University of Groningen
Modeling of MLL-AF9-rearranged pediatric leukemia Carretta, Marco
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Modeling of MLL-AF9-rearranged pediatric leukemia
Identification of mechanisms and potential targets
Marco Carretta
The studies described in this thesis were financially supported by the grant: Marie Curie Euro Cancer Stem Cell Training Network FP7-PEOPLE-2010-ITN-264361.
The publication of this thesis was financially supported by University of Groningen.
Cover design: Oscar ‘Odd’ Diodoro, odd-house.com Printed by: Ridderprint BV, www.ridderprint.nl
ISBN 978-94-034-0440-0 (printed) ISBN 978-94-034-0439-4 (e-book)
Copyright © 2018 by Marco Carretta. All rights reserved. No parts of this book may be reproduced or transmitted in any form or by any means without prior permission of the author.
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Modeling of MLL-AF9-rearranged pediatric leukemia
Identification of mechanisms and potential targets
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on Monday 5 February 2018 at 14.30 hours
by
Marco Carretta born on 7 May 1985
in Mantova, Italië
Supervisors Prof. J.J. Schuringa Prof. E. Vellenga
Assessment Committee Prof. S. de Jong
Prof. G. de Haan Prof. J. Jansen
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Alla mia famiglia
Paranymphs Vincenzo Terlizzi Bauke de Boer
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TABLE OF CONTENTS
CHAPTER 1 9
General introduction and scope of the thesis
CHAPTER 2 45
Modeling BCR-ABL and MLL-AF9 leukemia in a human bone marrow-like scaffold-based xenograft model
Leukemia. 2016 Oct;30(10):2064-2073
CHAPTER 3 89
Genetically engineered mesenchymal stromal cells produce IL-3 and TPO to further improve human scaffold-based xenograft models
Exp Hematol. 2017 Jul;51:36-46.
CHAPTER 4 123
BRD3/4 inhibition and FLT3-ligand deprivation target pathways that are essential for the survival of human MLL-AF9+ leukemic cells
PLoS One. 2017 Dec 14;12(12):e0189102.
CHAPTER 5 159
The cell of maintenance in MLL-rearranged B-ALL is multipotent and retains myeloid potential while MLL-rearranged AML is maintained by myeloid restricted LSCs
Manuscript in preparation
CHAPTER 6 183
Summary, general discussion and future perspectives
CHAPTER 7 211
Nederlandse samenvatting Acknowledgements
Curriculum Vitae List of publications
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CHAPTER
1
General introduction and scope of the thesis
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Introduction
Over the last decades, the continuous advancement of in vitro and in vivo models has greatly improved our understanding of normal and malignant hematopoiesis. The development of progressively more immunodeficient mice and xenotransplantation models has led to the identification of normal and leukemic hematopoietic stem cells, the detailed characterization of the hierarchical organization of human hematopoiesis, and also provided pre-clinical models for drug testing. Insights from these models also directly contributed to transplantation procedures and treatment options in the clinical practice. In this chapter, a general introduction on normal and malignant hematopoiesis is provided, with a particular focus on MLL-rearranged leukemias. Furthermore, the in vitro and the in vivo models that are being used to study leukemia are reviewed from an historical perspective. Lastly, the general scope and aim of this thesis are outlined.
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CHAPTER 1 11 Hematopoietic stem cells and their niche
Hematopoiesis is defined as the formation and development of blood cells. This essential process, which in adults occurs mainly in the bone marrow, ensures the daily generation of over 500 billion cells in response to the turnover of the short-lived red and white blood cells. The production of this impressive amount of new blood cells can even further increase in case of bleeding, infection or pathological loss, making the hematopoietic system one of the most actively regenerating tissues of the whole body 1,2. Despite the heterogeneity in both morphology and function of all the mature elements present in the blood, all these different cells originate from a rare population of primitive hematopoietic stem cells (HSCs). Adult HSCs possess multi- lineage potential, which is the ability to develop into any of the three types of blood cells: red cells, white cells and platelets. Furthermore, HSCs possess self-renewal capacity, which maintains the stem cells pool and ensures the continuous production of blood cells throughout the lifetime of an organism. Decades of studies on the hematopoietic system in mice and humans showed that stem cells are rare and quiescent and they display heterogeneity with respect to their self-renewal potential.
