University of Groningen The long road Hilgendorf, Susan

The long road

Hilgendorf, Susan

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Publication date: 2018

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Hilgendorf, S. (2018). The long road: The autophagic network and TP53/ASXL1 aberrations in hematopoietic malignancies. Rijksuniversiteit Groningen.

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Future Perspectives

• Nederlandse Samenvatting

• Acknowledgments

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SUMMARY

The development of MDS is a long-term multi-step process that is accompanied by genetic lesions, which can influence differentiation, proliferation, and self-renewal of HSPCs and can provide (pre-)malignant cells with clonal advantages. Understanding how single gene mutations and combinations of several mutated genes influence disease onset and progression and what their effect is on underlying molecular pathways, is essential for the development improved treatment strategies and patient prognosis. ASXL1 and TP53 have emerged as two frequently mutated genes in MDS and AML, but also in healthy individuals. Loss of ASXL1 function alters histone modification, possibly predisposing cells to malignant transformation. TP53 is a critical tumour suppressor. Its functional abrogation is essential for many tumours to allow for malignant progression. Many mouse and cell-line studies have been conducted to elucidate the role of ASXL1 and TP53 loss in disease development but research based on HSPCs is scarce.

In Chapter 2, we abrogated ASXL1 function with an RNAi approach and investigated its

effect on cord blood CD34+ _{cells under different lineage permitting growth conditions. The}

stem cell frequency was compromised and cell expansion along the myeloid lineage was reduced. The erythroid lineage revealed the most prominent phenotype with significantly increased apoptosis among differentiated erythroid progenitors, reduced cell expansion and accumulation of cells in the G0/G1 phase. While bone marrow stromal cells supported the growth of the most immature erythroid cells, it did not change the unfavourable phenotype that we observed upon ASXL1 knockdown. When we investigated underlying molecular pathways, we noted that no changes of H3K27me3 occurred within myeloid progenitors. However, loss of ASXL1 function within erythroid progenitors demonstrated a loss of H3K27me3 on the HOXA but also the p21 loci. This suggests that ASXL1 is necessary to balance the expression of p21 throughout erythroid development. Finally, we discovered that ASXL1 is necessary for development and differentiation of erythroid cells and that the observed aberrant differentiation is facilitated via trimethylation of H3K27, which is placed by PRC2 in interaction with ASXL1.

Many gene aberrations can take part in the development of MDS. TP53 is frequently mutated in high-/adverse risk MDS and therefore alleviates the apoptosis response of malignant cells. A combination of ASXL1 and TP53 gene abrogation is found in patients and may relieve the apoptotic phenotype induced by ASXL1 loss alone. In Chapter 3, we combined knockdown

of ASXL1 with loss of TP53 in HSPCs. As demonstrated in Chapter 2, ASXL1 loss impaired

cell proliferation and differentiation, particularly along the erythroid lineage. Moreover, colony formation and stem cell frequency were reduced. Upon additional knockdown of TP53, the stem cell frequency of ASXL1-compromised cells was restored to normal levels, as

well as colony formation and cell proliferation. A humanized mouse model revealed that the adverse effects of ASXL1 loss on HSPCs prevented engraftment, which could not be rescued by TP53 knockdown and therefore did not permit malignant transformation. We therefore concluded that combined knockdown of ASXL1 and TP53 gene expression is not sufficient for transformation. Loss of ASXL1 function does not recapitulate what is seen in the patients and therefore overexpression of mutant forms of ASXL1 should be considered.

In Chapter 4, we continued our studies on TP53 and focused on autophagy and evaluated

the efficacy of a autophagy inhibition on AMLs with mutant and wildtype TP53. Various studies have suggested that autophagy can be a survival mechanism for numerous types of cancers but how autophagy is involved in AML, especially in poor risk AMLs harbouring p53 mutations, is poorly understood. Therefore, we investigated whether autophagy inhibition could be used as a treatment strategy for AML. AMLs classified as poor-risk had a higher autophagic flux than favourable- and intermediate risk AMLs. The higher autophagic flux was associated with an increased expression level of different autophagic genes. Poor-risk AMLs are often associated with TP53 mutations but knockdown or overexpression of wild-type or mutant TP53 did not affect the flux. When using hydoxychloroquine (HCQ), an autophagy inhibitor, AML CD34+ _{cells were more sensitive than normal CD34}+ _{bone marrow cells.}

Additionally, knockdown of ATG5/7 inhibited autophagy and triggered apoptosis, which corresponded to increased p53 expression. Treatment of wild-type TP53 AML with HCQ triggered a BAX/PUMA-dependent apoptotic response, which was not observed in TP53 mutant AMLs. Separating AML CD34+ _{in to ROS}high _{and ROS}low _{revealed that ROS}low _cells

maintained higher basal autophagy with reduced survival upon HCQ treatment than ROShigh

cells. Finally, in vivo knockdown of ATG5 inhibited maintenance of AML CD34+ _cells,

revealing the importance of autophagy in AML. In conclusion, we discovered that autophagy is a critical contributor for AML maintenance and that autophagy inhibition in AML TP53 wildtype but not mutant could be a promising treatment strategy.

In Chapter 5, we describe the current understanding of autophagy signalling in in cancer

(stem cell) development and in drug resistance/response of cancer cells and we propose directions on strategies for targeting autophagy during cancer therapy. We discuss that defects or mutations in autophagy genes can drive transformation and that metabolic pathways are sustained by autophagy in order to facilitate tumour growth and survival. Further, we review several pathways and mechanisms that can be altered during treatment, leading to autophagy up-regulation and cancer cell survival. Consequently, inhibition of autophagy is being pursued as treatment strategy to augment/abolish therapy resistance in resistant cells. Moreover, we portray the combination of chemotherapeutic drugs with autophagy inhibitors and the results in clinical trials. Additionally, we discuss that in certain therapy settings autophagic cell death occurs, possibly aiding the efficacy of anticancer therapy. Then, we describe the role

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and reversely, the evasion of cancer cells of the immune response via autophagy. Finally, we give perspectives of selective targeting of cancer (stem) cells for therapy.

