Advances In Malignant Hematology (Hardcover)

Chapter One

Introduction

Hematopoiesis, simply stated, describes the regulated process of hematopoietic stem cell (HSC) self-renewal and differentiation into lineage committed progeny. Pluripotent HSC are rare cells (<1 of 10 000 bone marrow cells) specifically characterized by their proliferative capacity (though under steady state conditions >95% of HSC are quiescent, nondividing cells at any one time), pluripotency (they can regenerate the entire spectrum of mature blood derived cells), and self-renewal. The hierarchy of hematopoietic cell differentiation is depicted in Figure 1.1. HSC reside in close association with hematopoietic stromal cells within specific microenvironmental niches that function in concert with a variety of both multilineage and single lineage-specific hematopoietic growth factors, stromal cells, and extracellular matrix molecules to regulate their survival, cell cycle progression, proliferation, and differentiation. These processes of self-renewal, proliferation, differentiation, and cell death are tightly regulated under normal conditions throughout life. A normal individual maintains steady state numbers of blood cells within a very tight range with no more than a few percent variation from day-today, with constant production of the number of new cells required to replace the number of senescent cells that die. On average erythrocytes survive in the circulation for about 120 days, platelets for about 10 days, and neutrophils for about 6–12 hours. In order to replace senescent blood cells, the bone marrow of normal adult humans must produce about 180–250 billion erythrocytes, 60–100 billion neutrophils, and 80–150 billion platelets every day, or about 10¹⁶ (10 quadrillion) blood cells in a lifetime, with only minimal reduction in the bone marrow cell production capacity as a result of aging. The bone marrow can respond rapidly, in lineage-specific manner, to increase production of new blood cells by 6- to 8-fold over baseline under conditions of demand for each specific type of blood cells, such as in vivo destruction of erythrocytes, platelets, or neutrophils, infections requiring increased neutrophil production, and hemorrhage requiring increased erythrocyte production. Regulation of lymphocyte numbers is much less clearly understood, although it is known that some types of T and B lymphocytes may survive for many years. An understanding of these normal regulatory components in normal hematopoiesis is essential to unraveling the mechanisms that drive malignancy.

Isolation of hematopoietic progenitors

In 1961, Till and McCulloch isolated single cell-derived colonies of myeloid, erythroid, and megakaryocytic cells (CFU-S) from the spleens of lethally irradiated mice 1–2 weeks after rescue by bone marrow transplantation. These colonies were capable of extensive proliferation in vivo, exhibited some potential for self-renewal and, for the first time, conclusively demonstrated the presence of a multipotent hematopoietic progenitor cell. However, the lack of lymphoid colony development, as well as experiments in which 5-fluoruracil killed CFU-S without killing cells capable of replenishing CFU-S suggested that a more primitive "pre-CFU-S" must exist.

These data were further refined with the advent of flow cytometry, fluorescence activated cell sorting (FACS), in vitro hematopoietic progenitor cell systems, and xenotransplantation models, which revealed that long-term bone marrow repopulating HSCs were distinct from CFU cells, or multipotent progenitors (MPPs), and could be further subdivided into cells with short-term (ST-HSC) and long-term (LT-HSC) hematopoietic stem cell repopulation capacity. Specifically, LT-HSCs are defined by their extensive self-renewal capacity, allowing for full reconstitution of an irradiated host following transplantation of these cells. ST-HSCs, alternatively, have less capacity for self-renewal and instead more avidly differentiate into more committed MPPs. As such, ST-HSCs provide short-term hematopoietic cell reconstitution, but are incapable of permanently rescuing humans or other mammals with an aplastic bone marrow after lethal ionizing radiation.

Although some controversy exists, the most widely accepted model suggests that hematopoietic lineage commitment is both a stochastic and instructive process that occurs at specific branch-points, manifested at the time of cell division. During cell division, HSCs can either divide asymmetrically (a maintenance event with the production of one identical immature daughter cell and one differentiating daughter cell), symmetrically (an expansion/self-renewal event which serves to generate two identically immature daughter cells (self-renewal)), or terminally differentiate (an extinction event, in which both daughter cells are committed to terminal differentiation). The hierarchy of differentiation from HSC to mature end-stage hematopoietic cells is shown in Figure 1.1. As cells progressively differentiate into functional components of the hematopoietic system, they lose proliferative and multilineage differentiation capacity. Regulation of self-renewal, cell cycling, terminal differentiation, and apoptosis is therefore critically important to maintaining the production of hematopoietic elements over a lifetime. It is now clear that extrinsic and intrinsic systems act in concert to generate a network of events that govern HSC fate.

