Genetic defects in myeloid malignancies and preleukemic conditions Berger, Gerbrig

Genetic defects in myeloid malignancies and preleukemic conditions Berger, Gerbrig

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Berger, G. (2019). Genetic defects in myeloid malignancies and preleukemic conditions. Rijksuniversiteit Groningen.

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and preleukemic conditions

Printing of this thesis was financially supported by University of Groningen, UMCG Graduate School of Medical Sciences, Stichting tot bevordering der haematologie Groningen, MPN stichting and Onderzoeksfonds Hematon.

ISBN: 9789403416298

Cover and lay-out design: G. Berger

Printing: Ipskamp Drukkers BV, Enschede Copyright © 2019, Gerbrig Berger

All rights reserved. No part of this thesis may be reproduced, stored or transmitted in any form or by

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and preleukemic conditions

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de

rector magnificus Prof. Dr. E. Sterken en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op woensdag 22 mei 2019 om 16.15

door

Gerbrig Berger geboren op 5 juni 1989

te Heerenveen

Copromotor Dr. H. Schepers

Beoordelingscommissie Prof. Dr. G. de Haan

Prof. Dr. M.H.G.P. Raaijmakers

Prof. Dr. M.A.T.M. van Vugt

Aida Rodríguez-López

Chapter 1 General introduction p.9

Chapter 2 Early detection and evolution of preleukemic clones in therapy-related myeloid neoplasms following autologous SCT (Blood, 2018)

p.27

Chapter 3 Ringsideroblasts in acute myeloid leukemia are associated with adverse risk and result from an aberrant heme- metabolism gene program

(Submitted)

p.65

Chapter 4 Overexpression of TP53 is associated with poor survival, but not with reduced response to hypomethylating agents in older patients with acute myeloid leukaemia

(British Journal of Haematology, 2016)

p.95

Chapter 5 Hereditary hematological malignancies presenting in adulthood

(Leukemia, 2017 and British Journal of Haematology, 2018)

p.103

Chapter 6 CITED2-mediated human hematopoietic stem cell maintenance is critical for acute myeloid leukemia (Leukemia, 2015)

p.115

Chapter 7 CITED2 affects leukemic cell survival by interfering with p53 activation

(Cell, Death and Disease, 2018)

p.143

Chapter 8 Summary, discussion and future perspectives p.163

Appendices

Nederlandse samenvatting Dankwoord

Over de auteur Publicaties

p.181

              

              

General introduction 1.

1

The hematopoietic system Normal hematopoiesis and hematopoietic stem cells

The adult bone marrow is capable of the formation of all types of blood cells that harbor exclusive and distinct functions: platelets are involved in blood coagulation, erythrocytes in the transport of oxygen and white blood cells in immune function. Hematopoietic stem cells (HSCs) drive the process of hematopoiesis that results in the production of billions of cells per day. HSCs exhibit classical stem cell properties: they are able to self-renew in order to maintain a stem cell pool that can provide lifelong new blood cell formation and are pluripotent cells that have the capacity to differentiate into all types of mature blood cells. An estimated number of 11.000-22.000 functional HSCs is present in each individual, and this number decreases with age.

Hematopoiesis is regarded as a multi-step process involving different stages of multipotent progenitor cells that undergo expansion and differentiation and ultimately results in terminally differentiated mature blood cells. Cell fate determination is under control of transcription factors and epigenetic changes within the HSCs and progenitor populations. In the classical model, HSCs are at the apex of the hematopoietic hierarchy and lineage restriction towards the myeloid and lymphoid progenitors occurs at an early stage in the process ( Figure 1 ). However, recent findings have challenged this perspective and suggest a more versatile model in which different progenitor stages can also drive steady state hematopoiesis.

The bone marrow micro-environment Hematopoiesis is a tightly regulated process, for which the localization of HSCs in the hypoxic bone marrow micro-environment is essential.

The bone marrow microenvironment provides a protective niche consisting of different types of hematopoietic cells as well as non-hematopoietic components, including osteolineage cells, sinusoidal endothelial cells, mesenchymal stromal and stem cells, sympathetic neurons, and the extracellular matrix.

Communication takes place via excretion of cytokines and growth factors and direct cell-cell-contacts.

For a long time, the bone marrow niche has been considered to be only supportive for hematopoiesis. However, in recent years it has become clear that the functioning of supportive components within the bone marrow microenvironment can alter when subjected to inflammation, ageing or cytotoxic damage

and that a dysfunctional or damaged niche can contribute to hematopoietic disease progression.