DISCUSSION AND FUTURE PERSPECTIVES

A set of mutations, generally associated with leukemogenesis, has been discovered in healthy individuals. These mutations are thought to give cells survival advantages. The gene that was found most commonly mutated within several studies is DNMT3A1–3_{. Several other}

frequently mutated genes include ASXL1, TP53, TET2, and JAK21–3_{. In the first part of this}

thesis, we aimed at investigating ASXL1 and TP53 abrogation to elucidate whether these gene alterations can initiate or drive disease development and/or progression. This fundamental research is necessary to advance our understanding of MDS and AML, in an attempt to ultimately improve treatment strategies for patients and allow for more tailored therapy. Patients with ASXL1 mutations appear to have heterozygous loss of ASXL1. It was not known if the heterozygous loss of ASXL1 leads to loss-of-function or has a dominant-negative or a gain-of-function character. Many studies adopted deletion or knockdown of ASXL1 as strategy, as loss-of-function induced an MDS-like phenotype in mice4,5_{. Following}

this approach, we investigated how reduced ASXL1 expression influenced the development of hematopoietic stem and progenitor cells (HSPCs) under different growth conditions. We found that knockdown of ASXL1 was detrimental for HSPCs, with HSPCs revealing reduced stem cell frequency, deceased cell cycle and apoptosis in erythroid-permissive culture conditions. ChIP and qPCR data unveiled increased p21 expression as a possible conveyer of the observed phenotype. Additional knockdown of TP53 did not improve overall cell fate in vitro nor in vivo. However, recent studies found mutated ASXL1 protein at detectable levels in

cell lines, indicating that simply reducing the protein level cannot represent the full spectrum of MDS-development6_{. Furthermore, additional functions have been attributed to mutated}

ASXL1 proteins, suggesting gain-of-function activities. Balasubramani et al. reported that

ASXL1 mutation enhanced BAP1 activity thereby deubiquitinating H2AK119ub and losing H3K27 trimethylation7_{. Nonetheless, wild-type ASXL1 also increased BAP1 activity}7_.

In another study, Yang et al. determined that mice carrying a truncated ASXL1 protein in

the hematopoietic lineage developed many forms of myeloid malignancies8_{. They found}

an enhanced HSC pool with shortened survival of the mice but did not see changes in H3K27me3 or H2AK119ub. This is not surprising as Yang et al. attempted to determine

methylation and ubiquitination level changes only on western blot for a global approach8_.

Previously, we also attempted to visualize global methylation changes on western blot but failed to do so (data not shown). Nevertheless, Yang et al. discovered that mutated ASXL1

interacted with BRD4 and was sensitive to BET bromodomain inhibitors8_{. This opened a}

new therapeutic window for treating patients with ASXL1 mutations. Of note, it is possible that different ASXL1 mutations lead to different phenotypes, possibly explaining the different observations. For instance, in a KI frameshift model, no increased HSC pool was observed in

6

forced overexpression of truncated mouse ASXL1. A more humanized knock-in study could be beneficial for making more direct comparisons towards patients8_.

The studies discussed above focus primarily on development of MDS and AML, while disregarding the development of CHIP. CHIP is commonly seen as an age-related clonal expansion of certain hematopoietic stem cells and their progenies, which have acquired mutations that might give them survival advantages but without presenting any dysplasia10,11_.

While people presenting with CHIP are at an increased risk to develop MDS or AML, CHIP is not the only path cells can take to progress into malignant diseases. This can be seen by the different mutations that are present in CHIP and MDS/AML. In CHIP, the most common mutations are DNMT3A followed closely by ASXL1, TP53, TET2, and JAK2. These mutations are not the most frequently mutated genes in MDS or AML anymore. Mutations usually not observed in CHIP, such as in RUNX1, NPM1, and RAS, appear to make up a big part in MDS and AML clones12–15_{. Moreover, pediatric AML has a different mutational}

make-up than adult and these pediatric patients are unlikely to have encountered CHIP yet16_.

Interestingly, especially RAS mutations appear prevalent, while DNMT3A seems absent in children with AML. This suggests that programs were activated and additional events took place during disease onset and development in AML that were not present in CHIP. We can speculate that these events include inheritance of certain mutations, the acquired amount of mutations per clone, changes in the epigenetic and transcription factor landscape due to new mutations, and pathway alterations.

Many patients with TP53 mutations reveal p53 protein accumulation but until recently, it was not clear whether these proteins have additional or altered functions. Lately it was revealed that in mutated p53 cell lines, p53mut _{has different epigenetic binding sides than}

wild-type p5317_{. Various breast cancer cell lines containing different p53}mut _{were compared}

to p53wt _{breast cancer cell lines. It was discovered that these mutations had gain-of-functions}

by binding and up-regulating chromatin regulatory genes, such as MLL1 and MLL217_{. It is}

unclear whether these different binding strategies are context dependent and in which manner they are involved in malignant transformation. It is technically challenging to determine how mutated proteins can influence disease development once an MDS or an AML has been established. To study this more directly ASXL1 and p53 mutations need to be introduced into healthy HSPCs.

We have already established an overexpression model of p53, choosing one of the most common hot spot missense mutations: R273 (Fig 1A&B). We evaluated the stability of

mutant p53 by using cycloheximide and discovered that it remained stable for a period of at least two hours (Fig 1C). The stability of the wild-type protein is known to vary between five

to 20 minutes. Here, we confirmed that wild-type protein stability is less than 20 minutes.

Figure 1

DNA Binding Domain

Transcriptional Activation Domain Tetramerization Domain

G245 R249

N-

-C

A

B

D

Figure 1. Gain-of-func on p53 mutant promotes repla ng poten al of hematopoie c stem and progenitor cells. (A) Simplified protein structure and loca on of p53 muta ons in exons five to eight.

