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Target identification to selectively eradicate acute myeloid leukemia stem cells

Verhagen, H.J.M.P.

2018

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Verhagen, H. J. M. P. (2018). Target identification to selectively eradicate acute myeloid leukemia stem cells.

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C H A P T E R

1

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1 G E N E R A L I N T RO D U C T I O N

Leukemia

Leukemia is a malignancy characterized by the accumulation of aberrant leukocytes in the bone

marrow and peripheral blood (Figure 1). This increase in aberrant white blood cells hampers

the function and development of normal hematopoiesis, causing symptoms related to anaemia,

thrombocytopenia and dysfunctional granulocytes, which is insurmountable fatal when patients

are left untreated. Mid 1800s, John Bennet and Rudolf Virchow established the fundament for

the recognition of leukemia and proposed that the aberrant presence of white blood cells in

the patient’s blood was not due to inflammation but rather a result of transformation of the complete

blood system

1

_{. As leukemia was gradually accepted as a distinct disease and many novel cases}

were diagnosed, by studying morphology of the leukemic cells, a high degree of heterogeneity

among patients was observed. Although leukemia was generally considered to be a chronic disease,

the time between diagnosis and mortality for some patients was only a few weeks, therefore these

cases were referred to as acute leukemia. In the next decade, it became evident that the bone

marrow was the source for both the normal hematopoietic system and leukemia. In 1900, leukemia

was further categorised in myeloid and lymphoid neoplasms by detection of either myeloblasts

or lymphoblasts. This formed the basis of what we still recognize today as four primary types of

leukemia; chronic lymphoid leukemia (CLL), acute lymphoid leukemia (ALL), chronic myeloid

leukemia (CML) and acute myeloid leukemia (AML)

2

_.

Diagnosis and classification of AML

AML is the most common form of the myeloid malignancies, with an average incidence of 3.7

cases per 100.000 persons each year in Europe

3

_{. Diagnosis of AML occurs by the detection of >20%}

Figure 1. Illustration of a normal healthy and AML bone marrow aspirate. Healthy normal bone marrow (left panel) contains a wide variety of distinct differentiated myeloid cell types that all share a common ancestor, the myeloblast. The bone marrow of myeloid leukemia patients (right panel) shows accumulation of myeloblasts and the lack of differentiated cells which can result in symptoms like easy bleedings, pale skin, fatigue, weight

Normal hematopoiesis

Acute Myeloid Leukemia

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1 myeloblasts in bone marrow or blood, or the presence of myeloid cells with genetic abnormalities

that include t(8;21), inv(16)/t(16;16), t(15;17)

4

_{. AML can be divided into various subgroups (according}

to the WHO2016 classification) by integration of cell morphology, immunophenotyping, gene

expression, gene mutations and cytogenetics. Based on these features, AML cases can be classified

as either good, intermediate or adverse risk

5

_{. Elderly patients have in general a dismal prognosis}

due to a poor condition and co-morbidities, as well as the presence of additional poor prognostic

factors like complex cytogenetics

6,7

_{. Chromosomal abnormalities are detected in ~55% of the AML}

patients and have a strong impact on prognosis. These abnormalities include t(8;21), inv(16) and

t(15;17) which are good prognostic, and t(9;11), inv(3)(q21q26), t(3;3)(q21q26) del(7) or del(5) which

are poor prognostic. Patients lacking cytogenetic abnormalities are diagnosed as normal karyotype

(NK) and the presence of >3 abnormalities is referred to as a complex karyotype (CK). In addition to

chromosomal aberrations, somatic mutations are frequently found in AML. These include mutations

in genes like NPM1, FLT3, DNMT3A, CEBPA, MLL, RUNX1, TET2, WT1, KIT, RAS and p53. Some, such as

NMP1 and bi allelic CEBPA mutations are associated with a good prognosis, while others such as p53

mutations are associated with a poor prognosis. Moreover, high expression of genes such as ERG,

EVI-1, BAALC and MN1 are also linked to poor AML treatment outcome

8

_.

Aberrant expression of genes, caused by translocations, somatic mutations, or epigenetic

changes can result in a variety of functional consequences driving the development of AML.

