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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1. NORMAL AND MALIGNANT HEMATOPOIESIS
1.1 Normal hematopoiesis
Differentiation within the hematopoietic system cells is based on a hierarchal tree with immense regenerative potential. At the top of the hierarchy reside the most undifferentiated hematopoietic stem cells (HSC) with the greatest self-renewal potential (Figure 1). After
cell division they give rise to more differentiated progenitors, which constitute the entire blood lineage in a multi-step process. Constant cell differentiation and proliferation occurs to reconstitute the hematopoietic system but only a small amount of long-term HSCs is responsible for the total blood development. However, recent studies have also shown that during steady state hematopoiesis the multipotent and lineage-restricted progenitors are highly responsible to drive hematopoiesis. This suggests that dependent on the insult, e.g. transplantation vs steady state hematopoiesis, HSCs vs progenitors mainly drive hematopoiesis1–5. LT-HSC ST-HSC MPP LMPP CLP CMP GMP MEP T cells B cells NK cells dendritic cells granulocytes macrophages erythrocytes platelets
Figure 1: Schematic representation of the hematopoietic system Figure 1: Schematic representation of the hematopoietic system
The path into different lineages is under control of transcription factors and epigenetic changes within the hematopoietic stem and progenitor population (HSPC) in conjunction with the surrounding micro-environment6,7. Stem cells appear to have an open chromatin structure
to allow for multi-lineage differentiation and the activity of certain chromatin-related genes determines accessibility of lineage specific genes8. Mutations of genes within the primitive
hematopoietic cells can predispose individuals to develop myeloid malignancies.
1.2 Clonal hematopoiesis of indeterminate potential
Several studies have shown that with increased age, blood-specific mutations increase9–11. The
mutation frequency is low in the younger generation but is rising from 1.5% in those aged 50-59 to 19.8% in 90-98 year olds12. Many of the discovered genes play important roles in
leukemogenesis11. The term “clonal hematopoiesis of indeterminate potential” (CHIP) has
been proposed to describe the feature of clonal hematopoiesis with leukemogenic-related somatic mutations in the blood and bone marrow without displaying any dysplasia in the bone marrow (Figure 2)13,14. Many people can present with background mutations, which
are unrelated to hematopoietic expansion13. However, recurrently mutated genes may lead
to clonal hematopoiesis by awarding stem cells and their progenies with survival advantages and allow for clonal expansion but do not induce hematopoietic disease development13,14.
Mutated genes that are thought to contribute to pathogenesis of cancer, such as DNMT3A, TET2, and ASXL1, are part of CHIP and predispose individuals to develop hematological malignancies in particular when they are present in combinations. Nevertheless, persons with CHIP clones can remain healthy for years with clones being stable at low levels9,11. This
suggests that cell intrinsic changes are necessary but not sufficient for disease development and progression. Cell extrinsic factors, such as environmental stresses, inflammation, radio- and chemotherapy, might be important to drive preferential clonal expansion15,16. Evidence
is emerging that chemotherapy may lead to the expansion of an, until then, unrelated hematopoietic population with somatic mutations17,18. Consequently, to transform to overt
malignancy, cells need to acquire additional mutations in the background of cooperating genes. Additionally, the leukemogenic genes in CHIP have been found to lead to an increased risk incidence of ischemic stroke and coronary heart disease in patients11,19. In mice, mutated
TET2 has been demonstrated to induce clonal hematopoiesis and leads to an accelerated development of atherosclerosis with a striking increase in atherosclerotic plaque size due to an altered function of infiltrating monocytes and macrophages.
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Figure 2: Schematic representation of the development of hematological malignancies
CHIP MDS AML
Clonal size
Clonal haematopoiesis
Cytopenias & dysplasia
Excess blasts
Normal blood cell Mutation 1 Mutation 2 Mutation 3 Mutation 4
Figure 2: Schematic representation of the development of hematological malignancies
1.3 Myelodysplastic syndrome
Myelodysplastic syndrome (MDS) comprises a set of symptoms, with the most prominent ones being morphologic dysplasia, peripheral cytopenia and subsequently ineffective hematopoiesis20. MDS is characterized by ineffective hematopoiesis of one or several
lineages in the bone marrow21. Patients can present with reduced platelet production due to
enhanced apoptosis of immature megakaryocytes22,23. Evidence suggests that an abnormal
microenvironment can contribute to disease patterns within patients. Inflammation, oxidative stress, and abnormal stromal function can be instrumental to the pathogenesis of MDS24–27.