With the use of specific molecular surface markers, in mice HSCs were subdivided in long-term reconstituting HSCs (LT-HSCs) and short-term reconstituting HSCs (ST- HSCs) 1,3–5. Long-term HSCs are considered to be the least abundant and most quiescent population, only diving few times during life and able to reconstitute lethally irradiated mice for the lifetime of the animal. Analysis of the kinetics of BrdU incorporation revealed that over 99% of LT-HSCs incorporated BrdU after long periods of administration and it was postulated that LT-HSCs enter the cell cycle and divide on average every 57 days 6,7. In a more recent study by Wilson and colleagues
8, mathematical modelling of the results obtained from pulse-chase experiments
combined with the use of six different molecular markers phenotypically identifying LT-HSCs (Lin-, Sca+, cKit+, CD150+, CD48-, and CD34-) uncovered the existence of a subpopulation of dormant HSCs, which represented about one seventh of the studied population. These HSCs divide every 145 days, which is equivalent to five divisions per average C57/BL6 mouse lifetime 9.
The direct descendants of the LT-HSCs are the so-called short-term HSCs (ST- HSCs) and multipotent progenitors (MPPs). While these two cell populations still possess multilineage potential, they appear to have less self-renewal potential and are able to reconstitute lethally irradiated mice for only short periods of time.
However, all the evidence supporting these concepts has been obtained by using in vivo transplantation assays and therefore they might not reflect the real mechanisms
governing life-long blood cell production in unperturbed animals. Recent papers suggested that in a native setting without transplantation, where murine cells were uniquely and genetically labelled in situ, also committed progenitors can contribute to life-long hematopoiesis rather than classically defined hematopoietic stem cells 10,11. However, this concept of progenitor cells instead of LT-HSCs that sustain life-long hematopoiesis was again recently challenged by a study showing that, by genetic labelling of a subset of self-renewing LT-HSCs in the adult bone marrow, it is the most primitive HSCs that sustain endogenous hematopoiesis 12. No doubt, further future studies will be aimed at clarifying these different observations.
Upon progression down the hematopoietic tree, cells become more committed towards either the lymphoid (Common Lymphoid Progenitors; CLPs) or myeloid lineage (Common Myeloid Progenitors; CMPs). In the last stage of differentiation, CLPs generate B- and T-lymphocytes as well as Natural Killer (NK) and dendritic cells. On the other side, CMPs give rise to megakaryocyte erythrocyte progenitors
CHAPTER 1 13 (MEPs) that will terminally differentiate in erythrocytes and platelets, and give rise to granulocyte-macrophage progenitors (GMPs) that will differentiate into the mature granulocytes and macrophages. More recent studies have shown that the hierarchical organization of the hematopoietic tree might be different, whereby HSCs differentiate into a previously unidentified population of lymphoid-primed multipotent progenitors (LMPPs), which possess lymphoid, granulocyte and macrophage differentiation potential. Furthermore, an early MEP population would be able to give rise to megakaryocytes and erythrocytes 13–17.
Figure 1 Hierarchical organization of the hematopoietic tree Long Term Hematopoietic Stem Cells (LT-HSCs); Short Term Hematopoietic Stem Cells (ST- HSC); Multipotent Progenitor (MPP); Lymphoid-primed Multipotent Progenitor (LMPP); Common Lymphoid Progenitor (CLP); Common Myeloid Progenitor (CMP);
Granulocyte-Macrophage Progenitor (GMP); Megakaryocyte-Erythrocyte Progenitor (MEP).
In adults, HSCs are residing in a specific anatomical location in the bone marrow denominated the hematopoietic stem cell niche 18. These niches are constituted of various cell types, extracellular matrix and soluble chemical factors. The niche provides a specific physiological microenvironment that plays a key role in the regulation of the balance between differentiation and self-renewal of the HSCs. Early studies proposed that HSCs are mainly located in the endosteal region, but more recent studies have shown that quiescent HSCs are also perivascular and distributed throughout the bone marrow 19,20. In the perivascular space, several cell types have been associated with HSC regulation, including megakaryocyte 21, endothelial cells
22, arteriolar pericytes 23, sympathetic nerves 24 and non-myelinating Schwann cells
25. The perivascular space in the BM also includes perivascular mesenchymal stromal cells (MSCs) defined as CXCL12-abundant reticular (CAR) cells 26. CAR cells express high levels of niche factors such as CXCL12, also known as stroma derived factor 1 (SDF-1). By binding to the CXCR4 receptor, SFD-1 plays an important role in HSCs homing and retention in the BM 27. Furthermore, it has also been shown that CAR cells in the BM are producing stem cell factor (SCF), a crucial cytokine involved in the maintenance and proliferation of HSCs 26,28. In human BM, CD146-expressing cells might represent the counterpart of murine CAR cells 29. The role of osteolineage cells in HSC maintenance is still debated, but current evidence suggests that immature osteolineage cells rather than mature osteoblasts may contribute to the regulation of HSCs 30. Intriguingly, in a study published by Ding et al.31 it has been shown that deletion of CXCL12 from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation. Deletion of CXCL12 from osteoblasts instead depleted certain early lymphoid progenitors, but not HSCs or myelo-erythroid progenitors and did not
CHAPTER 1 15 mobilize these cells into circulation. The authors concluded that different stem/progenitor cells occupy distinct cellular niches in the BM: HSCs are residing in a perivascular niche and early lymphoid progenitors in an endosteal niche.