Mutations found in AML affect signalling pathways (59%), DNA methylation (44%), chromatin

modification (30%), and myeloid transcription factors (22%), and in lesser extend tumor suppressors,

the spliceosome complex and cohesins

9

_{. This aberrant gene expression can lead to aberrant}

immunophenotypes, including expression of lymphoid plasma membrane markers on myeloid cells

that normally lack these markers. The aberrant expression of several of these markers, including

CD25,CD2,CD7 and CD56, correlate to a poor prognosis

10–12

_{. Other aberrant immunophenotypes are}

the lack of a myeloid marker on myeloid cells, for instance the absence of the myeloid marker CD13

on CD33 expressing cells. Also aberrant expression of CD34 and MDR1 on myeloid cells is frequently

found for AML and both correlate to a poor prognosis

13,14

_.

A well-defined prognostic marker for AML patients that predicts a poor response to

chemotherapy and a poor overall survival, is high expression of the Ecotropic Viral Integration

site-1 (EVI-1) oncogene. Despite treatment with chemotherapy and a hematopoietic stem cell

transplantation if eligible, >95% of AML patients with high EVI-1 expression do not survive two

years after diagnosis

15

_{. EVI-1 was initially discovered as a common retroviral integration site for}

murine haematological malignancies

16

_{and enhanced expression of EVI-1 occurs in ∼10% of AML}

patients

17

_{. High EVI-1 expression is sometimes driven by 3q26 abnormalities, as a result of aberrantly}

positioned transcriptional enhancers

18

_{, however, in most cases EVI-1 is found to be overexpressed}

due to an unknown cause

19

_{. In healthy tissue, EVI-1 is predominantly expressed in embryonic and}

hematopoietic stem cells (HSCs) by acting as a multi-functional transcriptional regulator and is

essential for the proliferation and maintenance of hematopoietic stem cells (HSCs)

20,21

_{. Although}

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1 Chemotherapy response and resistance in AML

Although AML is a very heterogeneous disease, most patients are treated with a standard

combination chemotherapy that consists of cytarabine, and an anthracycline such as daunorubicin

and etoposide

5

_{. This treatment results in a complete remission (CR), defined as <5% myeloblasts, and}

recovery of normal hematopoiesis, in approximately 55-85% of adult AML patients

22–25

_{. If eligible,}

adult AML patients with an intermediate or adverse prognostic score are offered an allogenic

hematopoietic stem cell transplantation after achieving CR

4

_{. Despite these high initial CR rates, 50%}

of AML patients experience a relapse within 5 years post diagnosis

26

_{. Recurrent disease is difficult to}

treat and is the main reason for the in general poor overall survival rates (30%) of AML patients

27,26

_.

Once the patient has developed a relapse its 3-year overall survival is 12% and 0% for intermediate

and adverse risk groups, respectively

28

_{. Refractory AML patients, patients that still present >5%}

leukemia in the bone marrow after standard chemotherapy, can be offered alternative (salvage)

therapy. Commonly used salvage therapies include fludarabine, idarubcin and mitoxantrone, which

can potentially result in CR

5,29

_.

The large number of clinical trials (>200), that are currently testing the efficacy of tyrosine

kinase -, epigenetic - and many other inhibitors as well as immunotherapeutic modalities (Clinical

trial register.eu), suggest increased numbers of potential alternative therapeutic options for AML

patients in the next decades. Some of these novel therapeutic strategies are predicted to act

on subgroups of AML patients, for instance, inhibition of Disrupter of Telomeric Silencing1-like

(DOT1L). This histone 3 lysine 79 (H3K79) methyltransferase is highly active in DNMT3a mutated as

well as MLL rearranged AML and inhibition of DOT1L demonstrated to induce apoptosis and tumor

reducing activity in vivo

30,31

_{. Other promising options are targeted therapy for specific subgroups of}

AML patients with high FLT3 and SYK activity. Recently a dual inhibitor for both kinases in addition to

chemotherapy has demonstrated to improve survival of AML patients

32,33

_{. Other novel therapeutic}

options are predicted to have a broader applicability, for instance anti-CD33 antibodies that

potentially eradicate all myeloid cells

34

_.

Despite all the promising novel strategies, relapse is currently still the main cause for

the poor outcome of AML patients. Residual leukemic cells in the bone marrow of the patient after

chemotherapy treatment, referred to as minimal residual disease (MRD), initiate this relapse. MRD

can be detected using flow cytometric immunophenotyping and mutation analysis using molecular

techniques like PCR and next generation sequencing. High MRD levels strongly correlate with

increased relapse rates and measuring MRD is included in clinical studies as a novel risk parameter

to predict treatment outcome

35,36

_.