Hematopoietic stem cells as a source for stem cell therapy

Because of their self-renewal and pluripotent capacities, HSCs can be exploited for use in hematopoietic stem cell transplantation (SCT). Currently, hematopoietic SCT is the only stem cell therapy that is widely and successfully used in a clinical setting. Hematopoietic SCT is used in the treatment of a variety of hematologic diseases, other malignant conditions as well as auto- immune diseases.

Historically, HSCs were harvested from the bone marrow by repeatedly bone marrow aspiration.

However, nowadays, the peripheral blood is used as source for HSCs.

Normally, the HSCs cells reside in the

protected environment of the bone

marrow, but release into the peripheral

blood can be artificially initiated by

the administration of a variety of

agents, including cytokines and certain

cytostatic agents.

The application of

mobilized peripheral blood stem cells

(PBSCs) has been proven as a safe and

effective alternative for bone marrow as

1

a source of transplantation.

Hematopoietic stem cell transplantation

The transplanted cells can be either autologous or allogeneic; the latter is a potentially curative therapy for a variety of malignant and non-malignant hematological disorders in which a graft-versus-tumor effect mediated by donor T-cells is warranted. The outcomes of allogeneic hematopoietic SCTs have substantially improved over the past decades; an increasing proportion of recipients reach long- term survival. However, this increased life expectancy is associated with increased risk of development of late complications, including chronic graft-versus-host disease, development of solid malignancies and post- transplantation lymphoproliferative disorders.

Autologous SCT is most frequently applied in patients lymphoma, multiple myeloma and amyloidosis wherein autologous stem cell re-infusion following myelo- ablative regimens, including high

dose melphalan or BEAM combination therapy, is applied to prevent long-term hypoplasia. The transplant-related morbidity and mortality of autologous SCT is considerably lower as compared to allogeneic SCT.

Preleukemic stages of the bone marrow

Clonal hematopoiesis

Malignant transformation of healthy cells is a multistep-process that requires the acquisition of multiple genetic adaptations that are advantageous to cell proliferation and cell survival

Recently it was reported that low- frequent genetic mutations in certain genes are highly enriched in the bone marrow of otherwise healthy adults.

This phenomenon is related to ageing:

the incidence in people over the age of 65 is up to 10%, while over 20% of patients older than 90 years are affected.

Interestingly, these genes highly overlap with genes that are frequently mutated in myeloid neoplasms. The majority of the detected mutations were observed

HSC

CMP

GMP

MEP

macrophages

granulocytes

erythrocytes platelets MPP

LMPP

CLP

MkEP

dendritic cells T cells

B cells

NK cells

Figure 1. Schematic representation of the hematopoietic hierarchy

Abbreviations: CMP, common myeloid progenitor; CLP, common lymphoid progenitor; HSC, hematopoietic stem cell, MPP, multipotent progenitor, LMPP, lymphoid-primed multipotent progenitor; MkEP, early megakaryocyte erythrocyte progenitor;

MEP, megakaryocyte-erythrocyte progenitor; GMP, granulocyte-macrophage progenitor

1

leukemia. Such syndromes might be accompanied by organ dysfunction or platelet disorders, however prodromal symptoms may also be completely absent ( Table 1 ).

Several of these predisposing genes are also found to be recurrently mutated in sporadic AML, including RUNX1, GATA2, and CEBPA.

The pathogenesis of AML and MDS in the context of the different germline predisposition syndromes is poorly understood. A wide variability in both penetrance and latency is observed between and within different HHM syndromes, suggesting that additional hits are required for leukemic transformation in asymptomatic bone marrow cells.

Myeloid neoplasms Myelodysplastic syndromes

Genetic mutations, chromosomal abnormalities and epigenetic modifications in early hematopoietic cells can result in failure of faithful proliferation and differentiation.

Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal disorders that are characterized by morphological dysplasia and ineffective hematopoiesis that result in one or more blood cytopenias and risk of transformation to acute myeloid leukemia (AML).

Due to increased proliferation and incomplete differentiation of early hematopoietic cells, immature blasts may be present in the bone marrow up to 20 percent. MDS is a disease that mainly occurs in elderly people, with median age at diagnosis is 71-76 years

, and less than 10 percent of the MDS patients being younger than 50 years

. Based on specific morphological features, affected cell lineages and cytogenetic aberrations, MDS is classified into different subtypes ( Table 1 ).