Similarly, low-risk MDS erythroid-blasts reveal an overexpression of pro-apoptotic genes and are more vulnerable to programmed cell death when removed from the bone marrow environment28,29. With disease progression, a differentiation block of all myeloid lineages
can be observed and ultimately, a blast count of up to 19% can be detected within the bone marrow (Table 1).
Table 1: Classification of MDS
Name
Blast
BM PB
MDS with single-lineage dysplasia (MDS-SLD) <5% <1%
MDS with multi-lineage dysplasia (MDS-MLD) <5% <1%
MDS with ringed sideroblasts (MDS-RS) <5% <1%
MDS with excess blasts (MDS-EB) 5-19% 2-19%
MDS, unclassifiable (MDS-U) <5% =<1%
BM: Bone marrow; PB: Peripheral blood
A recent model suggested that the cells of origin are MDS-stem cells which arose from CHIP (Figure 2)30,31. With increased severity of MDS, patients appear to acquire more mutations
and are at greater risk to develop secondary acute myeloid leukemia (sAML)32,33. Patients with
sAML are more chemoresistant, have reduced event-free survival, and have an overall poorer outcome34. The genetic aberrations underlying MDS have been heavily investigated and 75
to 90% of patients have at least one known, recurrently mutated gene32,33. The spliceosome
machinery, epigenetic/DNA methylating machinery, and transcription factors are the most commonly affected34–38. Moreover, approximately half of all MDS patients present with
chromosomal rearrangements, such as del(5q),-7/del(7q), trisomy 839. These copy-number
alterations can drive MDS evolution as individual disease subset or by affecting several genes. For example, del(5q) can present with a distinct pathological and clinical disease subset in MDS patients as 5q- syndrome. It has been found that these patients respond very well to
lenalidomide40. EZH2, located at 7q, may present with deletion, missense and frameshift
mutations, leading to inactivating and loss-of-function mutations41,42. In mice, EZH2 loss in
hematopoietic stem cells resulted in myelodysplasia and MDS-like phenotypes43. Changes
in copy number alterations are more likely to occur in intermediate and high-risk MDS and predict for unfavorable outcome44,45. These genetic lesions could help to determine more
precise treatment options to manage MDS, as no known drug therapy is ultimately curative46–48.
Frequently, low-risk MDS patients are only seen in the clinic until symptoms worsen and disease progression occurs49. High-risk MDS patients may receive chemotherapeutic agents
and ultimately hematopoietic stem cell transplantation (HSCT). HSCT is considered the only curative option but is still associated with enhanced mortality rates50,51.
1.4 AML
AML is a disease that remains difficult to treat. Up to 70% of patients above the age of 65 that receive chemotherapy die within one year of diagnosis due to AML52. It manifests itself
as an accumulation of malignant myeloid cells that replace normal bone marrow cells and ultimately present in the peripheral blood53. AML can arise due to the genetic mutations
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that may lead to a block of differentiation and disturbed proliferation of clonal populations of the myeloid stem cells (Figure 2). The World Health Organization classified adult AML
with recurrent genetic aberrations into several separate groups, including the t(8;21) group, the inv(16) group, and the MLL fusion gene group54. By sequencing more than 1500 AML
patients, Papaemmanuil et al. established a new genomic classification with non-overlapping subtypes55. The second biggest subgroup established contained RNA splicing, chromatin,
and transcription factor genes. This chromatin-spliceosome group was composed of such complex patterns and co-mutations that no individual genetic aberration was defining this group55. This interplay of genetic lesions reveals the complexity of this AML subgroup.