Cytarabine, daunorubicin and etoposide are currently the most commonly used drugs to treat

AML, and mainly act on the DNA replication machinery by inhibition of DNA polymerase activity,

by cooperation into DNA during S-phase or by fixation of topoisomerase II to the DNA

37,38

_{. As}

a result, DNA replication is terminated and double strand breaks are generated that cannot be

sufficiently repaired, hence leading to the induction of apoptosis. Recently it has been shown that

anthracyclines induce histone eviction and changes the transcriptional landscape

39

_{, indicating that}

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1 of escaping chemotherapy induced cell death are illustrated in Figure 2 and include (I) enhanced

DNA repair, (II) enhanced drug efflux, (III) enhanced survival signalling, (IV) target modification, (V)

apoptotic block and (VI) quiescence

40–42

_.

_{Recently it has been shown that inactivation of the SWI/}

SNF chromatin remodeling complex, that is involved in loading the Topoisomerase II to the DNA,

can also mediate resistance to chemotherapy

43,44

_.

Figure 2. Mechanisms of resistance to chemotherapy in AML. (I) Genes that enhance DNA repair activity can prevent that cells undergo apoptosis44–47_{. High INPP4B expression is a biomarker for chemotherapy resistance}

and is associated with a poor overall survival of AML patients. IBPP4B regulates ATM dependent DNA repair by activating BRCA1and RAD5145,46_{. Another gene that provides enhanced DNA repair is CHEK1. High CHEK1}

expression is associated with a poor response to chemotherapy and when activated by ATR it still induces DNA replication upon cytarabine treatment47_{. (II) Chemotherapeutic agents that enter the cell can be efficiently}

exported by drug transporters like multi-drug resistance 1 (MDR1), which prevent that cytostatic drugs reach the nucleus and induce DNA damage48_{. High MDR1 expression, as well as functional efflux capacity,}

is observed in ~30% of the AML cases and correlates to a poor prognosis49_{. MDR1 is also frequently found}

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1 Leukemic and normal hematopoietic stem cells

AML is a clonal disease which is like normal haematopoiesis hierarchically organised

69

_{and originates}

from mutations that occur in a multipotent progenitor or a HSC

70,71

_{(Figure 3). The development}

of leukemia has been suggested to originate from environmental stress induced by chemicals and

radiation, but also genotoxic stress driven by the bone marrow microenvironment, both inducing

DNA aberrancies

72,73

_{. To date, the best accepted model describing the onset of AML is the “two}

hit model” hypothesis

74,75

_{. This hypothesis states that the cell of origin needs at least two impaired}

genes in order to transform into leukemia. The so called class I mutations can confer cells with

a proliferation advantage whereas the class II mutations direct a block in differentiation.

Within AML, tumor heterogeneity is observed since rare subpopulations are detected with

enhanced self-renewal capacity

69

_{. These cells are often referred to as leukemic stem cells (LSCs) and}

are functionally defined by their long term tumor-initiating capacity in immunodeficient mice using

serial transplantations, or by their colony forming capacity using long term colony forming unit

(CFU) assays in vitro

69,76–78

_{. Although the cell of origin can be a LSC, these two are not necessarily}

directly correlated to each other

70

_{. LSCs co-reside with HSCs in the bone marrow of AML patients}

and are initially identified as having the CD34

_{LSCs are mainly quiescent}

66

_{like HSCs}

80

_{, however, recently a novel non-quiescent}

LSC population was reported indicating the heterogeneity of the LSC pool

81

_{. The frequency of}

CD34

+

_CD38

-

_{LSCs at diagnosis and after chemotherapy treatment is predictive for overall survival}

and relapse

82,83

_{. Importantly CD34}

+

_CD38

-

_{LSCs show decreased sensitivity to chemotherapy and}

reside in the endosteal regions of the bone marrow

66,84

_{. The hypothesis that LSCs preferentially}

therapy-induced drug resistance50_{. (III) Activated signalling routes can trigger survival responses that}

overcome therapy induced cell death51–55_{. The best known example for AML is the FLT-3 receptor which is}

frequently found highly expressed due to internal tandem duplications in the DNA (FLT3-ITDs). FLT3-ITDs are associated with a poorer survival of AML patients and result in increased resistance to cytarabine54,56_{. Activated}