Dysplasia may coexist with peripheral blood monocytosis, which is a characteristic for myeloproliferative in the epigenetic modifiers DNMT3A,

TET2 and ASXL1, which are also recurrently mutated in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) ( Figure 2 ).

The presence of such leukemia-associated mutations in the absence of clear cytopenias and bone marrow dysplasia is referred to as clonal hematopoiesis of indeterminate potential (CHIP).

The presence of CHIP has been associated with increased risk of hematological cancers and increased mortality.

However the yearly risk for development of AML/MDS in CHIP carriers is only approximately 0.5-1 percent

, indicating that the presence of single mutations in these genes is insufficient to cause malignant transformation.

Instead, mutations observed in CHIP might be regarded as initiating event that primes the cells towards malignant transformation, but additional hits are required.

The paucity of other recurrent mutations in AML, including FLT3, NPM1 and IDH1, in CHIP supports the idea that these mutations are later events and are important for actual malignant transformation.

Germline predisposition

Predisposing germline mutations are implicated in 6-8 percent of the childhood cancers.

Although being considered as very rare, germline gene mutations can also predispose for development of myeloid malignancies.

(36)

Various inherited bone marrow failure syndromes, such as Fanconi anemia, Swachman diamond syndrome, and Diamond-Blackfan anemia, are well known to increase the risk for MDS/

AML development. In these conditions,

AML or MDS is usually diagnosed during

childhood or early adulthood.

Since

the introduction of next-generation

sequencing techniques, several other

single-gene hereditary hematological

malignancy (HHM) syndromes have

been identified to increase the risk for

1

Myeloproliferative neoplasms Acute myeloid leukemia and related neoplasms Chronic myeloid leukemia, BCR-ABL1 AML with recurrent genetic abnormalities

Chronic neutrophilic leukemia AML with t(8;21)(q22;q22.1)

Polycythemia vera AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22)

Primary myelofibrosis APL with PML-RARA

PMF, prefibrotic/early stage AML with t(9;11)(p21.3;q23.3) PMF, overt fibrotic stage AML with t(6;9)(p23;q34.1)

Essential thrombocythemia AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2) Chronic eosinophilic leukemia, not otherwise specified AML with t(1;22)(p13.3;q13.3)

MPN, unclassifiable Provisional entity: AML with BCR-ABL1

Mastocytosis AML with mutated NPM1

AML with biallelic mutations of CEBPA Myelodysplastic/myeloproliferative neoplasms Provisional entity: AML with mutated RUNX1 Chronic myelomonocytic leukemia AML with myelodysplasia-related changes Atypical chronic myeloid leukemia, BCR-ABL1 Therapy-related myeloid neoplasms Juvenile myelomonocytic leukemia AML, not otherwise specified

MDS/MPN with ring sideroblasts and thrombocytosis Pure erythroid leukemia MDS/MPN, unclassifiable Acute megakaryoblastic leukemia

AML with minimal differentiation Myelodysplastic syndromes AML without maturation MDS with single lineage dysplasia AML with maturation MDS with ring sideroblasts Acute myelomonocytic leukemia MDS-RS and single lineage dysplasia Acute monoblastic/monocytic leukemia MDS-RS and multilineage dysplasia Acute basophilic leukemia MDS with multilineage dysplasia Acute panmyelosis with myelofibrosis

MDS with excess blasts Myeloid sarcoma

MDS with isolated del(5q) Myeloid proliferations related to Down syndrome

MDS, unclassifiable Transient abnormal myelopoiesis

Provisional entity: Refractory cytopenia of childhood Myeloid leukemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasm Myeloid neoplasms with germ line predisposition

Myeloid neoplasms with germ line predisposition without a Acute leukemias of ambiguous lineage preexisting disorder or organ dysfunction Acute undifferentiated leukemia

AML with germ line CEBPA mutation Mixed phenotype acute leukemia with t(9;22)(q34.1;q11.2) Myeloid neoplasms with germ line DDX41 mutation Mixed phenotype acute leukemia with t(v;11q23.3) Myeloid neoplasms with germ line predisposition and Mixed phenotype acute leukemia, B/myeloid, NOS preexisting platelet disorders Mixed phenotype acute leukemia, T/myeloid, NOS Myeloid neoplasms with germ line RUNX1 mutation

Myeloid neoplasms with germ line ANKRD26 mutation Myeloid neoplasms with germ line ETV6 mutation

Myeloid neoplasms with germ line predisposition and other organ dysfunction

Myeloid neoplasms with germ line GATA2 mutation Myeloid neoplasms associated with BM failure syndromes Myeloid neoplasms associated with telomere biology disorders

JMML associated with neurofibromatosis, Noonan syndrome or Noonan syndrome-like disorders

Myeloid neoplasms associated with Down syndrome

Table 1: WHO classification myeloid neoplasms

1

100.000 in persons younger than 65 years old and increases to 12.2 cases per 100.000 persons in those older than 65.