Collectively, these findings are contributing to a better understanding of the hematopoietic BM niche but several aspects of HSC-niche interaction still remain to be elucidated 32.
Leukemic stem cells and their niche
In normal hematopoiesis, the differentiation of healthy HSCs into mature blood elements is a highly regulated and quantitatively controlled process. Disruption of these regulatory mechanisms by sequential acquisition of genetic alterations and/or epigenetic changes in stem or progenitor cells will first interfere with cell production and then lead to clonal expansion. Ultimately, such expanding clones may result in the development of leukemia.
Different types of leukemia can be distinguished according to their lineage, aggressiveness and dynamics. In acute leukemia, the disease evolves rapidly and aberrant blood cells are blasts that remain immature and cannot carry out their normal functions. Clinical signs of acute leukemia are derived from the infiltration of leukemic clones in the BM, which mainly causes cytopenia, impaired hemostasis and impaired immune functions. In chronic leukemia instead, the progression of the disease is slower and while some blast cells are present, most cells are mature and can carry out some of their normal functions. Both acute and chronic leukemia can develop along the myeloid or lymphoid lineages, giving rise to acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) or chronic lymphocytic leukemia (CLL) 33.
For these hematological malignancies there is experimental evidence supporting the concept of the cancer stem cell (CSC), which is defined as a subset of neoplastic stem cells that propagate malignant clones indefinitely and give rise to cancer 34. The existence of leukemic stem cells (LSC) was first shown in AML by John Dick, which represented the first direct identification of a cancer driven by cancer stem cells 35. In this study, non-obese diabetic, severely combined immunodeficiency disease (NOD/SCID) mice were used as a model to study the engraftment and proliferation of primary human patient-derived leukemic cells. By transplanting sorted CD34+CD38− cells, which often represents only 0.1 to 1% of the total AML cell population, the leukemic cell morphology found in patients was recapitulated, whereas the CD34+CD38+ and CD34− fractions contained no cells with these properties. This study conclusively established that, similarly to the healthy counterpart, also leukemia is hierarchically organized and sustained by a rare population of LSCs. However, consecutive studies have highlighted that LSCs may also reside in other compartments than the CD34+CD38- compartment. For example, LSCs were also found in the CD34+CD38+ cells as well as CD34- fraction in the majority of NPM1-mutated AML 36–38. In recent years, LSCs were found in even more mature fractions; for example, oncogenes such MLL fusion proteins can re-install aberrant self-renewing properties in committed MPPs and transform them into LSCs
39,40.
While for different AML subtypes the characterization of the LSCs has progressed significantly, the phenotype of LSCs in ALL is less clear. One study suggests that for pre–B cell lineage ALL the LSCs display an immature phenotype lacking the expression of CD19 41, whereas others showed that ALL-LSCs are more mature and do express CD19 42–44. Currently, little is known about similarities and differences
CHAPTER 1 17 between LSCs that maintain myeloid versus lymphoid malignancies, an aspect that is addressed in the context of MLL-rearranged leukemia in Chapter 5 of this thesis.
The experimental evidence so far suggests that leukemia, in analogy to normal hematopoiesis, is hierarchically organized and is produced by relatively few LSCs that are impaired in their differentiation potential. Regardless of the ‘cell of origin’
(HSCs or more committed progenitors), tumorigenic leukemic cells are able to give rise to phenotypically diverse progeny including a few LSCs with indefinite proliferation potential, as well as cells within the leukemia that may have limited or no proliferative potential 45. A better understanding of leukemia biology is fundamental to improve therapeutic strategies. Current approaches to cure leukemia tend in fact to target the bulk of the tumor cells, but are not effectively targeting the pool of LSCs.
