is reliant on signal transduction arising from genetic oncogenic events. Specifically, cancer cells can get ‘addicted’ to signaling induced by

activating mutations or overexpression of EGFR family members, and BCR- ABL translocations.^(4,5) Because tumor cells are ‘addicted’ to such oncogene signaling and healthy cells are not, this provides a therapeutic opportunity to kill tumor through targeting inhibition of the hyperactivated oncogene.

Conversely, some tumors become reliant on pathways that are not oncogenic, a process called ‘non-oncogene addiction’.

(6) For instance, tumor cells may be increasingly dependent on DNA repair or certain metabolic pathways, whereas these pathways are not oncogenic themselves, and therefore again provide a therapeutic window.

Another form of targeted therapy is based on the concept of synthetic lethality. In this concept, a mutation in a specific gene is lethal only when present in combination with another specific mutation. Such a combination of gene mutations is called synthetic lethal. This concept is highly relevant for cancer cells, in which a specific gene mutation makes these cells completely reliant for their survival on the function of another gene. This latter gene could thus serve as a therapeutic target.⁽⁷⁾ A well-known application of synthetic lethality is the use of PARP inhibitors in tumors with a defect in homologous-recombination (HR) DNA repair. Two important genes involved in HR are BRCA1 or BRCA2, mutations in which cause hereditary breast and ovarian cancer. Indeed, cells lacking functional BRCA1 or BRCA2 were shown to be extremely sensitive to inhibitors of the PARP enzyme.

(8,9) Conversely, healthy cells that

possess functional BRCA genes are not sensitive to PARP inhibition, allowing for tumor clearance without excessive side-effects. Unfortunately, however, tumors often develop resistance to PARP inhibitor therapy.⁽¹⁰⁾ Therefore, a better understanding of how PARP inhibitors work is required to further

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improve their efficacy by developing combination strategies. Additionally, only a relatively small subset of cancers has dysfunctional BRCA genes. The identification of other gene mutations that also cause a HR-defect could broaden the group of eligible patients for PARP inhibitor treatment. Yet, in spite of the clinical successes of PARP inhibitors, resistance mechanisms have been reported and, since only certain subgroups are eligible, additional mechanisms are required to selectively target all tumors

Additionally, owing to their frequent oncogene activation and consequent uncontrolled division, cancer cells regularly suffer from slowed or even stalled replication, a process called replication stress. Indeed, replication defects are thought to be an important underlying cause in the acquisition of mutations and genomic rearrangements that entail genomic instability. Notably, induction of replication stress or genomic instability in non-cancerous cells was shown to be lethal.⁽¹¹⁾ Apparently, cancer cells have evolved mechanisms to cope with high levels of replication stress and genomic instability. Therefore, targeting of these pathways could also be an interesting strategy to selectively kill cancer cells.

(12,13)

The aim of this thesis is therefore to:

1. Uncover the synthetically lethal mechanisms underlying PARP inhibitor- induced cell death in HR-deficient cancer cells, and to use these insights to develop new combination strategies.

2. Target replication stress to selectively kill cancer cells.

OUTLINE OF THIS THESIS In chapter 2 of this thesis, we discuss cancer-relevant factors that challenge the replication machinery and, how these factors induce replication stress. Furthermore, we explain how replication stress drives genomic instability in promoting cancer development. Lastly, we describe the mechanisms employed by cells to deal with replication stress, and how these mechanisms may provide therapeutic targets to kill cancers harboring replication stress. Special emphasis is placed on recent findings that show that, in conditions of replication stress, DNA replication can persist in mitosis, where it leads to the formation of chromatin bridges in anaphase, which can cause multinucleation and cell death when left unresolved.

Mutations in essential HR genes, including BRCA1 and BRCA2, cause susceptibility to cancer development.

Yet, BRCA1/2-mutant cancer cells have an elevated sensitivity towards PARP inhibition treatment. While PARP inhibitors show great promise as a treatment strategy, not all tumors respond to PARP inhibition.

Additionally, some tumors develop resistance, which causes tumor relapse.

More insight into the mechanisms-of- action of PARP inhibitors is therefore required to improve therapy outcome.

Specifically, it is still unclear how exactly PARP inhibitors kill HR-deficient cells.

To address this question, in chapter 3 we study the underlying mechanisms of PARP inhibitor cytotoxicity. To this end, we use multiple HR-deficient cancer models both in vitro and in vivo and follow the induction of PARP- inhibitor-induced replication stress throughout the cell cycle using both immuno-fluorescence microscopy and live-cell imaging. Lastly, we assess whether progression through mitosis

12 Chapter 1

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was essential for PARP inhibitor- induced toxicity. To this end, EMI1 depletion was used to bypass mitosis, while leaving the ability of cells to perform DNA replication intact.

In chapter 4, we further explore the mechanism underlying PARP inhibitor- induced cytotoxicity as discovered in chapter 3. Specifically, a possible combination strategy is tested, involving combined inhibition of PARP and ATR, a central orchestrator in the replication stress response. We determine the timing of mitotic entry by flow cytometric analysis of synchronized cell populations, in the presence of ATR/PARP inhibitors. Furthermore, we assess possible mitotic aberrancies and genomic instability using immuno- fluorescence and single cell sequencing.

The overexpression of certain oncogenes is a well-described cause of replication stress, which possibly fuels genetic instability and cancer. However, the resulting replication stress can also prove to be a therapeutically exploitable weakness in cancers. To test this hypothesis in chapter 5, we set out to develop inducible oncogene overexpression models in non- transformed RPE-1 cells. Subsequently, we investigated if overexpression of these oncogenes resulted in replication stress and whether the DNA lesions

caused by replication stress were transferred into mitosis. Additionally, we attempted to target cells with oncogene-induced replication stress by inhibition of the ATR and WEE1 checkpoint kinases. Lastly, the effects on mitotic aberrancies were studied in real- time using live-cell microscopy.

To allow for proper sister chromatid disjunction during anaphase, cells are equipped with topoisomerase II, Plk1- interacting checkpoint helicase (PICH) and Bloom’s helicase (BLM). This machinery resolves DNA catenanes, which are visible as ultrafine DNA bridges (UFBs) in anaphase. Since failure to resolve UFBs can impair genomic integrity, identifying novel players involved in this process is of great interest. In chapter 6, we studied the function of Rif1 in mitosis. Using immune-fluorescence and live-cell imaging, we determine whether Rif1 was involved in a mitotic DNA damage response. Furthermore, the localization of Rif1 and its role in maintaining genome integrity will be assessed.

Lastly, in chapter 7 the results and conclusions of all chapters will be summarized and discussed. We revisit the aims established in the introduction, and discuss implications and future directions.

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