(53)

Clinically, biologically and genetically, AML comprises a heterogeneous group of clonal malignancies. Ontogenetically, AML can be divided into three groups that are defined by the antecedent MPN or MDS phase (secondary AML), previous cytotoxic exposure (therapy-related AML) or neither of them (de novo AML).

Different disease characteristics that have been proven to be relevant for treatment and outcome are implemented in the WHO classification, but this is irrespective of leukemia ontogeny ( Table 1 ). In contrast to MDS and MPN, the risk stratification is exclusively based on cytogenetic and molecular profiles.

Therapy-related myeloid neoplasms Therapy-related myeloid neoplasms (t-MNs) comprise either acute myeloid leukemia or myelodysplastic syndromes that develop as late complication following cytotoxic chemotherapy and/

or radiotherapy.

The incidence of t-MN varies depending on therapy- intensity and the administered agents. In particular prior exposure to alkylating agents or topoisomerase II inhibitors is associated to increased risk for t-MN development.

Currently approximately 7% of all newly diagnosed AML cases are therapy-related; however the incidence of t-MN is expected to rise due to increasing numbers of cancer survivors.

(55)

A t-MN diagnosis is associated with inferior outcomes compared with de novo AML.

Patient-related factors that might contribute to poor prognosis are older age at diagnosis, decreased bone marrow reserves as resultant of prior treatment, or patients may have persistent disease or relapse of their primary malignancy. Also, disease- related factors, including the frequent occurrence of poor risk (cyto)genetic abnormalities and TP53 mutations neoplasms (MPNs).

MPNs comprise

a heterogeneous group of bone marrow disorders characterized by excessive proliferation of hematopoietic precursor cells that result in overproduction of mature blood cells and/or bone marrow fibrosis.

The World Health Organization (WHO) classification distinguishes several overlapping groups, which carry features of both MPN and MDS.

Currently, individual genomic mutations are not considered in the WHO classification for MDS, however this may change in future, as prognostic significance has been assigned for individual mutations.

(46,47)

With respect to therapy selection,

a distinction is established between lower-risk (LR) and higher-risk (HR) MDS patients. LR-MDS patients, have a lower risk for AML progression (0-8%) and hence a superior overall survival. Treatment in LR-MDS patients is focused on improvement of quality of life and usually supportive in nature and includes administration of hematopoietic growth factors and transfusions.

When unresponsive to this treatment, patients might be considered for allogenic SCT. In contrast, HR-MDS patients are at a high risk to progress towards AML (9- 40%) and treatment aims to alter the natural course of the disease to achieve prolonged survival.

Acute myeloid leukemia

In the case of acute myeloid leukemia

(AML), (epi)genetic events in early

hematopoietic cells result in a

misbalance in proliferation and a block in

differentiation causing an accumulation

of immature blasts, often accompanied

by hematopoietic insufficiency. The

diagnosis AML is defined by a blast

percentage in the bone marrow equal

to or above 20 percent or the presence

of certain AML defining karyotypes.

The incidence of AML increases with

age; the rate varies from ~1.3 cases per

1

more controversial, as this feature does not have independent prognostic significance.

Donor cell leukemia

The development of leukemia in donor cells following allogeneic SCT was first described in 1971 and is considered as a rare phenomenon; the incidence is estimated to be 1.2 in 1000 transplants.

(67)

The pathogenesis of donor cell leukemia (DCL) has not been fully elucidated: according to the multiple hit theory of oncogenesis, donor cells may contain a first hit and the transplantation and adaptation to the new micro environment of the recipient allow the acquisition of additional hits, finally resulting in leukemia development. This first hit may be somatically or acquired mutations that are present at low percentages in the donor

or a germline mutation

. Post-transplantation recipient factors, such as altered immune surveillance, replication stress and accelerated telomere shortening have been implicated in the disease pathogenesis.

(73)

However, such a first hit is not always detectable in the donor cells, suggesting a direct effect of the treatment and transplantation procedure in disease pathogenesis.

The steady increase that is observed in DCL incidence might be attributed to more sensitive testing of post-transplantation chimerism levels

(73)

, however also the more frequent use of older donors might contribute to this increase

.