Insulin Growth Factor 1 receptor (IGF1R) can also promote AML cell survival during chemotherapy treatment by facilitating resistance to chemotherapy52_{. (IV) Targets of chemotherapy can either be downregulated in}

expression levels or mutate during treatment, leading to therapy unresponsiveness40_{. Classically, etoposide and}

anthracyclines like daunorubcin target topo isomerase II activity, leading to double strand breaks in the DNA and consequently apoptosis. However, in vitro studies revealed that expression of topoisomerase II is reduced in leukemic cells that are resistant to etoposide57_{. Furthermore, topoisomerase II expression levels in AML cells}

have been shown to determine sensitivity to chemotherapy58_{. Moreover, a point mutation in topoisomerase II}

demonstrated to decrease sensitivity to cytotoxic agents more than 50 fold In AML cell lines59_{. (V) The activation}

or overexpression of anti-apoptotic proteins can overcome chemotherapy-induced cell death by blocking apoptosis60,61_{. BCL2 is a key protein in the regulation of apoptosis and its elevated expression is correlated to}

a poor response to chemotherapy62_{. Also loss of the apoptotic regulators Puma and Noxa decreased sensitivity}

to cytarabine and anthracyclines61_{. (VI) Quiescent (non-cycling) cells remain in the G0 cell cycle state and are}

therefore less vulnerable for chemotherapy and have more time to repair potential DNA defects63–67_{. The loss of}

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1 survive chemotherapy is confirmed by a study that shows that LSC frequency is increased at relapse

compared to diagnosis

85

_{. However, conflicting data shows no increase in the number of LSCs after}

chemotherapy treatment of AML in a xenograft mouse model

86

_.

Various molecular mechanisms can contribute to the presence of stem cell-like features

(“stemness”), such as self-renewing capacity and chemotherapy resistance in AML. “Stemness”

might be imposed on AML cells by (I) genetic changes, (II) epigenetic changes and by the (III)

tumor microenvironment

87

_{. Genetic changes, caused by irreversible somatic mutations, can result}

in distinct cell populations with different sensitivities to chemotherapy

88,89

_{. Epigenetically regulated}

events and the tumor microenvironment might cause dynamically regulated AML heterogeneity in

the sense of chemotherapy sensitivity. Soluble factors secreted by mesenchymal stromal cells, like

CXCL12 (SDF-1) and JAG1, but also fatty acids derived from gonadal adipose tissue have been linked

to the survival of LSCs and the facilitation of chemotherapy resistance

90,91

_{. Besides the induction of}

“stemness”, LSCs can also arise as a consequence of cell intrinsic changes

92,93

_{. Recently it has been}

shown that epigenetic reprogramming of H3K27m3 modifications is important for LSC survival

94

_.

The central role for LSCs in AML leukemogenesis, therapeutic responses and the initiation

of recurrent disease, drives the hypothesis that their elimination is key to improve AML therapy

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1 outcome. The development of therapies that eradicate LSCs is challenging since these should spare

healthy HSCs that are needed to restore normal hematopoiesis after therapy. To develop such

specific anti-LSC therapies, the identification of genes that are differently expressed between HSCs,

LSCs and the AML bulk is crucial. Several studies have already explored transcriptional differences

between LSCs and non-LSCs from AML patients and differences between LSCs and normal HSCs

derived from healthy donors (Table 1). Eppert and colleagues generated gene expression profiles

(GEPs) comparing functionally defined LSCs to non-LSCs fractions and showed that high expression

of a LSC signature in AML correlates to a dismal outcome

95

_{. Furthermore, in this study cord}

In the past decade, our lab identified biomarkers, that can distinguish LSCs from HSCs both

residing in the AML bone marrow. CD45RA

97

_{, CLL-1}

98

_{and other lineage markers such as CD11b,}

CD7 and CD56

99

_{can be present on LSCs, however, are always absent on HSCs. Also other labs have}

identified such markers, including CD123

100

_{and Tim3}

101

_{. Importantly, our lab has identified that HSCs}

can be functionally discriminated from CD34

+

_CD38

-

_{LSCs using aldehyde dehydrogenase activity}

(ALDH)

102

_{. ALDH was initially identified as a cancer stem cell (CSC) marker in solid tumors and high}

ALDH activity generally correlates to a poor prognosis

103

_{. In most FLT3-ITD and NPM1 mutated}

AML cases, ALDH

bright

_CD34

_{populations which gave myeloid leukemic}

engraftment in a xenograft mouse model.