Genetic aberrations are found in more than 97% of AML patients, affecting mostly the same pathways as found in high- risk MDS patients56,57. Drawing a line between high-risk
MDS and AML patients has been challenging as a 20% blast count in the bone marrow and peripheral blood is supposedly indicative for AML but at 19% for MDS. If applicable, AML and high-risk MDS patients will receive intensive chemotherapy. Distinguishing these two diseases and accurately classifying them is essential for patients to determine therapy but also to assess prognosis of outcome. A deeper understanding into genes leading to MDS and AML development may eventually help improve patient outcome by more targeted and individualized therapy.
2. THE ROLE OF ASXL1 IN NORMAL AND MALIGNANT
HEMATOPOIESIS
2.1 ASXL1
Originally discovered in Drosophila, a human homologue named ASXL1 (addition of sex-comb like 1) was later found at 20q11 by Fisher et al. Under normal conditions, ASXL1 is thought to recruit or stabilize the polycomb repressive complex 2 (PRC2) using its C-terminus-containing PHD finger to interact with chromatin and is therefore being classified as an epigenetic modifier (Figure 3, left). PRC2 controls gene expression patterns
in cells by controlling the chromatin structure. ASXL1 can either stabilize or recruit the PRC2 complex to the chromatin. Using its lysine methyltransferase capabilities, the PRC2 core subunit EZH2 can catalyse H3K27me3, compacting the chromatin further. ASXL1 is thus involved in the silencing of specific target genes, such as the HOXA family. Upon loss of the C-terminus, the interaction between ASXL1 and chromatin is disrupted and H3K27 trimethylation levels are reduced over time58. Therefore, genes such as the HOXA family may
become derepressed and are transcriptionally accessible. Most members of the HOXA cluster are found to be leukemogenic due to their control over the self-renewal and differentiation
potential of HSPCs59. Inoue et al. and Abdel-Wahab et al. demonstrated an upregulation of
HOXA9 after ASXL1 abrogation, suggesting that derepression of target genes via loss of H3K27me3 may play an important role in myeloid transformation58,60.
The ASX homology (ASXH) domain, which adjoins the N-terminal region of ASXL1, appears to be necessary for protein-protein interaction61. The most prominent interaction partner is
BAP1 with which ASXL1 forms the polycomb repressive deubiquitinase (PR-DUB) complex (Figure 3, right)62. BAP1 is a ubiquitin C-terminal hydrolase (UCH) with deubiquitinating
activity that possesses tumor suppressor functions63,64. Within the PR-DUB complex, BAP1
removes monoubiquitin from H2AK119Ub and prevents hyperubiquitylation of H2A and therefore balances gene expression62. Scheuermann et al. demonstrated in Drosophila that
deubiquitination of H2A requires ASXL1-BAP1 interaction, suggesting that loss of ASXL1 may lead to increased ubiquitination and therefore silencing of target genes62.
RbAp46/48 EZH1/2 SUZ12 Jarid2 EED ASXL1 BAP1 ASXL1
PRC2
PR-DUB
H3K27me3 H2AK119ubFigure 3: Schematic representation of ASXL1 within the PRC2 & PR-DUB complexes Figure 3: Schematic representation of ASXL1 within the PRC2 & PR-DUB complexes
2.2 ASXL1 in human and mouse hematopoiesis
Initially, ASXL1 was thought not to be required for stem cell and multipotent progenitor function65. However, later studies conducted by Wang et al. and Abdel-Wahab et al. revealed
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importance for hematopoiesis was established as a complete and tissue specific knockout in mice lead to myelodysplastic-like phenotype and developmental defects in vivo66,67. Moreover,
loss of ASXL1 was found to compromise the erythroid differentiation of human cord blood CD34+ cells and reduced the number of erythroid progenitors in mice68,69. In patients, ASXL1
abrogation is always heterozygous and most mutations occur as frameshift or missense mutations within exon 12 (Figure 4). This can lead to loss of the C-terminus, including the
PHD finger, and result in truncation of the protein. A deletion of ASXL1 gene expression and therefore loss of protein in mice has enabled myeloid leukemogenesis only in combination with other gene alterations, such as TET2 and NRAS58,67. Whether the truncated form of the
ASXL1 protein has any additional functions that may lead to cancer development is currently under investigation. Inoue et al. demonstrated that ASXL1 truncation mutants are detectable at protein levels, suggesting that the altered proteins may be stable for at least some time to facilitate other functions70. A mouse model overexpressing a mutant ASXL1 protein revealed
an MDS-like disease after a 12 month latency and some mice progressed towards leukemia60.