Most LSCs are quiescent the majority of the time, and are therefore resistant to standard chemotherapeutic options that target cells undergoing replication.
While we have learned a lot about the role of the niche in normal hematopoiesis, the exact role of the niche in leukemogenesis is still less clear and only in recent years experimental work has started to investigate specific roles of the microenvironment in the pathophysiological context. In spite of the technical limitations which still hinder our understanding of the BM niche physiology, it is becoming increasingly clear that BM stromal changes and the formation of a self-reinforcing malignant niche is more than a mere bystander effect of disease development and can directly contribute to myeloid malignancies 46. Stromal abnormalities and structural changes in the BM cavities due to impaired angiogenesis and bone-loss are documented in both AML and Myelodysplastic Syndrome (MDS) 47. A growing body of literature has investigated how leukemic cells remodel their niche into an aberrant environment that provides support to malignant cells at the expense of normal hematopoietic cells.
BM-derived MSCs from AML patients have been shown to have a decreased production of CXCL12, which could potentially affect also the maintenance of healthy HSCs 48. More recently, intra vital imaging and xenograft transplantation were used to show how human leukemic cells drive the formation of specialized malignant niches that directly impair healthy HSC function due to overproduction of Stem Cell Factor (SCF) 49,50. Another concept that has emerged is the possible existence of different niches between CML and AML. For example, experimental evidence suggests that while parathyroid hormone impaired CML growth, it augmented MLL- AF9-induced AML proliferation 51. Furthermore, Schepers et al. 52 showed that CML cells stimulate MSCs to proliferate and adopt an abnormal differentiation program resulting in the overproduction of functionally altered osteoblastic cells, which accumulate in the BM cavity as inflammatory myelofibrotic cells. For AML instead, it has been reported that leukemic cells create neuropathic changes in the BM niche, which affect the activity of perivascular MSCs and alter the function of the HSC niche
53,54. It is thus clear that the disease can be the initiator of niche changes but, conversely, genetic alterations of the niche may also represent driving mutations during malignant transformation. In support of this concept, it has been shown that targeted deletion of the miRNA-processing endonuclease Dicer1 from osteoprogenitor cells disrupts the integrity of the hematopoietic system, recapitulating key features of human myelodysplastic syndrome. This included the propensity to develop, although in low frequency, AML55,56. Lastly, but importantly, the leukemic niche in the BM is shown to support LSC in the maintenance of their quiescent state, thus contributing to protect the cells from chemotherapeutic agents 57–59.
Taken these pieces of evidence together, it immediately becomes clear that manipulating the leukemic niche might represent an advantageous therapeutic
CHAPTER 1 19 strategy, particularly in malignancies for which targeting the hematopoietic cells has proven inefficient.
Characteristics of MLL-rearranged leukemias
Acute leukemia is a highly heterogeneous disease and comprises a wide variety of cytogenetic aberrations and molecular mutations. To date, hundreds of genetic markers have been identified, serving as basis for the categorization of cases, prediction of prognosis and therapeutic strategies.
Due to their unique biological and clinical features, MLL-related leukemias are of particular interest. The MLL (Mixed Lineage Leukemia) gene on chromosome 11q23 encodes for a Histone-Lysine N-methyltransferase, an epigenetic regulator which plays an essential role in early development and hematopoiesis. Chromosomal translocations bearing the MLL gene define a unique group of leukemias, which can give rise to AML, ALL or biphenotypic (mixed lineage) leukaemia (BAL). MLL-related leukemias account for approximately 70% of infant leukemia 60, 10% of adult AML and are frequently found in therapy-related acute leukemia cases that arise following treatment with topoisomerase II inhibitors 61. Patients with MLL-rearranged AML have a similar poor prognosis as others AML, but children with MLL-rearranged ALL have a particularly poor outcome compared with children with other forms of ALL. At a molecular level, two main aberrations of the MLL gene are found. One of these creates a short repeat within the N-terminal MLL coding sequence which results in an internal partial tandem duplication (PTD). Thus, an extra amino-terminus is added in- frame to full-length MLL, resulting in MLL-PTD driven myeloid dysplastic syndrome (MDS) or AML. In the other type of disruption of the MLL gene, the genomic sequences encoding the N-terminal portion of MLL are fused to sequences encoding
the C-terminus of another translocation partner. These types of translocation are the most common and to date, nearly 100 different chromosome bands have been described in rearrangements involving chromosome 11q23 and more than 70 fusion genes have been characterized at the molecular level 62.
