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2.2.1 Treatment of AML

AML is an aggressive malignancy, usually associated with a rapid progression of symptoms from the time of diagnosis, often within a few months. Research over the past 30 years has significantly improved outcome for patients under 60, but less so for older patients. For younger patients, the standard treatment procedure is induction chemotherapy, which involves a combination of two strong chemotherapeutic agents administered over seven days – cytarabine on all seven days and an anthracycline such as daunorubicin or idarubicin on only the first three days216_{. This treatment typically}

destroys most bone marrow and leukemic cells. Biopsies taken a month after treatment are considered successful if blast frequency is <5%. Then begins the consolidation phase of treatment consisting of high dose cytarabine therapy, with the intention of eliminating all blasts and leading to complete remission. For younger patients otherwise in good health with intermediate or high risk cases of AML, this therapy is typically followed by HSPC transplant216_.

Chemotherapy is highly intensive, destroying healthy cells and causing serious side effects. Older people are therefore not ideal candidates for this treatment. They instead receive milder drugs with fewer side effects, with periodical treatment rather than in intense induction and consolidation phases. The development of drugs that could specifically target leukemic cells without side effects would therefore be highly beneficial to older patients. This is currently an active area of research, with some drugs in early clinical trials. Alternately, the potential for targeted immunotherapies using the patient’s own immune system to eliminate leukemia is being investigated in both leukemia and solid tumors217_.

2.2.2 Animal models of AML

Primary human AML cells are an invaluable resource for studying leukemia, but important research has also been conducted using mouse models of AML. There are two primary methods used in murine AML

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models – introducing mutations or oncogenic genes into mice corresponding to those in human AML, or by directly transplanting human AML cells into recipient mice218_.

As discussed in Section 2.3, the WHO classification system categorizes AML based on the primary “driver mutations” underlying the disease. Some of these aberrations are fusion proteins formed by chromosomal translocation and others are mutations of certain genes in karyotypically-normal cells. Expression of oncogenic genes in mouse cells is possible using retroviral expression systems, which introduce permanent alterations to DNA. Examples of leukemic models developed using this system include BCR- ABL, MOZ-TIF2219_{, PML/RARα}220_{, NUP98-HOXA9}221_{, and AML1-ETO (corresponding to human}

RUNX1-RUNX1T1)222_{. Importantly for this thesis, retroviral models also exist for MLL-rearranged}

leukemias, which comprise approximately 10% of AML. MLL-AF9 is the most common MLL fusion protein, and the MLL-AF9 mouse model is used extensively in leukemic research223_{. Retroviral transduction of HSCs}

with MLL-AF9 immortalizes the cells, allowing for expansion in vitro in the presence of SCF, IL-6, and IL-3. When transplanted into irradiated recipient mice, blasts accumulate in the bone marrow and spread to other hematopoietic tissues, leading to symptoms mirroring AML. Survival is approximately 60-70 days. MLL-AF9 transduction of MPPs or CMPs also leads to immortalization and leukemogenesis, though with an increased latency compared to HSC-transformed cells224_{. Certain oncogenic fusions, such as BCR-ABL,}

can only transduce HSCs, which may offer insight into malignant mechanisms of different driver oncogenes225_{. Murine models also exist for commonly mutated AML genes, including FLT3-ITD and}

NPM1226_{. Some of these models recapitulate AML differently than in the human disease. For example,}

mice transplanted with FLT3-ITD-expressing cells develop a malignancy more closely resembling MDS, although leukemic latency is similar to other AML models227_.

Xenotransplantation of primary human AML cells into immunodeficient mice is another technique used to model human AML. This has both benefits and limitations compared to retroviral oncogene models. The downside of this technique is that the leukemic phenotype is incomplete. Recipient mice typically

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present some characteristics of AML, including organ infiltration and blast cell proliferation. However, they do not usually develop a lethal disease or show significant leukocytosis within the blood228_.