Underlying epigenetic changes clearly involved reduction of H3K27me3 of the HOXA family members. A study conducted by Balasubramani suggested that ASXL1 mutations could have gain-of-function properties. Overexpression of several mutants together with BAP1 in a mouse cell line enhanced PR-DUB activity and reduced H2AK119Ub and H3K27me3 levels greatly. ASXL1 mutant activity seemed to be completely dependent on BAP1 binding or interaction, suggesting that H2AK119Ub depletion is the main cause of H3K27me3 loss71.
N PHD EXON 12 EXON 11 Frameshift Nonsense G646fs*12 Missense E1102D R693*
ASXH ASXM1 ASXM2
Mutations
Figure 4: Schematic representation of ASXL1 mutations
Figure 4: Schematic representation of ASXL1 mutations in hematopoietic and lymphoid tissue)2.3 ASXL1 in MDS and AML development and progression
According to a study conducted by Jaiswal et al., ASXL1 is the third most commonly mutated gene in healthy individuals predisposing these individuals to an increased risk of hematological cancers. However, the absolute risk of developing malignant hematopoiesis remains small11. Interestingly, ASXL1 appears to be mutated mainly in the oldest of the healthy
individuals10. Once an MDS or AML is diagnosed, ASXL1 singularly predicts for adverse
outcome37,72–75. The frequency of ASXL1 distortion varies between myeloid malignancies. In
MDS, approximately 16% of patients have ASXL1 alterations, while this is 6% for de novo AML and 30% for sAML patients75.
It is still unclear whether ASXL1 is a cancer-initiating gene or a mutation contributing to disease development and progression. Initially, studies suggested that ASXL1 alterations occur primarily in high-risk MDS and AML patients33,74. However, new studies conducted
on low-risk MDS found ASXL1 mutations to be present and also predicting for worse overall survival35,76,77. Moreover, ASXL1 mutations appeared to be retained throughout disease
progression and relapse. Nevertheless, mouse models failed to reveal a clear relationship between initiation of malignant myeloid development and ASXL1 functional abrogation, raising the question whether mutations in ASXL1 are initiators of disease37,78,79. ASXL1 could
be a second- or third hit to help shape the epigenetic landscape and thus allow for genes to induce transformative effects. Furthermore, ASXL1 mutations could be acquired throughout disease development. Evidence for these theories comes from the frequencies of co-mutation. Up to 85% of MDS patients with ASXL1 mutation present with at least one additional gene alteration. Patients appear to retain the concurrent gene alterations, suggesting that ASXL1 does not convey initiating events on its own77. Often, ASXL1 is co-mutated with genes
typically occurring in a distinct set of pathways, including transcription factors (RUNX1, TP53), spliceosomes (U2AF1, SRSF2), epigenetic modifiers (TET2, EZH2), and signaling (NRAS)35,74,76,77,80,81. The variety of possible co-mutations and the high percentage of sAML
suggest that ASXL1 alone cannot initiate and drive MDS/AML development and progression.
2.4 ASXL2 and ASXL3
All three ASXL genes are expressed in different tissues and have been found mutated in a variety of cancers82,83. ASXL2 mutations in AML are mainly found in t(8;21)-AML patients
and are mutual exclusive with ASXL1 mutations84. ASXL3 is expressed at rather low levels
in bone marrow cells compared to the other two genes and reports about its mutation in adult AML are rare. The three genes share up to 70% homology between their ASXN, ASXH, ASXM1, ASXM2 and PHD domains83. Sahtoe et al. discovered that the DEUBiquitinase
Adaptor (DEUBAD) domain of ASXL2/3, containing all of ASXH and more, comprises >60% sequence identity to ASXL185. They demonstrated that all three ASXL proteins could
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stimulate BAP1 activity to similar extends in E. coli bacteria. In a study conducted by Daou et al., it was found that BAP1 forms two mutually exclusive PR-DUBs with ASXL1 and -286.