Genetic defects in myeloid neoplasms

As in other malignant diseases, genomic instability is one of the hallmarks of cancer in myeloid neoplasms.

A diverse spectrum of genomic mutations and chromosomal aberrancies has been identified in myeloid neoplasms and the combinations of different genetic defects account for the inferior outcome.

(56,58-60)

With an incidence of 5-15

percent, t-MN is a relatively frequent complication following autologous SCT.

(61)

The risk for t-MN development is higher and the latency time is shorter following autologous SCT as compared to conventional chemotherapy.

In contrast to autologous SCT, t-MN development following allogeneic SCT is extremely rare suggesting a role for the pretreatment that the transplanted autologous HSCs have undergone.

Besides therapies given prior to SCT, transplantation-conditioning regimens, age at transplantation and factors related to stem cell harvesting have been identified to increase the risk for t-MN development following autologous SCT.

(62,63)

Secondary acute myeloid leukemia When AML occurs following antecedent myeloid disease, including myelodysplastic syndrome (MDS) or myeloproliferative neoplasia (MPN), the disease is defined as secondary AML (sAML).

Equally to t-MNs, patients with secondary AML, especially non- MDS-sAML, have dismal outcomes as compared to de novo AML patients.

(64)

In the WHO classification, sAML

is classified within the group of ‘acute

myeloid leukemia with myelodysplasia-

related changes’ (AML-MRC). Besides

AML occurrence following antecedent

myeloid disorders, also de novo AML with

MDS-related cytogenetic abnormalities

and/or multilineage dysplasia upon

bone marrow examination are

considered as AML-MRC.

Supportive

for the inclusion of AML with MDS-

related cytogenetic abnormalities is

the genetically resemblance between

a subset of de novo AML patients and

sAML, indicating that apparent de

novo AML might be preceded by an

unrecognized MDS prodrome.

The

inclusion of multilineage dysplasia

as single criterion for AML-MRC is

1

DNMT3A EZH2 BCOR IDH2 IDH1 BCORL1

STAG2 CTCF SMC3 SMC1A

SF3B1 SRSF2 U2AF1 ZRSR2 ATRX

RAD21

LUC7L2 U2AF2 PRPF8 SF1 RUNX1 CUX1 ETV6 NPM1 GATA2 WT1 CEBPA TP53 JAK2 NF1 NRAS CBL MPL KRAS FLT3 GNAS KIT PTPN11 GNB1

Frequency

in CHIP Frequency

in MDS Frequency

in sAML Frequency in AML Gene

Category Epigenetic

Cohesins

Splicing

Transcription factors

p53 Signaling

Frequency

High Low

No mutations found

Figure 2 Recurrent mutations in CHIP, MDS and AML

Mutations are categorized into different functional groups. Frequencies are based on percentages for MDS, sAML and AML and on absolute mutation counts for clonal hematopoiesis of indeterminate potential (CHIP). Abbreviations: ASXL1, additional sex combs-like 1; BCOR, BCL6 co-repressor; BCORL1, BCL6 co-repressor-like 1; CEBPA, CCAAT/enhancer binding protein alpha; CTCF, CCCTC-binding factor; CUX1, cut-like homeobox 1; DNMT3A, DNA methyltransferase 3A; ETV6, ETS variant 6; EZH2, enhancer of zeste 2; FLT3, fms related tyrosine kinase 3; GATA2, GATA binding protein 2; GNB1, G protein subunit beta1; IDH1, isocitrate dehydrogenase 1; JAK2, Janus kinase 2; LUC7L2, LUC7 like 2, pre-mRNA splicing factor; MPL, gene that encodes the thrombopoietin receptor; NF1, nuclear factor 1; NPM1, nucleophosmin; PRPF8, pre-mRNA processing factor 8;

PTPN11, protein tyrosine phosphatase, non-receptor type 11; RUNX1, runt related transcription factor 1; SF1, splicing factor

1; SF3B1, splicing factor 3b subunit 1; SMC1A, structural maintenance of chromosomes 1A; SRSF2, serine and arginine rich

splicing factor 2; STAG2, stromal antigen 2; TET2, tet methylcytosine dioxygenase2; U2AF1, U2 small nuclear RNA auxiliary

factor 1; WT1, Wilms tumour 1; ZRSR2, zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2. Figure adapted

from Sperling, Nature reviews oncology, 2017 .

1

signaling, chromatin-modifying genes, myeloid transcriptional regulating genes, cohesion-complex genes and spliceosome-complex genes ( Figure

2 ).