Cytokine regulation

Cytokines/growth factors include interleukins, lymphokines, monokines, interferons, chemokines, colony-stimulating factors (CSFs), and other hematopoietic hormones. These secreted factors interact with receptors on both pluripotent stem cells and committed hematopoietic progenitor cells to affect their survival, proliferation, and differentiation. The stages of differentiation from pluripotent HSC to fully mature hematopoietic cells of all lineages and the growth factors that play roles in these differentiation events are shown in Figure 1.1. Kit-ligand (also known as stem cell factor (SCF), and Steel factor (SF)) and Flt3 ligand, which function to drive proliferation by binding to the Kit and Flt3 tyrosine kinase receptors, respectively, on CD[34.sup.+] CD[38.sup.-] progenitors are important regulators of the early stages of hematopoietic differentiation from HSC. SCF, in particular, cooperates with multiple cytokines and cytokine receptors to influence differentiation, as well as upregulating BCL-2, BCL-X_L, and perhaps other antiapoptotic molecules to promote target cell survival. These receptors are downregulated during normal differentiation. Colony-stimulating factors, including erythropoietin (EPO), thrombopoietin (TPO), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), and macrophage-CSF (M-CSF; CSF-1), induce the differentiation and function of specific hematopoietic cell lineages. These factors accordingly are named for the lineages that they predominantly stimulate, although several also have effects on multipotent hematopoietic progenitors and perhaps even on pluripotent HSC. Alternatively, TGFβ (tumor growth factor-β), TNFα (tumor necrosis factor-α), and IFNs (interferons) all tend to negatively influence hematopoiesis.

Although cytokine-receptor interactions would appear to generate a level of specificity with regards to transcriptional and genomic regulation and, hence, lineage-specific cell differentiation, the convergence of similar molecular pathways upon genomic targets makes it difficult to delineate this. What can be said, however, is that cytokine receptors appear to fall into specific families based upon their signal transducing subunits (see Table 1.1), and that these signaling subunits rely on three major pathways to ultimately influence transcription. These pathways include the JAK-STAT pathway, the MAPK pathway, and the PI3/AKT pathways, although other pathways involving NF-κB, TGF/ SMAD, and protein kinase C pathways also play roles in the regulation of hematopoiesis. Importantly, mutations that affect these pathways are well described in lymphomas, myeloproliferative neoplasms, and leukemias.

Mechanistically, growth factors and cytokines act as ligands for transmembrane receptors that are located on the surface of hematopoietic cells, with differing receptor expression on HSC, multipotent progenitors, single lineage precursors and mature hematopoietic cells of different lineages. Dimerization (or conformational change) of receptors occurs following ligand binding. This receptor dimerization and conformational change leads to autophosphorylation of the intracellular portion of the receptors and recruitment of signaling molecules to docking sites on the activated receptors. This leads, in turn, to recruitment, phosphorylation, and activation of a broad range of cytoplasmic effector signaling molecules, such as STATs, Src-kinases, protein phosphatases, Shc, Grb2, IRS1/2 and PI3K via binding at the conserved SH2 domains and phosphorylation sites on the receptors themselves. For example, phosphorylation of STATs leads to the generation of STAT homo- and heterodimers, which are then translocated to the nucleus, where they can bind specific nucleotide sequences in the regulatory regions of specific genes to influence transcription of those genes, which determines the proliferation, survival, differentiation, and function of those cells. Similarly, phosphorylation of Grb2 facilitates the activation of SOS, which in turn, influences transcription via activation of the Ras/Raf/Mek/Erk, and the Rho/Mlk-Mekk/Mek/ p38-JNK pathways. Activation of phosphatidylinositol-3 kinase (PI3K), either directly or indirectly via RAS or IRS 1 and 2, generates PIP3, which in turn activates PKC, SGK, RAC1/CDC42 and AKT. Activation of AKT is particularly relevant to both normal and malignant hematopoiesis, as it can phosphorylate multiple transcription factors, leading to activation of mTOR, MDM2, and NFκB and inhibition GSK3β, FKHR, and BAD. Notably, multiple related proteins and isoforms of many of the signal transduction molecules exist (including JAK, STAT, Mek, Mlk, Mekk, Erk, p38, JNK, PI3K, PIP3, and AKT), and appear to have different nuclear targets depending on the cell type in which activation occurs.

Transcriptional regulation

Transcription factors are proteins that interact with the regulatory region of genes, either alone or in protein complexes, to increase or decrease expression of genes that contain specific sequences of nucleotides in these regulatory regions, which are recognized by the specific transcription factors. Transcriptional networks play a central role in the intrinsic regulation of HSC and lineage-committed progenitor cell survival, proliferation, and differentiation. Accordingly, these pathways are commonly perturbed in hematopoietic malignancies. Unfortunately, our knowledge in many cases is limited to non-human and in vitro models, which may not accurately reflect human hematopoiesis. Nonetheless, these experimental approaches have helped to define several important concepts in transcriptional regulation, including timing, autonomous and antagonistic pathways, cofactor regulation, and cellular signaling-related changes to transcription factor activity/function. A summary of relevant transcription factors thought to be involved in varying steps in the hematopoietic differentiation pathways is provided in Figure 1.2 and the transcriptional regulatory factors involved in each of the specific lineages of hematopoietic differentiation are described in more detail in the sections below on each of those lineages.