White and blue circles indicate in-frame and frameshi� muta ons. Red circles are missense muta ons with the reported six hot spot muta ons. (B) Overexpression of R273H TP53 mutant on mRNA and protein level (N=2). (C) Protein stability with 15uM cycloheximide. (D) Clonogenic poten al of sorted CD34+ cord blood control and p53 mutant overexpressing cells (N=3). Coun g and repla ng occured a�er 10-14 days.

Error bars represent standard devia on; **p<0.01; ***p<0.001

Time (min) 0 5 10 15 20 30 60 120 pRRL-R273H pRRL-WT p53_b-actin p53 b-actin

C

Figure 1. Gain-of-func on p53 mutant promotes repla ng poten al of hematopoie c stem and progenitor cells. (A) Simplified protein structure and loca on of p53 muta ons in exons five to eight. White and blue circles indicate in-frame and frameshift muta ons. Red circles are missense muta ons with the reported six hot spot muta ons. (B) Overexpression of R273H TP53 mutant on mRNA and protein level (N=2). (C) Protein stability with 15uM cycloheximide. (D) Clonogenic poten al of sorted CD34+ cord blood control and p53 mutant overexpressing cells (N=3). Coun g and repla ng occured after 10-14 days.

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rapidly, the continuous stability of the mutant protein might allow for additional functions independent of cellular stress. Following this data, we transduced CD34+ _{cells with either a}

lentiviral vector containing an overexpression of R273H (pRRL-R273H) or an empty control (pRRL-EV). These cells were then sorted for GFP+ _{and plated into methylcellulose assays}

(CFC) (Fig 1D). Initially, overexpression of the mutated protein led to reduced erythroid

colony formation. Upon replating, colony formation was increased due to mutant protein expression and stable for two replates. It appeared that an enhanced presence of mutant p53 induced replate potential of colonies, suggestion a survival benefit for stem cells. Further, we also reduced TP53 gene expression with a hairpin in CD34+ _{and sorted GFP}+ _{cells for}

CFC assays. We observed a slight reduction in erythroid colony formation when compared to an empty control. Upon the second replate, enhanced colony formation was observed, indicating that stem cells also benefit from a loss of TP53 (data not shown).

To establish if stem cells really benefit from enhanced p53mut _{expression, we conducted a}

long-term culture initiating colony assay (LTC-IC), which is considered a surrogate in vitro

assay to determine stem-cell frequency (Fig 2A). In line with data obtained in CFC assays,

an enhanced stem-cell frequency was noted upon overexpression of R273H. Interestingly, we did not observe growth advantages of CD34+ _{cells transduced with the mutant protein}

on MS5 bone marrow stromal cells (Fig 2B). However, when we added methylcellulose

to the MS5 co-culture after a five-week period, we noted that the mutant protein enhanced colony formation of cells under the MS5 stroma (Fig 2C). This suggests that while there

is no apparent growth advantage, altered signaling pathways are probably activated due to mutant overexpression, allowing for immature stem-cell like cells to reside under the stroma more easily. When transduced CD34+ _{cells are grown on stroma with cytokines, cells with}

mutant protein demonstrate significant growth advantage (Fig 2D). Moreover, cells taken

from suspension and grown in CFC assays revealed significantly enhanced colony forming potential. In order to establish which pathways are affected and whether mutant p53 has different binding sides, chromatin immunoprecipitation and RNA sequencing needs to be conducted. However, we tested endogenous p53 antibodies and found them to be inefficient. Therefore, we created overexpression constructs containing p53 mutant and wild-type fused to GFP. We now can precipitate the constructs with an exogenous GFP antibody and determine which signaling routes are turned on and off. Zhu et al. demonstrated in breast

cancer cell lines that p53 mutants bind and upregulate methyl- and acetyltransferases17_{. Our}

first screens of gene expression in HSPCs with R273H overexpression did not reveal similar expression patterns as shown by Zhu et al (data not shown). This is not too surprising, as they investigated p53 mutations within established breast cancer cell lines, while we are investigating leukemia. Our goal is to determine which genes are targeted by mutant p53 in AML and how these genes could be involved in malignant transformation.

0 5 10 15 20 25 30 35 0.1 1 10 100 1000 10000 pRRL-EV pRRL-R273H days

Cumulative cell count MS5 + cytokines

* 0 1 2 3 4 5 0.1 1 10 100 weeks

Cumulative cell count MS5

pRRL-EV pRRL-R273H LTC-IC in bulk BFU-E CFU-GM pRRL-EV pRRL-R273H 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80 90 pRRL-EV pRRL-R273H Colonies / 10000 cells pRRL-EV pRRL-R273H Day 28 CFU-M CFU-G ** *** 0 1 10 100 0 500 1000 1500 % NEGA TIVE WELLS PLATED CELLS LTC-IC pRRL-EV pRRL-R273H 1 in 723 1 in 361 0.000 0.001 0.002 0.003 0.004 LTC-IC frequency * pRRL-EV pRRL-R273H

Figure 2

A

B

C

D

Figure 2. Overexpression of TP53 mutant preserves stem cell frequency. (A) Long-term culture-ini a ng cell (LTC-IC) in limi ng dilu ons, CD34+ cord blood (CB) control and TP53 overexpressing mutant cells (N=2). (B) Expansion of CD34+ CB transduced control or TP53 mutants cells on MS5 stromal co-culture (N=3). (C) LTC-IC in bulk, stem progenitor cells from transduced CB CD34+ cells (N=3). (D) Cummula ve cell count of transduced CD34+ CB cells on MS5 stromal co-culture with CFC assay of suspension cells (N=2).