On average, each AML patient

harbors 10-13 mutations in the coding genomic regions, including 3-5 that affect recurrently mutated genes.

Frequently, patients have mutations in several genes belonging to different functional classes. Among several of the genes and categories, specific patterns of cooperation and mutual exclusivity can be determined.

For many gene mutations, the exact contribution to leukemic development individually and in the context of cooperating mutations is poorly understood. The presence of clonal genomic diversity within individual patients adds on to the complexity of this matter.

Genetic signatures define different entities

Although mutational landscapes of myeloid neoplasm show quite some overlap, also distinct genetic signatures can be determined. Mutations in genes involved in JAK-STAT signaling, including JAK2, CALR and MPL, are highly specific for MPNs.

In MDS the most frequently mutated genes belong mainly to the group of epigenetic modifying genes and members of the spliceosome, including TET2, SF3B1, ASXL1, SRSF2, DNMT3A, RUNX1, ZRSR2, STAG2, U2AF1 and TP53.

In AML, the mutational spectrum is more diverse and the ten most recurrent genes with defects include FLT3, NPM1, DNMT3A, CEBPA, IDH1, IDH2, TET2, NRAS, PML/RARA and AML1/ETO.

Within AML a distinction in ontogenic subtype can be based on the genetic defect, with mutations in NPM1, FLT3, IDH1, N/

KRAS, RUNX1, CEBPA, WT1, PTPN11 and c-KIT being more often observed in de novo AML.

In contrast, mutations in SRSF2, SF3B1, U2AF1, ZRSR2, underlie disease heterogeneity and

account for clinical variability within the different types of disease.

Especially in recent years, major progress has been made in the determination of genetic defects underlying myeloid neoplasms.

The implementation of novel techniques in routine diagnostics, such as next- generation sequencing and single nucleotide polymorphism (snp)-arrays, has enabled further discrimination between myeloid neoplasm subtypes.

Although being classified as distinct entities based on clinical and biological features, genetically a considerable degree of overlap has been shown between the different myeloid neoplastic entities .

Therefore, disease classifications are subject to change and warrant regular revision.

Recurrent genetic defects in myeloid neoplasms

Chromosomal abnormalities have been recognized in malignant hematopoiesis for a long time. Defects can be either numerical, resulting in aneuploidy, or structural including deletions, duplications, insertions, uniparental disomy, inversions or translocations.

(77)

The latter two groups results in

fusion proteins or repositioning of

promotors or enhancers that induce

abnormal gene expression and are

almost exclusively present in AML

patients.

Other frequently observed

chromosomal abnormalities include

autosomal monosomies and complex

karyotypes (defined as three or more

cytogenetic abnormalities), which are

both associated with inferior outcomes

in AML, MDS as well as primary

myelofibrosis.

Besides chromosomal

defects, multiple genomic mutations

have been identified to play a role in

malignant myelopoiesis. These mutated

genes can be subdivided into several

functional classes, including tumor

suppressor genes, DNA-methylation

related genes, genes involved in

1

impact cancer progression.

Loss of TP53 via deletion of its chromosomal locus, 17p, is also observed in AML.

However, the result of this deletion is even more detrimental, suggesting that deletions of tumor suppressor genes around TP53 play are of significance.

Also, the combination of 17p deletion with a TP53 mutations results in a more unfavorable prognosis than only TP53 mutations.

Together TP53 mutations and deletions are recognized as distinct disease entity, defined by aggressive disease, chemo resistance and dismal treatment outcomes.

SF3B1 mutations in myeloid neoplasms

The gene encoding splicing factor 3b subunit1, SF3B1, is one of the most recurrently mutated genes in MDS.

(99-103)

Presence of SF3B1 mutations

is usually observed in low-risk MDS, characterized by a stable clinical course and low risk of transformation to AML.

(99,104)

In contrast to MDS, mutations

in SF3B1 are infrequent in AML.

(76,101,105)

Together with other members

of the spliceosome-complex, SF3B1 is essential for faithful pre-RNA splicing.

(106)

Mutations in genes involved in

the spliceosome-complex give rise to specific patterns of missplicing, thereby affecting gene expression.

The presence of mutated SF3B1 has a positive predictive value of 98 percent for the specific MDS phenotype with ringsideroblasts.

Ringsideroblasts are erythroid precursor cells that contain abnormal accumulation of iron in their mitochondria that form a circle around the nucleus. The strong correlation between ringsideroblast phenotype and the presence of SF3B1 mutations is also present in MDS/MPN variants.