Moreover, they found that loss of BAP1 destabilizes ASXL2 but not ASXL1. In murine hearts, knockout of ASXL2 was seen to disrupt PRC2-chromatin interaction due to PRC2s’ failure of trimethylating H3K27me2. Additionally, loss of ASXL2 H2Aub levels increased noticeably82.
Consequently, the three ASXL proteins appear to have similar functional mechanism but might exert them on different target regions due to tissue dependency. How and to what extent ASXL2 and 3 confer their activity in myeloid malignancies remains to be seen.
3. TP53 MUTATIONS IN HEMATOPOIETIC MALIGNANCIES
3.1 Normal and mutant functions of TP53
The normal functions of the tumor suppressor p53 have been extensively studied for more than 30 years in cell cultures and animal models. It has been widely established that this gene plays a pivotal role in many different pathways, such as differentiation, cell cycle, and apoptosis. Located on chromosome 17p13.1, the encoded p53 protein has a half-life of 5-20 minutes, which is tightly regulated via a negative feedback loop involving the N-terminus87,88.
P53 induces MDM2 transcription, which in turn binds to the N-terminus of the p53 protein and marks it for degradation89. Additionally, the N-terminal is necessary for transcriptional
transactivation and repression, and the induction of apoptosis90. The C-terminal region
contains an oligomerization and a DNA damage recognition domain, which is necessary for conformational changes and transactivation of p5388. The central region of the gene contains
the DNA binding domain that can distinguish target sequences for binding88. A wide amount
of genes are under the control of p53, such as autophagy genes (ATG7, FOXO3), DNA repair genes (DDB2, MLH1), and senescence genes (CDKN1A, PML)91. Stressors such as DNA
damaging agents may transiently stabilize and therefore lead to accumulation of the p53 protein, resulting in increased transcriptional activation of downstream targets92. Mutant p53
protein may accumulate in mutant cells via several pathways. Accumulation may occur due to the inability to bind MDM2, due to failed induction of transcription of MDM2, or due to differences in folding and degradation, thereby keeping mutant p53 constitutively stabilized and expressed at augmented protein levels87,93.
Half of all cancers have a deregulation of p53, with many tumors revealing an accumulation of mutant protein94,95. Many p53 mutants are unable to activate the same targets as wild-type
p53 and therefore lose the p53 tumor suppressor function. Additionally, these mutations may confer a dominant-negative effect over the remaining wild-type p53, abolishing wild-type functions over time96. Increased presence of p53 mutant protein can lead to the
resistance97. Furthermore, mutant proteins that lost their apoptotic functions can interrupt
p53-independent apoptotic pathways to enhance cell survival and diminish apoptosis98.
Taken together, this may lead to tumor initiation and progression.
3.2 TP53 mutations in AML and MDS
Knowledge of the role of TP53 mutations in leukemia and its involvement in leukemia onset and progression is scarce. Overall, only five to ten per cent of hematopoietic malignancies have TP53 dysfunctions, while up to 50-70% are found in solid tumors (for example in ovarian cancer and head and neck cancer)99,100. Rücker et al. conducted a study into complex
karyotype AML (CK-AML), a subgroup of AML101. They demonstrated that TP53 alterations
were the single most important prognostic factor for CK-AML with 70% of the cases having a TP53 deletion and/or mutation. Additionally, Wong et al. recently established the importance of TP53 in therapy related AML/MDS18. By sequencing the genomes of patients
and establishing mouse models with TP53-mutated HSPCs, they uncovered the preferential expansion these clones after chemotherapy. This was recently confirmed in patients who had obtained an autologous stem cell transplantation and were followed over time to determine the evolution of preleukemic clones16. Present data suggests that a TP53 clone may undergo
clonal evolution due to the selective pressure of cytotoxic therapy16,18,102. Generally, AML and
MDS patients presented with TP53 alterations predict for chemotherapy resistance101,103,104.