Error bars represent standard devia on; *p<0.05; **p<0.01; ***p<0.001

Figure 2. Overexpression of TP53 mutant preserves stem cell frequency. (A) Long-term culture-initiating cell (LTC-IC) in limiting dilutions, CD34+ cord blood (CB) control and TP53 overexpressing mutant cells (N=2). (B) Expansion of CD34+ CB transduced control or TP53 mutants cells on MS5 stromal co-culture (N=3). (C) LTC-IC in bulk, stem progenitor cells from transduced CB CD34+ cells (N=3). (D) Cummulative cell count of transduced CD34+ CB cells on MS5 stromal co-culture with CFC assay of suspension cells (N=2).

Error bars represent standard devia on; *p<0.05; **p<0.01; ***p<0.001

So far, we did not observe indefinite expansion or transformation in p53 mutant overexpressing CD34+ _{cells. While in some cultures conditions a clear growth advantage was observed, all}

cultures exhausted within weeks. Several factors can play a role in expansion of cells harboring mutations, such as the microenvironment the cells reside in and mutation load. For instance, Wong et al. demonstrated that cytotoxic therapy results in expansion of TP53 carrying clones18_{. Clearly certain cellular stressors can contribute to the increase of clonal pool.}

Additionally, Wong et al. showed that these clones persisted after autologous transplantation with stable chimerism, with TP53 mutants revealing higher propensity towards evolution of leukemia than other clones18_{. Another reason for not observing enhanced expansion of}

cells could be the missing mutational load, with the need for additional mutations beside aberrant TP53 for full leukemic transformation. Studies suggest that development of MDS and AML require several mutations. Mouse models with overexpression single ASXL1 and TP53 mutations have shown that development of myeloid malignancy-like diseases are

6

it is unlikely to see a patient with only one mutation that presents with MDS or AML. In our hands, double knockdown of ASXL1 with TP53 did not lead to transformation possibly due to lack of additional mutations. Berger et al. showed that patients, who had received autologous hematopoietic stem cell transplantation (ASCT), carried several mutations before developing treatment-induced MDS or AML19_{. However, it took up to seven years}

until disease developed. Likely epigenetic factors, ChIP, aging, and other environmental cues can influence disease development. Nevertheless, these therapy-related myeloid neoplasms (t-MNs) were characterized by a significant increase of mutational load when compared to de novo MDS. Also, Silva-Coelho demonstrated that treating (de novo) MDS could influence clonal evolution20_{. It appeared that after treatment, sub-clones carrying mutations in the}

RAS-family emerged long before patients experienced clinical symptoms20_{. It seems that}

selective pressure, by cytotoxic therapy with or without ASCT, favors the expansion of certain clones, especially of TP53-mutated cells18,19,21_{. However, the data observed so far indicated}

that current in vitro and in vivo models are inadequate to represents studies focusing on tumor

development. It is therefore important to establish studies that use patient material and to develop adequate models in order to determine which factors are relevant for tumor initiation and maintenance.

A study focusing on human cell models, which can combine the complexity of human disease in mice, has been created by Tothova et al. by using the CRISPR/Cas9 system22_{. In this study,}

adult CD34+ _{cells were gene edited for frequently occurring leukemic gene mutations, such}

as RUNX1, TP53, and ASXL1. Single-gene editing of CD34+ _{cells and transplantation in to}

mice took place in order to imitate human ChIP.

Consistent with that as has been observed in humans, some clonal selection was detected for DNMT3A, TET2 and ASXL1 but no changes in the bone marrow, spleen or blood cell counts were observed. However, multiplex gene editing, thus the abrogation of several genes per cell, led to expansion of clones in vivo, with 80% of the mice harboring clones with at least

two mutations22_{. Clones with multiple gene alterations engrafted during serial transplants}

successfully, while single gene editing limited transplant capabilities of clones. The CRISPR/ Cas9 genomic engineering model may be more reliable alternative to study human myeloid malignancies in a mouse environment.

Several studies have demonstrated that one fifth of low-risk MDS patients with del(5) carry TP53 mutations in sub-clones23,24_{. Presumably, when these patients are treated, they might}

be at risk to progress to more severe MDS or sAML due to outgrowth of the TP53 sub-clone. Moreover, in hematological malignancies, TP53 mutations are more common than TP53 deletions25_{. It was found that AML patients carrying one or more TP53 mutations}

have a significant shorter overall survival, while this was not the case for patients with TP53 deletions. Additionally, TP53mut _{appeared to be correlated to higher age, while deletions}

were not. In MDS, patients with TP53 mutations or deletions were associated with more aggressive disease and poorer outcome similar to AML. TP53 mutations did not appear to be associated with age, however25_.

In a study conducted by Gibson et al., patients who had CHIP before undergoing ASCT for lymphoma were more likely to develop therapy-related myeloid neoplasms (t-MNs) with inferior survival26_{. Also in this study, higher frequencies of TP53 mutations were observed.}

30% of the included patients had two or more mutations at an early median age of 61 years. In the general population at this age, this would commonly be one mutation1_{. A possible}

explanation for the enhanced mutational burden is the predisposition of lymphoma patients to the administration of chemo- and radiotherapy, and the use of growth factor mobilization26_.

Moreover, chemotherapy has been shown to induce bone marrow injury that is irreversible and can lead to reduced engraftment of autologous stem cells following myelo-ablative chemotherapy 27_{. Presumably cells with CHIP may outgrow within the damaged bone}

marrow, as these cells may carry mutations that give them survival advantages. The conditions under which we observed a temporary growth benefit were with addition of growth factors (Fig 2D). Another experimental approach can be to treat cells with a chemotherapeutic agent

in order to facilitate expansion.

It is challenging to model all possible factors that may play a role in malignant transformation. For instance, attempting to model the percentage of variant allele frequency (VAF) in vitro

or in vivo to imitate human disease is almost impossible. Several studies have shown by now

that mutations that were detected at low VAF had the potential to outgrow after cytotoxic therapy18,28_{. Moreover, it has recently been observed that non-leukemic clones have the}

potential to expand during treatment of the leukemic clones20,28,29_{. This presents a new}

challenge as treatment intent on curing MDS or AML may now lead to an “evolutionary bottleneck”20_{. Small, pre-existing clonal populations may now arise once the initial MDS or}

AML clonal population has been reduced. Previously existing sub-clones may disappear20_.