The presence of SF3B1 mutations is mutually exclusive with mutations in other spliceosomal genes, which suggests that these different mutations have similar roles in MDS ASXL1, EZH2, BCOR and STAG2 are

determined to be highly specific for sAML as compared to de novo disease.

(54)

The overlap between these so called

‘secondary-type’ mutations in sAML and recurrently mutated genes in MDS reflects the ontogenetic background of sAML.

Specific for t-MN are frequent mutations in TP53, but also

‘de novo-type’ and ‘secondary-type’

mutations are observed in this group.

TP53 mutations in myeloid neoplasms

The tumor suppressor protein p53 is

a transcription factor that has many

important functions in hematopoiesis,

including preservation of genomic

integrity and maintenance of quiescence

and self-renewal in HSCs.

TP53,

the gene encoding p53 protein is the

most frequently mutated genes cancer

wide, with 42 percent of all cancers

being affected.

In contrast to solid

tumors, TP53 mutations in AML are

relatively rare; around 8 percent of de

novo AML cases do contain mutations in

TP53.

The rate of TP53 mutations

is more frequent in high risk groups,

including elderly de novo AML patients,

sAML following myeloproliferative

disorders and therapy-related MNs

(21-33 percent).

AML with TP53

mutations are characterized by

complex cytogenetic defects, including

monosomies, and a paucity of other

leukemic driver mutations.

TP53

mutations are most often heterozygous

point mutations,

however inactivation

of the second allele is frequently

induced by loss-of heterozygosity.

More than 80 percent of the TP53

defects are missense mutations that

give rise to a stable full-length p53

protein

and immunohistochemistry

can be used for the detection of protein

accumulation in the nucleus.

Missense mutations can result in loss of

function, resulting in loss of wild type

p53 functions, but can also introduce

gain of function activities that can

1

successful.

Hypomethylating agents

Relatively novel agents in treatment of myeloid malignancies are azacitidine and decitabine, which are both cytosine analogs that can be incorporated into the DNA during replication. The irreversible binding of DNA methyltransferases to these cytosine analogs results in depletion of these epigenetic modifying enzymes, leading to a genome wide decrease in methylation and subsequent reversal of gene silencing

, hence the name hypomethylating agents (HMAs). Additionally, HMAs can induce DNA damage similar to certain chemotherapeutic drugs.

Because of the reduced side-effects, these agents are especially of interest in the group of AML/MDS patients that are not eligible for intensive treatment. Both azacitidine and decitabine are administered in cycles and the response to HMAs tends to be slow and is short-lived; many patients require three or more cycles to achieve maximal clinical response and remissions usually last less than one year.

However, beneficial effects regarding survival using HMA’s have been shown for both high-risk MDS and AML patients.

Interestingly, this survival advantage was also observed in patients with adverse risk karyotypes, which usually show a very poor response to conventional treatment.

Currently, the potential beneficial use of decitabine as single agent for induction therapy in preparation for hematopoietic SCT is being investigated (NCT02172872).

Scope of this thesis

Despite the progress that has been made in treatment of myeloid malignancies, disease outcomes of patients are generally still very poor, mainly due to disease relapse. In recent years, research has put more and more emphasis on the pathogenesis.

However, mutations

in other spliceosomal genes have not been implicated in the RS phenotype, indicating that this specific form of dyserythropoiesis might specifically be due to altered SF3B1 function.

Current treatment modalities Conventional chemotherapy

In AML patients, treatment with intensive induction chemotherapy is administered aiming to rapidly restore normal bone marrow function.

For many years, anthracycline-based and cytarabine-based chemotherapy combinations are used for induction therapy.

With the commonly used

‘7+3‘ regimen, remission rates are 60- 80 percent in younger AML patients and 40-60 percent in AML patients that are 60 years and older.

Biologically and clinically, high-risk MDS and AML show great resemblance; as in AML, high risk MDS patients most often die from bone marrow failure, frequently without progression to AML.

These observations justify to treat high risk MDS patients similar to AML patients.

In MDS, complete remission is achieved in 55 percent of the patients following intensive induction therapy.

(112)

Following induction therapy,

post-remission consolidation therapy is generally administered aiming to reduce the risk of disease relapse following initial remission. For high- risk and intermediate-risk patients that are under the age of 75 years, patients usually proceed with allogeneic SCT.

Post remission options for patients with

low-risk AML include additional cycles

of intensive chemotherapy or high-

dose therapy followed by autologous

SCT.