The median survival among these patients can be as low as four month in comparison to 11 month with TP53 unaltered function101. As conventional chemotherapy does not increase
patient survival, new treatment strategies are currently being exploited. The most prominent success so far has been achieved by the use of hypomethylating agents, such as Decitabine and Azacytidine. Both drugs may lead to favorable clinical responses and to (although incomplete) mutation clearance105,106. However, different types of TP53 mutation may require different
treatment strategies.
TP53 mutations in advanced MDS and sAML have been discovered to be mainly hemizygous or homozygous due to a copy number-neutral loss of heterozygosity (CN-LOH) or deletion of 17p107. Patients with loss of heterozygosity (LOH) of 17p and other forms of TP53
mutations are generally presented with complex karyotypes101,105,108. P53 can present with
many co-mutations, such as DNA methylators (DNMT3A, TET2), spliceosome machinery (U2AF1, SRSF2), transcription factors (ETV6, CEBPA), fusion and gene rearrangements (MLL and EVI1 translocations) and more 57,105,109–111.
In myeloid malignancies, most TP53 mutations are missense substitutions and are located within the DNA binding domain. These mutations can exert an oncogenic gain-of-function (GOF), presumably altering the binding profile of mutant p53 versus the wild-type but still allowing for full-length expression of the protein112. In a panel of breast cancer cell lines, Zhu
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et al. discovered that GOF p53 appears to be conveyed via post-translational modification of histones113. Specifically the MLL family of DNA methyltransferases appeared to be under
tight control of GOF p53. It is therefore not surprising that the DNA hypomethylating agent Decitabine revealed a favorable clinical response among AML and MDS patients with TP53 mutations105. A study into several, non-hematopoietic cell lines revealed the
interaction of GOF p53 and the proteasome machinery114. In concert with NRF2, p53
missense substitutions led to the resistance against the proteasome inhibitor carfilzomib. The resistance was overcome by combining carfilzomib with APR-246, a small molecule that restores TP53 wild-type conformation115. Post-translational changes of mutants may bestow
additional treatment options. Activity of wild-type and mutant p53 can be influenced by acetylation and phosphorylation, with the latter shielding the proteins from degradation via MDM2116,117. In triple negative breast cancers, Wang et al. demonstrated that certain histone
deacetylase inhibitors (HDACIs) could decrease phosphorylation, protein, and mRNA levels of mutant p53 in vitro118. Usage of the HDACI SAHA destabilized mutant p53 but not
wild-type p53 levels, killing tumour cell lines in vitro119. A phase 1 study in AML and MDS
patient with SAHA showed first promising results with some patients achieving complete response120. Another encouraging approach is the selective degradation of mutant p53 via
the mevalonate pathway. Parrales et al. demonstrated that statins could reduce mevalonate-5-phosphate, thereby leading to misfolding of mutant proteins by disrupting the binding of the heat-shock protein DNAJA1 to the mutant p53 protein121. The mutant proteins are then
marked for degradation. Using statins based on mutant p53 status might provide additional treatment options.
Unraveling the differences in downstream targets between wild-type and mutant p53 may also play a pivotal role in overcoming negative effects of mutant p53. Some mutants, but not wild-type p53, have been found to enhance NF-ĸB activation thus promoting cancer progression via a chronic inflammatory pathway122. Moreover, mutant p53 proteins may bind
and inactivate the two p53 family member p63 and p73123,124. Mouse models demonstrated
that, when wild-type p53 function is abrogated, p63 and p73 could partially compensate125.
However, their binding by mutant p53 disrupts their tumor suppressive functions. If this p53-p63-p73 axis is targetable remains to be elucidated. More studies into p53 mutants and their role in hematopoietic malignancies are currently conducted and may pave the way for more efficient (and novel) therapeutic agents.