Figure 2 Distribution of major MLL fusion partner genes in de novo childhood and adult leukemias Mixed lineage leukaemia (MLL) rearrangements are found in approximately 5% of acute lymphoblastic leukaemias (ALL), approximately 5–10% of acute myeloid leukaemias (AML) and virtually all cases of mixed lineage (or
biphenotypic) leukaemias (MLL). Adapted from 63
Among the numerous fusion translocation partners, four out of the five most frequent MLL rearrangements belong to a family of serine/proline rich nuclear proteins (AF4, AF9, AF10, ENL). Different MLL fusion can give rise to different leukemia subtypes
CHAPTER 1 21 and the same translocation can give rise to different leukemia in pediatric or adult patients. For example, the MLL-AF9 fusion gene is of particular interest since it is found in pediatric cases in similar proportions between AML (33%) and ALL cases (18%) however, for adults this translocation is mainly found in AML (32%) compared to ALL (2%) (Fig.2). MLL fusions are transcriptional regulators that take control of targets normally controlled by MLL. In the wild type form of the MLL gene, the SET domain confers H3K4 methyltransferase activity, allowing transcription initiation by polymerase II. When the MLL gene is fused with one of its partners, the SET domain is in most cases lost together with its catalytic activity. However, MLL fusion proteins gain the ability to methylate H3K79, which results in an aberrant gene expression of homeobox genes such HOXA9 and MEIS1 64. The H3K79 methyltransferase DOT1L and the MLL-interacting protein Menin have emerged as important mediators of MLL fusion-mediated leukemic transformation. In work by Okada et al. 65, a unique function of an MLL fusion (MLL–AF10) was identified for the first time. The recruitment of the H3K79 methyltransferase DOT1L by AF10 is in fact shown to be indispensable for transformation and responsible for the aberrant H3K79 methylation at the HOXA9 locus. Other MLL fusion partner genes, for example the ones involved in transcriptional elongation such AF4 and AF9, were also reported to interact directly with DOT1L 66,67. Furthermore, fusion partner genes such ELL, ENL and others have been reported to interact with DOT1L via common binding proteins 68,69. Although inhibition of DOT1L is currently under clinical investigation, downstream signalling is still far from clear and a more detailed characterization of molecular mechanisms underlying MLL-fusion leukemia is still greatly needed. In Chapter 4 of this thesis, by taking advantage of state of the art molecular techniques, we aimed to investigate
the downstream targets in human MLL-AF9 leukemias and identify targetable signaling networks.
As briefly mentioned before, MLL fusions offer a valuable tool to study LSCs biology.
By transducing the MLL-ENL fusion oncogene in BM-isolated GMP cells, Cozzio et al. demonstrated that murine myeloid leukemia can also originate from committed
myeloid progenitors that have no intrinsic self-renewal capabilities, at least in the mouse. Despite the fact that 10 times more GMP-transduced cells were needed compared to HSC-transduced cells to generate AML, this experimental evidence shows that committed myeloid progenitor cells are also capable of being transformed into LSCs. The same concept was demonstrated also upon transduction of GMPs with MLL–AF9 fusion 40,70. Gene expression analysis revealed that these LSCs preserve the overall GMP expression profile, but the MLL fusion is capable of re- conferring a self-renewal programme normally present only in the HSCs compartment. However, another study focused on the malignant transformation initiated by the fusion gene MLL-AF9 and compared when expressed at physiologic levels in a knock-in model or at supraphysiologic levels in a retroviral model. The data from the physiologic model showed highest levels of MLL and MLL-AF9 in the most transformable HSCs and lower levels in the more resistant committed myeloid progenitor GMPs. The authors conclude that while it is possible that the transforming human MLL-AF9 translocation may take place at a maturation stage later than the HSC, murine studies suggest that this is much less likely to be functionally meaningful than a “hit” within the HSC population 71. Future studies will be necessary to further define this issue, but similar conclusion were also found by Horton et al. 72. Transcriptome analysis identified enrichment of HSC but not progenitor gene signatures in MLL-AF9-expressing cells and highlighted the differences in MLL-AF9-
CHAPTER 1 23 mediated immortalization in neonatal cells versus adult cells, reinforcing the notion that intrinsic properties of the cell of origin, in addition to extrinsic cues, might dictate lineage of the immortalized cell.
Modeling of human leukemia in vivo
The in vitro and in vivo models available for studying normal and malignant hematopoiesis have constantly improved over the last four decades, directly contributing not only to a better understanding of human biology but also significantly impacting on drug design and the development of therapeutic strategies.