Furthermore, they require human cytokines (IL-3, SCF, GM-CSF) for survival, making experiments more challenging than retroviral models. However, these studies have yielded important insight into the nature of leukemic stem cells, a topic that will be discussed in the next section229_.

2.2.3 Leukemic stem cells

The existence of cancer stem cells was first demonstrated for human AML, and the currently accepted model is that leukemia organizes itself in a hierarchical manner akin to normal hematopoiesis, with a malignant version of an HSC termed a leukemic stem cell (LSC) which sustains the blast population through self-renewal and limited differentiation (diagram in Figure 2-1). LSCs have been defined similarly to HSCs – they are cells capable of completely reconstituting AML and generating downstream leukemic blasts230_.

The LSC model has significant clinical implications, suggesting that the destruction of LSCs would be necessary and sufficient to cure leukemia, and that elimination of only blasts will not cure the disease as any remaining LSCs will eventually regenerate leukemia in the patient.

Early xenotransplantation studies using human AML cells demonstrated that there were cells capable of LSC activity. Virtually all LSCs were found in the CD34+_CD38- _{population, with a much lower frequency in}

the double positive population229_{. One exception has been observed in karyotypically normal,}

NPM1-mutated AML where there is a significant subset of CD34- _LSCs231_{. Further refinement of LSC}

experiments has elucidated two LSC subtypes that may exist in AML patients. One resembles a more primitive MPP (Lin-_CD34+_CD38-_CD90-_CD45RA+ _{) and another more closely resembles a committed myeloid}

precursor (Lin-_CD34+_CD38+_CD123+_CD45RA+₎232_{. Patients with a high frequency of CD34}+_CD38- _{cells, or}

frequency of calculated LSCs in xenotransplantation experiments, have a worse prognosis and shorter overall survival, confirming the clinical significance of LSCs233,234_.

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Figure 2-1: Leukemic stem cell model in AML. In normal hematopoiesis, HSCs differentiate into

downstream progenitors with progressively lower self-renewal potential and multipotency, resulting in mature cell production. In acute myeloid leukemia, HSCs or other progenitors undergo mutations, epigenetic alterations, or chromosomal translocations which cause enhanced self-renewal capability and a block in differentiation resulting in a decrease in multipotency. The bone marrow compartment begins to fill with blast cells resembling myeloid progenitors (CMPs or GMPs) but with little or no capacity to differentiate into mature cells. This loss of mature cell production is responsible for the symptoms observed in AML.

Different methods have been proposed to specifically target and eliminate LSCs, which would represent a major step forward in AML treatment. LSCs could be targeted with cytotoxic antibodies if they exhibited a specific surface expression pattern. In humans, CD123235_{, CD47}236,237_{, TIM3}238_{, and IL1RAP}239 _are

overexpressed in LSCs relative to healthy HSPCs and may serve as potential targets. LSCs also have a distinct biology from HSCs, and therefore targeting intracellular pathways inherent to LSCs could eliminate them, leaving healthy progenitors intact. The proinflammatory NF-kB pathway is upregulated in CD34+

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AML cells compared to CD34+ _{HSCs and is important for their survival, as inhibition of NF-kB activity by}

preventing degradation of IkB inhibitors causes cell death in LSCs with no effect on HSCs240_{. LSCs also have}

significantly altered epigenetics, possibly a result of mutated epigenetic regulators including TET2, MLL, and DNMT3A, described in Section 2.3. These changes may induce leukemogenesis by blocking normal differentiation and enhancing self-renewal241_{. Inhibition of BRD4, a “reader” of modified histones,}

promotes terminal differentiation of AML cells and reduces leukemic burden in mouse models of AML242_.

Lastly, another aspect of LSC biology believed to be important for their survival is the maintenance of low reactive oxygen species (ROS) levels. LSCs have high expression of the anti-apoptotic BCL-2 protein and require its expression for their survival. Inhibition of BCL-2 elevates oxidative phosphorylation and ROS levels in LSCs, leading to cell death243,244_{. These pathways illustrate some of the important differences}

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