The consistent change and turnover of clones may lead to clinical progression and can make treatment challenging29_{. Therefore, when investigating genes and their potential to induce}

MDS or AML, many factors have to be taken into account. And ultimately, the search for a therapy that does not kill one clone and paving the way for another to arise is essential. Finding new treatment strategies could be aided by identifying key pathways that underlie certain mutations, such as TP53. Using RNA sequencing, Huang et al. discovered that TP53mut were significantly associated with PI3K-Akt signaling pathway30_{. The continually}

and uncontrolled activation of PI3K-Akt pathway is known to affect survival and therapy resistance of leukemic cells31_{. Moreover, mutation of TP53 also has been found to influence}

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apoptosis32_{. Additionally, it has been suggested that mutant TP53 disrupts the DNA damage}

response32_{. It is not clear if all TP53 mutations exert the same functions. However, if a majority}

of different TP53 mutations affect the same signaling pathways, treating downstream targets within TP53 clones might be an additional treatment alternative.

A new treatment strategy has been established that aims at restoring normal protein functions of mutated proteins. Specifically re-activating p53 wild-type is of interest. As described above and in the introduction of this thesis, TP53 is considered the most frequently mutated gene in cancer. Previous mouse models have shown that restoration of functional wild-type p53 can lead to apoptosis of tumor cells, cellular senescence, and increased mouse survival33–35_.

Pharmacological studies established a list of possible small molecules that reactivate missense-mutated p53. These small molecules may restore original folding and allow for DNA binding of p53 target genes36_{. Several clinical phase I/II trials are currently underway to mainly establish}

safety of these drugs. APR-256 was the first pharmacological molecule tested37_{. At high}

doses, patients tolerated the drug well with all showing reversible side effects. Lehman et al observed increased upregulation of pro-apoptotic genes NOXA, PUMA, and BAX, all known downstream targets of wild-type p5337_{. Further, tumor reduction in two patients was noted}37_.

In another trial, the same molecule was tested on patients with hematological malignancies38_.

One AML patient showed reduction in blasts of more than 25%. Another AML patient went into complete remission. However, this patient had received prior treatment and follow-up treatment, making it difficult to interpret if APR-256 was responsible for this favorable outcome. Overall, five of six patients with confirmed TP53 mutations demonstrated signs of clinical activity38_{. In conclusion, APR-256 is a well-tolerated drug and should be further}

investigated with addition of chemotherapy.

Autophagy is essential for adult hematopoietic stem cells (HSCs) to protect them from stressors such as nutrient deprivations and fluctuations in cytokines, which are naturally occurring in the bone marrow microenvironment39_{. Efficient autophagy is required for}

the preservation of healthy stem cells, as well as for control of malignant transformation40_.

Furthermore, autophagy appears to sustain the metabolism and possibly function of old and young stem cells41_{. Compared to HSCs from young mice, HSCs from aged mice require high}

basal levels of autophagy in order to survive39_{. Ho et al. discovered that knockout of Atg12 or}

Atg5 in mice was myeloid biased and led to a phenotype which resembled premature blood ageing41_{. In another study, mouse HSCs lacking ATG7 led to severe myeloproliferation, loss}

of HSC function and death of mice within weeks42_{. In human cord blood, HSCs had a higher}

autophagic flux than myeloid and erythroid progenitor cells and knockdown of ATG5 or ATG7 caused a reduction of HSC frequencies in vitro and in a xenograft model43_{. Therefore,}

towards the myeloid lineage. This raises the question if autophagy contributes to age-related development of myeloid malignancies39_{. Based on these findings, we aimed at investigating}

whether autophagy inhibition is a treatment strategy in primary AML samples. We discovered that there is a great variety in autophagic flux among different leukemic cell lines. Primary leukemic stem cells, specifically AMLs with complex karyotype, adverse risk, and TP53 mutations, have a significantly higher autophagic flux than normal karyotypes or AMLs with favorable or intermediate risk. Furthermore, we discovered that TP53mut _{AML cells were}

not overly sensitive to autophagy inhibition, while TP53wt _{AMLs responded more strongly.}

We noted that the upregulation of apoptotic genes was impaired during HCQ treatment. At this point it is unclear what the underlying genetic factors are. Presumably, mutations of TP53 do not allow for proper activation of pro-apoptotic targets upon HCQ treatment and therefore diminish apoptotic responses, leading to better survival of AML TP53mut _{cells than}

TP53wt _{AML cells. In the future, we want to dissect what the connection is between TP53}

and autophagy within AML. We overexpressed one TP53mut _{in an acute myeloid leukemia}

cell line and CD34+ _{cord blood cells and compared it to overexpression of TP53}wt_{. We did}

not observe changes in autophagy flux, while primary AMLs with TP53mut _{have a higher flux.}

We therefore are interested to know if the enhanced flux is due to TP53mut _{or due to other}

underlying genetic factors.

Furthermore, research has shown that autophagy is reduced during cancer initiation and can take part in driving malignant transformation40_{. For instance, without sufficient autophagy,}

dysfunctional mitochondria remain in the cells, which are prone to produce ROS, a pro-inflammatory agent40_{. However, we noted that a subset of AMLs have reduced survival upon}

treatment with HCQ, suggesting a therapeutic benefit. In the future, we need to investigate the impact of autophagy inhibitors on non-malignant hematopoietic cells as healthy stem and progenitor cells require certain levels of autophagy for their survival.