Progressively improved xenograft mouse models supporting the engraftment and development of human hematopoietic cells have been generated and they now represent essential tools in the field, making the disease accessible to experimentation73–77. In this section, a historical review of the most relevant mouse xenograft models and in vitro assays to study leukemia are provided.
In order to generate humanized mice in which successful xenotransplantation can be achieved three major requirements need to be met. First, the recipient mouse needs to be immunodeficient in both its adaptive and innate immune compartments in order to tolerate the human graft and prevent xeno-rejection. Second, an appropriate spatial location (or niche) needs to be provided in the bone marrow, in which transplanted human cells can home and develop. Lastly, the host needs to provide the appropriate cross-reactive human growth factors, cytokines and major histocompatibility complex molecules that can act on human cells 76.
The development of mouse strains that support human hematopoiesis and AML in vivo started in the late 1960’s and a schematic historical overview of the development of the different immunodeficient mouse models is shown in Figure 3.
Figure 3 History of the development of immunodeficient mice for establishing leukemia mice models (Adapted from Goyama, S., Wunderlich, M., & Mulloy, J.
C. (2015). Xenograft models for normal and malignant stem cells. Blood, 125(17), 2630-2640.)
The nude mice (nu) were first generated in 1962 and they represent the first immunodeficient mouse strain ever described and utilized for xenotransplantation 78. These mice are athymic and lack functional T and – only partially - B cells 79. Initial attempts to engraft human AML consisted in the subcutaneous injection of BM derived leukemic cells. However, even when the nude mice were immunosuppressed with radiation, transplantation of leukemic tissue proved to be inconsistent and limited to the formation of localized myelosarcomas with little evidence of BM engraftment
80Moving to the late 1980s, in an attempt to further improve immunodeficient models, beige mice (bg) and X-linked immunodeficiency (xid) models were generated 81–83. In the bg/nu/xid model, mice are athymic and have a reduced number of natural killers (NK) and so-called lymphokine activated killer cells. Engraftment of human myeloid
CHAPTER 1 25 cells indicated better seeding, proliferation and differentiation of human stem cells, but attempts to engraft peripheral blood derived human cells proved difficult84,85. In 1983 Bosma et al. described the SCID (severe combined immunodeficiency) mice, which are deficient in mature B and T cells. In this model, germ line DNA segments encoding immunoglobulin and T lymphocyte antigen receptor molecule fail to undergo rearrangement resulting in lack of functional antigen receptors 86. The first successful xenotransplantation of AML into SCID was reported in 199187, followed by several other studies demonstrating the superiority of this model over the be/nu/xid
88,89. In spite of the clear benefits of the SCID model, the lack of cross-reactive human cytokines and innate host resistance still posed major practical limitations in modeling AML in vivo 87,90–96. Researchers have tried to overcome these issues by injecting endogenous human cytokines and growth factors such as IL3, IL6, GM-CSF and EPO into the SCID recipients 97–99. However, although this approach was simple and resulted in enhanced engraftment, it is expensive, time-consuming and not suited for long-term experiments. Furthermore, antigen-presenting and NK cells were not affected in the SCID strains, and moreover, SCID mice occasionally express mature B- or T-cells indicating a certain degree of ‘leakiness’ of the system
100,101. Further improvements of this immunodeficient strain led to the generation of the NOD/SCID mouse strain.
The NOD/SCID mice were generated by backcrossing the SCID gene onto the non- obese diabetic (NOD) background and displayed impaired innate and adaptive immunological functions. Besides lacking mature B- and T-cells, NOD/SCID mice possess impaired NK and antigen-presenting cell functions. Moreover, these mice are insulitis- and diabetes-free throughout their lifespan 102. These mice showed appreciably higher engraftment rate also when a limited amount of cells were injected
103,104. Importantly, the NOD/SCID model allowed the formal demonstration of the concept of leukemic stem cells (LSCs) 104. In fact it has been shown that only a rare subset of the most immature cells termed SCID-Leukemia Initiating Cells (SL-ICs) is capable of initiating and sustaining leukemic growth in vivo. SL-ICs are able to recapitulate the morphological, genotypic and biological characteristics of the donor and, as demonstrated in serial transplantation experiments, possess high self- renewal capacity 104,105. Despite the clear advantages of the NOD/SCID model, major limitations are represented by the frequent occurrence of thymic lymphoma and spontaneous NK cells activity, which frequently hindered long-term enduring engraftment 102.