LSCs in AML patients BM are heterogonous in multiple ways. Accumulation of mutations in LSCs

and hence Darwinian selection during disease progression and after chemotherapy treatment, can

lead to a complete different clone that is present at relapse compared to diagnosis

88

_{. The same}

holds true for aberrant immunophenotype which is in some AML patients different at MRD or

relapse than at diagnosis

105

_{. Importantly, cancer cells can show different sensitivities to therapy that}

is regulated by non-genetically induced events, thereby adding another complexity to intratumoral

heterogeneity

106

_{. For example, small small populations of EGF receptor tolerant cells, with a stem}

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1 Insulin-like Growth Factor-1 (IGF-1) signaling

The IGF-1 receptor (IGF1R) is frequently found overexpressed in cancer cells and can regulate a wide

variety of cellular processes including survival, differentiation, proliferation, quiescence and therapy

resistance

114

_{. Although the role of the IGF-1 system has been extensively studied in solid tumors, less}

is known about this pathway in hematological malignancies. In multiple myeloma (MM), IGF-1 has

been shown to stimulate the growth and survival of leukemic cells

115

_{and several anti-IGF1R therapies}

were shown to inhibit MM proliferation

116

_{. In AML, expression of IGF-1 and the IGF1R was detected in}

a panel of human AML blasts and cell lines and IGF-1 could promote the growth of AML blasts, AML

cell lines as well as AML progenitors

in vitro

117–119

_.

_{For AML, inhibition of the IGF-1 system has been}

performed by neutralizing anti-IGF1R antibodies and IGF1R tyrosine kinase inhibitors (NVP-AEW541

and NVP-ADW742) which both inhibited cell proliferation and induced apoptosis

51,120

_{. The effect of}

IGF-1/IGF1R inhibition on the survival of AML LSCs is currently unknown. Anti-IGF-1/IGF1R strategies

seem to mimic the molecular effects caused by calorie restriction, which is known to decrease IGF-1

signaling. Combining calorie restriction (starvation) with chemotherapy treatment demonstrated

to specifically reduce the survival of cancer cells, while enhancing that of normal cells

121,122

_{. IGF1R}

signaling can be regulated by expression of the components of IGF1R pathway, ligand bioavailability

and by receptor trafficking

123

_{. Moreover, IGF1R activity can be regulated by insulin-like growth}

factor binding proteins (IGFBPs)

124

_{. To date, ten IGFBPs have been identified from which IGFBP1-6}

are considered to have a function in inhibition of IGF1R signaling by binding IGF-1 and thus affecting

ligand bioavailability. IGFBP 7-10 do not bind with high affinity to the IGFs and might have additional

functions next to inhibition of IGF1R signaling

125

_{. An important role of IGFBP7 in IGF1R signaling is}

Table 1. Gene expression profiles of HSCs, leukemic progenitors and LSCs. NBM = normal bone marrow, CB = Cord Blood, PB = peripheral blood.

Source Sorted fractions Reference

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1 shown in multiple studies

126,127

_{. The loss of secreted IGFBP7 by tumor associated endothelial cells}

contributes to the transformation of naïve tumor cells to chemotherapy resistant stem cells

127

_.

Importantly, treatment of breast cancer and melanoma cells with purified recombinant human

IGFBP7 (rhIGFBP7) led to the induction of senescence and apoptosis and reduced tumor growth

in vivo

126,128

_.

Retinoic acid based therapy for AML patients

AML cases that harbor a t(15;17) translocation, leading to expression of the fusion gene between

promyelocytic leukemia (PML) and the retinoic acid receptor alpha (RARA), are a separate entity of

AML cases, referred to as acute promyelocytic leukemia (APL). APL patients are treated with all- trans

retinoic acid (ATRA) differentiation therapy in combination with arsenic trioxide (ATO), resulting in

overall survival rates around 95%

129

_{. ATRA is a vitamin A derivative that belongs to the best known}

examples of a successful targeted treatment and turned APL from a poor prognostic malignancy

into a curable disease

129,130

_{. In APL patients, expression of PML-RARA results in a differentiation}

block by repression of genes that regulate differentiation

131

_{. RARs function as molecular switches}

by recruiting N-Cor, SMRT and a histone deacetylase (HDAC), resulting in closed chromatin

and transcriptional silencing. Application of ATRA leads to conformational changes of RARs and

release of the transcriptional repressors and recruitment of transcriptional activators such as

the histone acetyl transferase p300. The increase of histone acetylation results in decompressed

chromatin leading to active gene transcription facilitating myeloid differentiation. Various clinical

trials have explored the applicability of ATRA for the non-APL AML subgroups. however, in general