Additionally, it needs to be investigated if HCQ could be used in AML as single treatment or as adjuvant therapy to currently existing chemotherapy. It might be used in a pre-treatment setting followed by intensive chemotherapy for priming the AML cells to the death pathway. In solid tumors it was seen that treatment of cancer cells with chloroquine (CQ) as single agent confers treatment resistance by activating the NF-κB pathway44,45_{. There is a risk that}

also AML patients benefit only temporarily from HCQ treatment before becoming resistant. Moreover, it is possible that, with addition of HCQ, TP53 mutated leukemic cells have a selective advantage over wild-type cells due to reduced activation of apoptotic downstream targets (this thesis). Treatment resistance in leukemia and solid cancers is a great issue and upregulation of autophagy is at last in part responsible for it. Primary AML treated with the bromodomain inhibitor BET have been found to become therapy resistant due to enhanced autophagy indcution46_{. Further, primary chronic myeloid leukemic (CML) cells treated}

6

autophagy47_{. Similarly, treatment of colorectal and pancreatic cancer cell lines with bortezomib}

led to therapy resistance due to elevated autophagy levels48_{. Pharmacological inhibition of}

autophagy or knockdown of essential autophagy genes has led to near elimination of cancer in CML stem cells47_{. Additionally, preventing activation of autophagy in BET-resistant}

AMLs induced apoptosis46_{. In the future, combination therapy in clinical trials might be}

an auspicious treatment strategy in at least a subgroup of AML patients. Currently, several phase I and II clinical trials with combination treatment of chemotherapy and adjuvant HCQ treatment are on-going, but these mainly focus on patients with solid tumors49–53_{. Promising} in vitro and in vivo studies with co-treatment haven been conducted in leukemia and clinical

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27. Lucas D, Scheiermann C, Chow A, et al. Chemotherapy-induced bone marrow nerve injury impairs hematopoietic regeneration. Nat Med 2013;19(6):695–703. 28. Wong TN, Miller CA, Klco JM, et

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V, et al. An open-label phase I dose-finding study of APR-246 in hematological malignancies. Blood Cancer J 2016;6(7):e447. 39. Warr MR, Binnewies M, Flach

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NE, et al. Phase I study of the combination of sorafenib and temsirolimus in patients with metastatic melanoma. Clin Cancer Res Off J Am Assoc Cancer Res 2012;18(4):1120–1128.

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NEDERLANDSE SAMENVATTING

De voortdurende aanmaak van bloedcellen in het menselijke lichaam wordt hematopoëse genoemd. Binnen de hematopoëse kan er onderscheidt gemaakt worden tussen verschillende groepen bloedcellen waaronder: rode bloedcellen, witte bloedcellen en bloedplaatjes. Rode bloedcellen of erytrocyten, vervoeren zuurstof vanaf de longen naar verschillende weefsels in het lichaam en verzorgen daarnaast de afvoer van koolstofdioxide. Witte bloed cellen ook wel leukocyten genoemd, spelen een belangrijke rol in de afweer van het lichaam en bloedplaatjes spelen een essentiële rol in bloedstolling. Bloedvormende stamcellen in het beenmerg zorgen ervoor dat er voortdurend stapsgewijs nieuwe bloedcellen worden gevormd. Dit proces word ook wel differentiatie genoemd. Daarnaast kunnen bloedvormende stamcellen zichzelf vernieuwen waardoor de populatie aan stamcellen vitaal blijft. Gedurende een mensenleven kunnen er afwijkingen (ook wel mutaties genoemd) in het DNA van de bloedvormende stamcellen ontstaan. Soms leiden deze afwijkingen ertoe dat een bepaalde stamcel een groeivoordeel krijgt, ten opzichte van andere stamcellen. Dit wordt klonale hematopoëse genoemd en wordt met name gezien op oudere leeftijd. Echter, het optreden van meerdere DNA mutaties kan er uiteindelijk toe leiden dat het zelfvernieuwings en uitrijping proces in stamcellen verstoord raakt. Er is dan sprake van leukemogenese, waarbij een functionele stamcel transformeert in een kwaadaardige (leukemische) stamcel welke uiteindelijke bloedkanker of leukemie veroorzaakt. Er zijn verschillende typen leukemie. Bij het myelodysplastisch syndroom (MDS) vormen leukemische stamcellen misvormde bloedcellen, ook wel dysplasie genoemd. Deze afwijkende cellen sterven sneller af voordat ze het beenmerg verlaten, waardoor er een tekort ontstaat aan functionele uitgerijpte bloedcellen. Een meer agressief type leukemie is acute myeloide leukemie (AML). AML kan voortkomen uit MDS, maar kan ook uit zichzelf ontstaan. Hierbij ontstaat een woekering van abnormale, niet goed uitgerijpte bloedcellen, welke uiteindelijk gezonde cellen kunnen verdringen uit het beenmerg. In dit proefschrift hebben we specifieke DNA mutaties bestudeerd welke sterk worden geassocieerd met leukemogenese.

Mutaties in het ASXL1 gen komen vaak voor bij patiënten met AML of MDS, wat suggereert dat deze mutaties een rol kunnen spelen bij het ontstaan van leukemie. In een muizenmodel waar het ASXL1 gen genetische is verwijderd zagen onderzoekers een fenotype dat sterk lijkt op MDS. Om de functie van ASXL1 beter te begrijpen hebben we de expressie van ASXL1 genetisch geremd (ook wel knockdown genoemd) in gezonde en normale humane hematopoiëtische cellen. Verlies van ASXL1 expressie leidde tot een afname van het aantal stamcellen en de mogelijkheid om myeloïde cellen te vormen. Daarnaast was de vorming van rode bloedcellen sterk verlaagd doordat voorlopercellen niet goed konden overleven. Dit

negatieve fenotype kon niet ongedaan worden gemaakt door de cellen te kweken in een meer “beenmerg omgeving” d.w.z in de aanwezigheid van stromale cellen. Het bleek dat ASXL1 genen beïnvloed die belangrijk zijn voor celgroei en uitrijping van bloedcellen.