MicroRNA regulation

MicroRNAs (miRNA) have been recently implicated in the control of gene expression in hematopoiesis (Figure 1.2). miRNAs are small non-coding RNAs that bind to the 3'-untranslated regions and destabilize messenger RNAs (mRNAs) leading to their rapid degradation or, less commonly, may bind to the coding region of targeted genes and inhibit transcription of those genes. To date over 700 miRNAs have been identified in humans, with over 33% of human genes identified as potential targets of these miRNAs, based on identification of sequences in those genes that are reverse complements of specific miRNAs. A thorough review of the involvement of miRNAs in hematopoiesis is beyond the scope of this review; however, interested readers are referred to several recent reviews highlighting the importance of miRNA in both normal and malignant hematopoiesis.

Hematopoietic microenvironment

HSCs are most likely generated independently in the yolk sac and aorta-gonad-mesonephros (AGM) region in the developing embryo, after which they migrate to the placenta, attaching via VE-cadherin, and subsequently to the liver and spleen via b1 integrin-dependent interactions with the extracellular matrix (ECM). Mesenchymal cell development in the liver and spleen creates a unique microenvironment that fosters HSC survival and expansion. During most of human fetal development the liver is the primary source of hematopoietic cell production, with erythrocyte production predominating, and the spleen contributes a small proportion of fetal hematopoiesis. Shortly before birth, HSCs migrate to the bone marrow, presumably under the influence of CXCL12/CXCR4, c-Kit/SCF, CD44/hyaluronic acid, and a4b1 integrin (VLA-4)/ECM and stromal cell interactions. At that point, hepatic and splenic hematopoiesis virtually ceases, and essentially all subsequent human hematopoietic cell production is restricted to the bone marrow. It is now well accepted that stem cells routinely circulate into and out of the bone marrow niche throughout life, although the purpose of circulating hematopoietic stem and progenitor cells is not known. The same molecules that are involved in movement of HSCs to the bone marrow during development appear to play similar roles in HSC homing and marrow engraftment throughout adulthood. Curiously, in adults, CD44 is fucosylated, converting it to an E-selectin ligand, and accordingly facilitates binding and retention by bone marrow endothelial cells [12, 13]. CD44/hyaluronic acid and CD44/E-selectin interactions, which serve redundant roles in normal stem cell homing and engraftment, also have been found to be required for both human CML and AML leukemia cell growth in mouse xenograft models.

HSC and early hematopoietic progenitors tend to predominantly lodge into endosteal niches near N-cadherin-expressing osteoblasts, where they tend to remain quiescent, perhaps under the influence of osteoblast-secreted Angiopoietin-1, active at HSC TIE2 receptors. Increasing the osteoblast population via conditional inactivation of bone morphogenic protein receptor type 1A (BMPR1A) or administration of PTH leads to an increase in the number of HSCs in the marrow. PTH also increases CXCL12 expression by osteoblasts, and indeed CXCR4 appears to retain its importance in HSC repopulation even after homing. SCF and extracellular calcium-ion concentration (sensed via the calcium receptor, CaR) also may play a role in localization to the endosteum (reviewed in).

Interestingly, a separate population of HSCs is also found adjacent to endothelial cells, where N-cadherin expression is lower. Endothelial interactions likely play a role in HSC retention and egress, and may also facilitate HSC expansion and differentiation. For example, Tie2 is also expressed on endothelial cells, and blocking of this receptor impairs neoangiogenesis and delays hematopoiesis following myelosuppression. Angiopoietin-1, conversely, can rescue hematopoiesis in TPO-deficient mice. Together these data suggest that two pools of HSCs may exist, a quiescent fraction adjacent to osteoblasts in the endosteal niche, and a more rapidly proliferating and differentiating fraction adjacent to blood vessels.

Adhesive interactions via osteopontin/CD44 and b1 integrins, N-cadherin, c-Kit/SCF, CXCL12/ CXCR4, Jagged1/Notch and TIE2/Angiopoietin-1 all play roles in maintenance of the bone marrow niche and in HSC quiescence. These adhesive interactions are commonly altered in hematologic malignancies. Increased expression appears to confer a more aggressive and more drug-resistant "stem cell" phenotype, while decreased expression, as seen with AML1/ETO translocations, appears to confer a more migratory phenotype (reviewed in). CXCL12 is particularly important in HSC retention, and interestingly has been found to be expressed at a higher level among a subset of stromal reticular cells. These CXCL12-abundant reticular cells, or CAR cells, are found throughout the marrow, generally surrounding sinusoidal endothelial cells. Rhythmic noradrenaline secretion via local sympathetic nerves modulates CXCL12 expression via β3 adrenoreceptor-mediated regulation of Sp1 levels. HSC egress is commonly provoked using high doses of G-CSF, which acts on neutrophils to facilitate proteolytic cleavage of these adhesive interactions, and may also regulate CXCL12 expression via CSF receptors found on sympathetic nerves. Importantly, the marrow niche also critically regulates more mature cells as well. Osteoblast and endothelial cell niches play a role in both myelopoiesis (via G-CSF secretion) and B-cell lymphopoiesis (via IL-7 secretion and VCAM-1/cannabinoid receptor 2 expression). On the other hand, erythroid maturation is critically dependent on specialized bone marrow macrophage interactions.

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