ATRA treatment is not superior over chemotherapy treatment

132,133

_{. Previously, it has been shown}

that inhibition of the H3K4 demethylase LSD1 (KDM1A), using tranylcypromine (TCP), can unlock

the ATRA differentiation pathway for non-APL AML indicating the importance of the epigenetic

landscape of AML cells in ATRA sensitivity

134

_{. The combination TCP and ATRA results in expression}

of the myeloid differentiation marker CD11b in HL60 cells and reduced primary AML engraftment in

vivo, indicating a novel therapeutic opportunity to extend ATRA based therapy for AML. Recently,

several studies have explored the susceptibility to ATRA in smaller subgroups of AML patients and

reported a partial response to ATRA in vitro as well as in vivo for IDH1 mutant AML

135

_{, NPM1 mutant}

AML

136,137

_{and FLT3-ITD}

+

_AML

138

_{. Altogether these data indicate that ATRA sensitivity is determined}

on a transcriptional or epigenetic level and treatment of AML patients might work for well-defined

subgroups of AML patients.

O U T L I N E O F T H I S T H E S I S

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1 enhancing insulin-like growth factor binding protein 7 (IGFBP7), by overexpression or addition of

recombinant human IGFBP7 (rhIGFBP7), induces apoptosis and enhances chemotherapy sensitivity,

similar as the inhibition of IGF1R activity by a tyrosine kinase inhibitor. The tumor reducing effect of

IGFBP7 is at least partly explained by an IGF1R independent mechanism. Furthermore we report that

low IGFBP7 expression levels are more indicative for a poor overall survival compared to high IGF-1

or high IGF1R expression levels in AML patients.

Since LSCs play a central role in the treatment outcome of AML, the eradication of these cells

at diagnosis, while sparing HSCs, is of critical importance for the development of novel therapeutic

strategies. In chapter 3 we have studied GEPs of LSCs relative to HSCs that are both derived from

the bone marrow of an AML patient. Moreover, we compared GEPs of CD34

+

_CD38

-

_{leukemic cells}

with those of leukemic CD34

+

_CD38

+

_{cells and identified IGFBP7 as differently expressed. IGFBP7}

expression levels correlate with chemotherapy sensitivity and self-renewal capacity. Importantly,

we show that rhIGFBP7 induces differentiation and apoptosis in primary AML cells in vitro and

eradicates LSCs while sparing HSCs. Treatment of AML with rhIGFBP7 in patient derived xenograft

models reduces engraftment and LSC activity, this in contrast to HSCs derived from healthy BM

which remain unaffected by rhIGFBP7 treatment.

In chapter 4 we have studied anthracycline resistance in a subpopulation of myeloid leukemic

cells residing within a sensitive leukemia cell line. This small subpopulation of leukemic cells

possesses stem cell-like features and a reversible drug tolerant state. Using GEP we identified

KDM6B as a potential regulator of this drug tolerant state. Inhibition of KDM6B activity using GSK-J4

resulted in the elimination of the drug-tolerant cells, indicating that KDM6B inhibition might be

a potential successful therapeutic strategy to eliminate stem cell-like chemotherapy resistant

AML cells.

In chapter 5 we explored the effect of ATRA on AML patients that have high expression of

EVI-1. Here we show that part of the EVI-1 positive AML cases respond to ATRA by induction of

differentiation and in some cases induction of apoptosis in vitro. Importantly, we show that EVI-1

positive AML can be successfully treated with ATRA in vivo, suggesting that treatment with ATRA

might be a promising strategy for EVI-1 positive AML patients.

In chapter 6 we have reviewed the current knowledge on susceptibility of non-APL AML for

ATRA. Non-APL AML cases are less sensitive for ATRA therapy as compared to APL, however,

epigenetic reprogramming as well as the identification of biomarkers that predict ATRA response

can potentially change this dogma.

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