Andere genmutaties kunnen ook een rol spelen bij het ontstaan van MDS waaronder het TP53 gen. ASXL1 en TP53 mutaties komen regelmatig samen voor bij patiënten met het ziektebeeld AML of MDS. Mogelijk zijn de cellen door de additionele TP53 mutatie minder kwetsbaar geworden. Daarom hebben we zowel ASXL1 als TP53 tegelijkertijd geremd in hun functie waarbij we gebruik hebben gemaakt van humane hematopoietische stam- en voorloper cellen. Deze onderzoekingen hebben aangetoond dat het verlies van TP53 functie gedeeltelijk het door ASXL1 opgelegde negatieve fenotype kan opheffen. Echter het verlies van beide genen veroorzaakte geen ontwikkeling van leukemie in deze cellen. Dit kan mogelijk verklaard worden door de beperkte periode dat de cellen zijn bestudeerd of dat uiteindelijk er toch een verschil ligt in de functies van ASXL1 en TP53 als gewerkt wordt met een verminderde expressie versus de aanwezigheid van een mutatie in beide genen.

Autofagie is een cellulair aanpassing mechanisme waarbij eiwitten en structuren worden opgeruimd of hergebruikt. In het Grieks betekent het zelfvertering. Als onderdelen van een cel beschadigd raken, kunnen deze beschadigde onderdelen door middel van autofagie worden afgebroken om vervolgens de bouwstoffen te hergebruiken. Autofagie is dus belangrijk voor het goed functioneren van een cel. Verschillende onderzoeken hebben aangetoond dat autofagie ook zorgt voor betere overleving van kanker cellen. Daarom hebben we gekeken of remming van autofagie een nadelig effect heeft op de overleving van leukemische cellen. In onze studie hadden AML cellen van patiënten met de meest agressieve vorm van leukemie, een verhoogde autofagie activiteit. Uit andere studies blijkt dat bij deze patiënten groep vaker mutaties in het TP53 gen optreden. Echter verhoogde expressie (overexpressie) of knockdown van TP53 expressie in AML cellen veroorzaakte geen verandering in autofagie activiteit. Het is dus niet duidelijk of TP53 mutaties een rol spelen in de regulatie van autofagie in AML. Wel bleek dat AML cellen met het normale TP53 gen gevoeliger waren voor het remmen van autofagie t.o.v AML cellen met TP53 mutaties. Remming van autofagie had een nadelig effect op de uitgroei van AML cellen uitgetest in verschillende model systemen. Samengevat duiden deze gegevens dat autofagie cruciaal is voor de overleving van AML cellen en dat remming van de autofagie celdood veroorzaakt in AML cellen met het normale TP53 gen. Naast deze laboratoriumonderzoekingen, hebben we literatuuronderzoek gedaan naar de rol die autofagie kan spelen bij het ontstaan en overleven van kwaadaardige cellen en welke gevolgen dit ook heeft voor de gevoeligheid voor celdodende middelen of chemotherapie. Samenvattend maken deze studies duidelijk dat tijdens de ontwikkeling van kanker autofagie geremd wordt terwijl bij bestaande kanker de cellen vaak gebruik kunnen maken van autofagie om zo te ontsnappen aan de chemotherapeutische behandeling. Verder

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ook voor kankercellen om te kunnen ontsnappen aan de immuunrespons van deze immuun cellen. Ten slotte bespreken we de mogelijkheden ten aanzien van het selectief remmen van autofagie in kanker (stam) cellen.

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ACKNOWLEDGMENTS

Naturally, I would have never accomplished all this research without the input of so many; the women who donated their placentas so I could isolate the healthy stem cells for research, the patients that contributed their blood and bone marrow despite the disease they were battling, and my supervisors and colleagues that influenced my research.

Dear Edo, thank you for giving me the opportunity to follow a PhD career in your lab. I have come to greatly appreciate your knowledge about literature, your management skills, and most of all your reliability. Your supervision has made a great impact on my development as researcher and I thank your for it. JJ, your input and comments during the meetings were a major benefit for my research and many times I left the meetings with enthusiasm to try what you suggested. I also want to thank the members of the reading committee for taking the time to read and assess my thesis.

A big thank you to my two paranymphs Mylène and Julia. Mylène, I am very glad I could win you for my p53 mutant project. Without you, it would still only be two lonely figures but now we are so close to publishing it! I am happy you took over. Thank you so much for the talk and the laughter in ‘your’ primary lab ;)

Liebe Julia, vielen Dank das du an meiner Seite bist in dieser schweren Zeit. Auch wenn wir uns als Kinder manchmal nicht so gut verstanden haben, bin ich froh, dass du meine Schwester bist. Ich bin unglaublich stolz auf dich, wie du alles meisterst: Haus, Hof, Familie. Ich bin sehr gespannt, wie sich deine Jungs entwickeln!

My thanks also goes to all my old colleagues. Dear Bart-Jan, I consider you one of the backbones of the lab. Your seemingly infinite knowledge of assays and techniques, and your scientific input during our meetings have left an irreplaceable mark on my research, my papers, and my education as a researcher. Thank you for always sharing your wisdom and being up for a joke (or two). The same holds true for you, Annet. You are incredibly resourceful and smart and I have come to rely on your knowledge and experience during my PhD. I will never forget the parties we had in which we would cuddle up to you. You made my PhD time so much more enjoyable. Hendrik, my cheerful conference buddy. You always had an open ear and a smile for me. Your friendship means a lot to me. Thanks for the great time in Orlando at the ASH. Your cooking skills are awesome and please, never stop singing “Let it go” on top of your voice. I am happy to share several publications with you. And I think we are all still waiting on this mysterious Sing Star evening you have been talking aboyt for so long… Jenny, thank you for giving a hand with my mice (meaning implanting of scaffolds, injection of cells, babysitting, and finally sacrificing). We all know I could not have done it alone.