In more recent years, in order to overcome the limitations of the previous models, NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) 106 and NOG (NOD/Shi-scid/IL-2Rγnull)107 mice have been developed. In these mice the complete deficiency of NK cells is achieved by disrupting the signalling downstream of IL-15, which is essential for the NK-cell development and maturation. The common gamma chain of cytokine receptor (IL2Ry) is shared by multiple members of the IL2 family of cytokines, including IL15. Thus, deficiency in the IL2Ry gene results in the complete absence of NK cells and limits the spontaneous development of murine thymoma, a phenomenon often observed in NOD/SCID mice 57,108–110. Several reports have highlighted the superiority in engraftability of AML cells compared to previous models
110–112 and to date the NSG model is still considered the ‘golden standard’ for xenograft models. The generation of immunodeficient mouse strains like the NSG allowed researchers to functionally define hematopoietic stem cells and their malignant counterpart and also to serve as preclinical models for drug testing.
CHAPTER 1 27 Despite the ability of engrafting different subtypes of primary acute lymphoblastic leukemia samples, NSG mice still possess major limitations for myeloblastic leukemia, since the engraftment of at least 30-40% of AML is not achieved 113 and overall this model is clearly lymphoid biased 72,114. This may be explained by the high influence of the murine microenvironment and the absence of species-specific human factors over AML. In order to overcame these limitations, in the last decade researchers have developed several immunodeficient mice where the transgenic expression of cytokine-encoding genes is under the control of a strong constitutive promoter in order to provide support for human hematopoietic (or leukemic) cell development 115–119. For example, NSG transgenic mice expressing human factors such SCF, GM-CSF, IL-3, and TPO have been developed, showing an increase in the engraftability rate of primary tumors. However, one limitation of this approach consists of the non-physiological levels of human cytokines expressed by these transgenic mice that eventually lead to myeloid biasness or exhaustion of self- renewal capacity 115. Thus, novel approaches are greatly needed in order to improve the current xenotransplantation procedures and in particular the ones that consider the importance of providing multiple human factors at physiological concentrations.
During the time I performed the studies presented in this thesis, an innovative mouse model called MISTRG has been generated in which human versions of four genes encoding cytokines important for innate immune cell development (M-CSF, IL-3, GM- CSF and TPO) are knocked into their respective mouse loci 120. These mice support the development of human NK cell, myelo-monocytic cells and show a strong human innate immune response in response to viral and bacterial infections. The possible implications of the use of this mouse model for leukemia research will be discussed in the final chapter of this thesis.
Modeling of human leukemia in vitro
Although the in vivo models (in particular the NOD-SCID/NSG models) have been very instrumental, in most cases these systems have limitations such as their cost- effectiveness and duration. Thus, in order to further understand the differences in molecular mechanisms that are involved in leukemic transformation of hematopoietic stem cells, accessible assays are also required, in which gene-function analyses of the leukemic stem cell can be performed and in which phenotypes such as long-term expansion, self-renewal, and apoptosis can be monitored. It is clear that self-renewal of normal stem cells heavily depends on extrinsic cues they obtain from the bone marrow microenvironment. Culture systems aiming to recapitulate the bone marrow microenvironment are thus essential to study niche-mediated regulation of normal and malignant hematopoietic stem cells at a molecular level and much effort has been put in the development of such in vitro systems.
Over three decades ago, Dexter et. al developed a 2D co-culture system where murine HSC where cultured on a basal cell layer comprised of mixed BM stromal cells obtained by flushing out the marrow from mouse femurs and sub-culturing the adherent cell fraction121. Viability and stemness of murine HSCs cultured on stroma were improved and cultures could be maintained over longer periods of time (7–12 weeks) compared to pure HSC cultures, especially when the culture medium was supplemented with cytokines. The Dexter model paved the way for many HSC and AML ex vivo culture models that have been developed subsequently.
AML cocultures with bone marrow stromal and supplemented with human cytokines could also be established, in which a long-term leukemic expansion for 7 to >24 weeks could be achieved122–132. In these assays, Leukemic Cobblestone Area Forming Cells (L-CAFCs) readily form and self-renewal capacity can be addressed
CHAPTER 1 29 by serial replating of cultures onto new stroma, whereby new L-CAFCs are generated. This in vitro assay also allows for the evaluation of leukemic stem cell markers that identify populations with long-term self-renewal potential 122,128,131. Moreover, it is also possible to introduce or downmodulate genes in the AML LTC- ICs by lentiviral (RNAi) approaches 122,125–127,130,131,133.