4. AUTOPHAGY
Autophagy is a process used by cells to degrade and recycle cellular substrates. Three different types of autophagy exist, with macroautophagy being the most intensely studied so far. Eukaryotic cells have a consistent basal level of macroautophagy that can be enhanced under stress conditions. Macroautophagy takes place in several steps. At first, the ULK1/2 complex induces the nucleation of a phagophore126–128. Then, with the help of complexes
and proteins such as LC3-II and ATG9, the phagophore with a double membrane becomes elongated and ultimately matures enough to close around its cargo, which is then known as autophagosome128–130. Thereafter, the outer membrane of the autophagosome fuses with an
endosome and/or lysosome to form an autolysosome. The content within the autolysosome is then degraded and transported through the permease back into the cytoplasm for re-use128,131,132.
While it is widely acknowledged that autophagy is a general housekeeping process necessary to remove misfolded proteins and damaged organelles, evidence has been emerging that autophagy is an important regulator of HSPC maintenance. In mice, Warr et al. discovered that autophagy is essential for maintaining young and old HSCs via FOXO3A induced autophagic gene program133. Gomez-Puerto et al. found that CD34+/CD38- immature HSPCs
appear to have a higher autophagic flux than the more progenitor-like CD34+/CD38+ cells,
as well as differentiated erythroid and myeloid cells134. Upon knockdown of the autophagy
genes ATG5 and ATG7, which are required for elongation and closure of the phagophore double membrane, reduced HSPC frequencies could be observed in vitro as well as in vivo134– 136. Additionally, Mortensen et al. discovered that ATG7-deficient Lin-Sca-1+ c-Kit+ (LSK)
cells were not able to reconstitute the hematopoietic system in lethally irradiated mice137.
Moreover, production of myeloid and lymphoid progenitors was impaired when ATG7 was absent. These findings collectively suggest that autophagy is an important player in regulating HSPC/HSC maintenance. Increasing evidence also points towards autophagy being involved in cancer development and therapy resistance in cancer treatment. Folkerts et al. discovered that leukemic cell lines and primary human CD34+ AML cells have a large variability in
basal autophagic flux (Chapter 3, this thesis)138. Especially poor-risk AMLs, which are
frequently associated with mutations in TP53, have higher autophagic levels than favorable- or intermediate risk AMLs. Knockdown of the autophagy genes ATG5 or ATG7 initiated apoptosis and coincided with increased expression of p53138.
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5. SCOPE OF THE THESIS
The aim of the first part of this thesis was to investigate the role of ASXL1 in HSPCs and whether malignant transformation can occur upon combined knockdown of ASXL1 with TP53. Mouse studies into ASXL1 functions suggested that a complete loss of ASXL1 leads to an MDS-like phenotype accompanied with pronounced anemia, a clinical manifestation often observed in MDS and AML patients66,67. However, studies into the function of
ASXL1 and its abrogation in human cells are missing. In Chapter 2 of this thesis, we aimed
to elucidate the role of ASXL1 during human stem and progenitor development in vitro and investigated its effect particularly in erythroid development. It has also been shown that ASXL1 is co-mutated with different transcription factors in MDS and AML patients. Following the detrimental effects of ASXL1 loss, we investigated in Chapter 3 if additional
loss of the transcription factor TP53 could rescue ASXL1 mutated cells and lead to malignant transformation in vitro and in vivo.
Autophagy can be used as a survival mechanism in times of energy crisis or other forms of stress. In treatment of cancer, autophagy has been found to play a pivotal role in therapy resistance. The aim of the second part of this thesis was to highlight the present understanding of the autophagy pathway in cancer, its role in transformation, disease progression and therapy resistance (Chapter 5) and how targeting autophagy might provide a new treatment
strategy in AMLs (Chapter 4). The aim of Chapter 4 was to determine if autophagy
inhibition could be used as a treatment strategy for patients with AML and which underlying genes and pathways are involved. In Chapter 5, the goal was to review the multi-faceted
role of autophagy in regulating cancer initiation and cancer (stem) cell maintenance, how autophagic flux in cancer affects therapeutic outcome, and the role of autophagy in the tumor microenvironment. Moreover, the aim was to give perspectives on selective targeting of cancer (stem) cells for therapy.
Finally, in Chapter 6, a summary of the results and a discussion of future perspectives are
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