Special thanks to my office people Antonella, Matthieu, Alan, Gerbrig, Sonya, Maurien, and Aysegül. We had a great office spirit with good laughs. I appreciated the work and off-topic discussions and I will miss that. Martijn, my stubborn and rebellious neighbor, it was a pleasure working with you. Marco, our characters didn’t always see eye-to-eye but when they did, we had one hell of a time. Harm-Jan, Lieke, Bauke, Kathi, Shanna, and Roos all the best in the future. Pallavi, I can’t believe how good your German has become! I miss our spontaneous food trips and I hope you feel at home in my native country. Henny, we had fun at “Uut de Hoogte” and I wish you all the best for your future career. Aida, thanks for sharing an apartment with me. We didn’t always have it easy with the landlord but we sure didn’t go down quietly. Thank you Vincent, Fiona, Robin, and Marjan for the fun and interesting conversations in the lab. Also thank you for the ones that left before me: Hein, Francesco, Rikst-Nynke, Jeanet, Patrick, Carolien, Carin, Janine, and Chloe. Edwin, thank you for giving me the opportunity to work with you. Technically speaking, you gave me my first “real” job and I am grateful for that. Gerwin, thank you for your contributions during the meetings. Yuan, Nienke, Tom, Manu, Douwe and Iris, we had a short but fun run together and I wish you all the best. Valerie, thank you for the great team work to get the review done. Another very special thank you to the sorting people Henk and Roelof-Jan, Theo, Geert, and Johan. Henk and Roelof-Jan, I learned things from you that I never thought I would. How innocent I was before I worked with you. I miss our conversations and I hope you are both doing well. To my old CRCG committee members: thanks for the being an enthusiastic and reliable team. Additionally, I am grateful for you Else, Sylvia and especially Gerda. Thank you for all your support in the bureaucratic department. Special thanks goes also to all the members of the Pediatric Oncology lab.

Dear Marta, znamy się juz od dłużej niż 6 lat. Twój spokojny i rozsądny sposób był ważną część mojego życia robotnego a - poza wszystkim - mojego życia prywatnego. Dla mnie jesteś prawdziwą przyjaciółką. Bardzo dziękuję ci za spędzone z tobą swietnie czas i mam nadzieję że będą mieć więcej. Napoli 2028! Jodie, our friendship is incredible. We knew each for only a few months before you went onto your adventures and yet we have stayed friends throughout. I managed to visit you in (almost) all the countries you have lived in. When I see you it feels like we have never been apart. Clément, you were with me through most ups and downs in my PhD career and personal life. Always full of advice and with a foot ready to kick my behind. I have learned so much from you and I miss our time together. Don’t be a stranger! The same holds true for you Jelena, Milica, Thomas, Henri, Katja, Helgi, Sara, Manel, Katia and Evgenia. We were a fantastic group! Rebecca, you have helped me through heart ache and personal loses. Walking with you and Niño was the highlight of many dark days. Corka, thanks for your input in my acknowledgment section and the spontaneous drinks. Dave and Jules, thanks so much for your hospitality at Pacific and now at Block and Barrels. Big thank you also to my bouldering ladies Irena and Laura. Claudia und Antje, wir kennen uns seit über

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es als wären wir nie getrennt gewesen. Ein großer Dank geht an meine Familie, insbesondere meine Mama. Es tut mir leid das ich es nicht rechtzeitig geschafft habe zu verteidigen. Ich weiß, wie gerne du dabei gewesen wärst. Auch vielen Dank an den Rest meiner lieben Familie, die mich so gut es ging unterstützt hat. Ich dürft euch jetzt Familie Dr. nennen.

Susan Hilgendorf

PhD in Medical Science

I am a result oriented, dedicated professional with strong analytical skills and excellent communication and social skills. Colleagues describe me as enthusiastic, motivated, and disciplined. I aim to contribute to the development and advancement of medical treatments.

analytical reliable considerate pragmatic organized Professional Experience Contact Details Professional Skills 04/2017 -

03/2018 Scientist/Postdoc for KAHR Medicals Ltd, Israel at the UMC Groningen

Bispecific antibody research & testing for co-stimulation of T cells to (re)activate anticancer immune responses

ELISA FACS qRT-PCR Retro-/lentiviral production T cell proliferation/killing assay Primary/cell line culturing Cloning

Western Blotting ChIP Protein stability/pulldown(IP)

12/2011 -

11/2016 PhD in Medical Science, Rijksuniversiteit Groningen,UMC Groningen

Research into mutated genes and their role in leukemia development at the Experimental Hematology Dept. Education

09/2009

-08/2011 MSc in Molecular Life Sciences, Maastricht UniversityMaster in Oncology & Developmental Biology Grade: 7.8 out of 10

11/2010

-06/2011 Internship at the Ontario Cancer Institut, Canada_{Identifying a biomarker for tumour hypoxia and potential}

therapeutic target at the Radiation Oncology Dept.

01/2010

-07/2010 Internship at the UMC MaastrichtInvestigating hypermethylated tumour suppressor genes in cutaneous malignant melanoma at the Pathology Dept.

09/2006

-08/2009 BSc in Health Science, Maastricht UniversityMajor: Bioregulation Minor: Policy and Management

Study was conducted in Dutch

06/2009

-08/2009 Internship at Maastricht UniversityStudying dietary glycemic index, protein, and 24h glucose pro�iles at the Human Biology Dept.

Language Skills German - native English - pro�icient Dutch - pro�icient Computer Skills MS Word/Excel/Power Point Adobe Illustrator/Photoshop Clone Manager Corel Draw

08/2003

-07/2006 A-level Kopernikus Gymnasium, GermanyHigher education at Kopernikus Gymnasium in Blankenfelde

Koningin Wilhemina Fond Kankerbestrijding - Scholarship Radiotherapy & Oncology, Experimental Hematology, Cell Death &

Disease, Haematologica _Bouldering

Extracurricular/Hobbies Co-founder of the CRCG PhD council making furniture Awards/Publications susanhilgendorf@hotmail.com Kwinkenplein 14 9712GZ Groningen The Netherlands (prepared to move) Tel.: +31-648982779 Mindfulness training