Besides using patient samples to gain further insight into LSCs, model systems have been generated as well in which oncogenes such as MLL-AF9114,134,135, BCR-ABL
125,136, FLT3-ITDs137, KRAS138, STAT5 139 and NUP98-HOXA9 137 are introduced into healthy human HSCs. For many of these studies fetal cord blood (CB) CD34+ cells have been used, but distinct phenotypes can be obtained when oncogenes are introduced into fetal versus adult human CD34+ cells135. Myeloid and lymphoid transformation of transduced human CD34+ can be achieved upon changes of composition of the culturing media 72.
Scope of the thesis
As discussed in this introductory chapter, mouse xenograft models have significantly contributed to a better understanding of normal and malignant hematopoiesis but major limitations still remain in the currently available and widely used xenograft models. In Chapter 2 of this thesis, we describe a novel in vivo approach aimed to reconstruct a humanized BM microenvironment in immunodeficient mice. We hypothesized that the presence of a human niche would provide human niche- specific factors that might still be missing in the murine recipient and would also provide interaction of the hematopoietic cells with various niche components. To test this hypothesis, we focus our studies on two leukemic CB models that possess distinct biology: BCR-ABL and MLL-AF9. For BCR-ABL, a serially transplantable CD19+ B-ALL could be induced in NSG mice only when additional hits were provided, such as co-expression of BMI1. In this study, we wished to evaluate if in the humanized BM xenotransplant model would allow BCR-ABL-only expressing cells to engraft and induce leukemia. Also for MLL-AF9, a serially transplantable CD19+ B- ALL could be induced in NSG mice while the AML transformation was less frequently observed. Since the susceptibility of MLL-AF9 to microenvironmental cues had already been shown 140, we wanted to evaluate if the presence of a human BM niche would favor myeloid engraftment. Furthermore, we also set out to test the engraftment efficiency of primary material derived from patients with the same mutations. Lastly, we investigated whether this model could also be used to evaluate the efficacy of inhibitors in vivo.
The ectopic implantation of a humanized BM niche in immunodeficient mice allowed the faithful recapitulation of some of the key features of BCR-ABL and MLL-AF9 leukemia. Furthermore, in parallel studies from our lab, we showed that the BM
CHAPTER 1 31 humanized model allowed the engraftment of leukemic samples that failed to engraft in NSG mice without humanized niches and resulted in a better preservation of leukemic stem cell self-renewal properties 141. Although the presence of an ectopic human BM niche presented clear advantages compared to normal NSG mice, some key issues still remain such for example the engraftment of myeloid transformed cells in secondary recipients. Transcriptome studies of primary human BM-derived MSCs revealed that a variety of cytokines and growth factors are produced, but some critically important cytokines such IL3 and TPO are not. In Chapter 3, we investigated whether we could genetically engineer MSCs to produce such factors and evaluated these modified MSCs in vitro and in vivo in humanized scaffold xenograft models.
In Chapter 4, we aimed to identify targetable signaling networks by taking advantage of our in vitro and in vivo models for human MLL-AF9 leukemia. Progress in DNA- sequencing technologies has reinforced the notion that cancer is initiated and maintained by alterations in the genome and it has also become more evident that epigenetic regulators are among the most frequent aberrancies in hematopoietic malignancies 142. Here, we show that MLL-AF9 cells critically depend on FLT3-ligand induced pathways as well as on BRD3/4 for their survival. We evaluated the in vitro and in vivo efficacy of the BRD3/4 inhibitor I-BET151 in various human MLL-AF9 (primary) models and patient samples and analyzed the transcriptome changes following treatment
Switches between lymphoid and myeloid lineages can occur during treatment or during relapse of pediatric leukemia patients bearing MLL-translocations. The mechanisms behind these phenomena have remained elusive, but published studies suggest that both intrinsic and environmental cues may inspire changes in cell fate
decisions of leukemic stem cell clones. Although these complete lineage switches occur infrequently, it is relevant to evaluate whether the leukemic blasts originate from the same leukemic clone or whether new clones emerge as a consequence of the treatment. In chapter 5, by making use of a lentiviral MLL-AF9 CB model as well as primary MLL-rearranged pediatric patient samples, we aimed to model lineage switching in vitro and in vivo in xenograft mice. Furthermore, we used ligation mediated PCR (LM-PCR) and RNA-sequencing to investigate clonality of leukemia and gene expression programs.
We conclude in Chapter 6 with a summary of the results and a discussion on the future perspectives of modeling of human leukemia in vitro and in vivo.
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