One exception to conventional

AML treatment is the AML subtype

acute promyelocytic leukemia, for which

specific therapy using all-trans retinoic

acid-based therapy and arsenic trioxide

(ATO) has been proven to be highly

1

contrast to MDS-RS patients, frequently have poor risk characteristics including adverse karyotypes and an absence of SF3B1 mutations. In Chapter 3 the underlying basis of the presence of ringsideroblasts in AML patients is investigated based on DNA and RNA analyses.

Next to conventional chemotherapy, hypomethylating agents (HMA’s) are being used increasingly frequent in the treatment of AML and MDS. It was shown that patients with adverse risk karyotypes may benefit from HMAs to the same extend as patients with more favorable risk disease.

The presence of adverse risk karyotypes in patients is strongly associated with mutations in TP53, which confers a very poor response to treatment with conventional chemotherapy. In Chapter 4, the response of patients to HMA treatment is studied using p53 protein overexpression as marker for the presence of TP53 mutations.

Unlike t-MNs, most myeloid neoplasms represent sporadic cases for which no causative factors can be identified. Rarely, the disease can have an early onset and/

or occur more frequent within families, which may suggest the presence of an inherited predisposition. To date, fewer than half of the familial AML and MDS cases can be explained by known genetic factors.

However the availability of next generation sequencing techniques continues to contribute to the discovery of novel genetic associations. Chapter 5 contains two reports on families with aggregation of MDS/AML, for which whole exome sequencing was performed to identify underlying genetic defects.

To maintain normal hematopoiesis and prevent the development of leukemia, the fragile balance between self-renewal and differentiation needs to be carefully regulated, a process in which various importance of genetic defects that are

frequently identified in patients with myeloid malignancies. Gaining insight into the molecular pathogenesis of the disease may help to identify new targets for therapy and allow improved subdivision of this heterogeneous group of diseases. The first part of this thesis aims to get insight in the mutational composition and its consequences for both premalignant and malignant hematopoiesis.

Therapy-related myeloid neoplasm (t-MNs) is a relative frequent complication following treatment with autologous hematopoietic SCT.

Patients that develop t-MN following autologous SCT show impaired peripheral blood cell regeneration after the transplantation in comparison to those who do not

, indicating that the defects underlying t-MN development may already be present and detectable at an early stage. Chapter 2 aims to get insight into the mechanism of t-MN development following autologous SCT.

By using next generation sequencing techniques, the mutational landscape of t-MN patients is studied. Furthermore, the determination of the mutational burden in graft material and consecutive pre-leukemic bone marrow and/or peripheral blood samples provides insight into the clonal dynamics preceding leukemic transformation.

The classification of myeloid neoplasms is based on both genetic and morphological disease characteristics.

A highly specific disease-identifying morphological aberrancy in MDS is the presence of ringsideroblasts (MDS- RS).

Mutations in spliceosome gene SF3B1 are ubiquitous in MDS-RS patients, and the disease is considered as low-risk MDS with only a very small risk for transformation towards AML.

However, ringsideroblasts can also be

observed in certain AML cases that, in

1

type and context, CITED2 might act as oncogene or tumour suppressor gene.

Interestingly, a subset of AML patients exhibits enhanced CITED2 expression.

In Chapter 6 the importance of CITED2 in human AML is investigated. Furthermore, a novel interaction is established between CITED2 and PU.1, a hematopoietic transcription factor that is frequently inactivated in AML. The inactivation of CITED2 dramatically impacts cellular survival in both normal hematopoietic and leukemic cells.

Chapter 7 aims to get insight into the downstream mechanisms that play a role in CITED2 depletion-mediated apoptosis by using an RNA interference approach.

Finally, Chapter 8 summarizes and discusses the research described in this thesis.

transcriptional players are involved.

The second part of this thesis focusses on CITED2, a CBP/p300-interacting- transactivator-with-an-ED-rich-tail 2, is a transcriptional modulator that has been shown to be essential for the maintenance of normal hematopoiesis in both mice and humans.

CITED2 itself doesn’t have typical DNA-binding domains, but instead, the physical interaction of CITED2 with other proteins modulates their transcriptional activities.

CITED2 is a target gene of several transcription factors, including HIF-1a, FOXO3a, STAT5 and MYC.

(123)

Downstream targets of CITED2

include the polycomb transcription factor BMI1 and transcription factor GATA2, which are both essential for HSC biology.

Also, CITED2 has been described in connection to p53 biology.

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The role of CITED2 in oncogenesis

is ambiguous, dependent on the cell

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