Self-Renewal Pathways in Acute Myeloid Leukemia Stem Cells

*Jonason Yang, Nunki Hassan, Sheng Xiang Franklin Chen, Jayvee Datuin and Jenny Y. Wang*

### **Abstract**

Acute myeloid leukemia (AML) is a difficult-to-treat blood cancer. A major challenge in treating patients with AML is relapse, which is caused by the persistence of leukemia stem cells (LSCs). Self-renewal is a defining property of LSCs and its deregulation is crucial for re-initiating a new leukemia after chemotherapy. Emerging therapeutic agents inhibiting aberrant self-renewal pathways, such as anti-RSPO3 monoclonal antibody discovered in our recent study, present significant clinical potential that may extend beyond the scope of leukemogenesis. In this chapter, we provide an overview of normal and malignant hematopoietic stem cells, discuss current treatments and limitations, and review key self-renewal pathways and potential therapeutic opportunities in AML.

**Keywords:** acute myeloid leukemia, leukemia stem cells, self-renewal, signaling, WNT, β-Catenin, G protein-coupled receptor, GPR84, LGR4, RSPO3, targeted therapy

### **1. Introduction**

AML is a heterogenous clonal disorder characterized by blocked differentiation and increased proliferation of hematopoietic progenitors. The heterogeneity of AML can be attributed to diverse driver mutations that may be present in combination with multiple cells of origin and epigenetic abnormalities [1–3]. The main treatment for AML is chemotherapy, which kills rapidly dividing leukemic blasts but is ineffective against quiescent, self-renewing LSCs leading to relapse and poor clinical outcomes. The discovery of LSCs not only as the origin but also the culprit of therapeutic resistance in AML is a milestone in our understanding of malignancy and thus targeting LSCs is a critical and challenging step in developing anticancer therapy [4]. Recent evidence has shown that aberrant activation of self-renewal pathways, such as WNT/β-catenin and RSPO3-LGR4 pathways, is essential for the initiation and development of LSCs [5, 6], unveiling a potential target for curative therapies in AML.

#### **2. Normal and malignant hematopoietic stem cells**

#### **2.1 Hematopoietic stem cells**

Normal hematopoiesis is a hierarchically organized process where hematopoietic stem cells (HSCs) can self-renew to produce new copies of themselves via symmetric division or differentiate into lineage-committed progenitors via asymmetric division [7], which ultimately give rise to all blood lineage cells (**Figure 1**). A tight balance between self-renewal and differentiation is critical for sustaining the functional integrity of hematopoiesis, which prevents HSC exhaustion or hematologic malignancies such as leukemia. HSCs preferentially reside in a hypoxic microenvironment within the bone marrow in which they are maintained in a quiescent state [8]. Quiescence is important to preserve the genetic integrity of HSCs during adult homeostasis as frequent DNA replication may incur oncogenic mutations. Therefore, dysregulation of key HSC properties is essential for leukemia initiation as they enable the accumulation of genetic lesions and promote malignant transformation.

## **2.2 Leukemia stem cells**

The discovery of the first cancer stem cell in AML two decades ago has led to a paradigm shift in our understanding of cancer cell biology and the way cancer can be treated and cured [9]. Cancer stem cells have been subsequently identified in a variety of tumors. Similar to normal HSCs, LSCs are a subpopulation of leukemic cells, which reside at the apex of a malignant hierarchy and possess the ability to self-renew and to differentiate into non-LSC bulk blasts [10]. LSCs with unlimited self-renewal capacity and chemoresistance are responsible for disease initiation and progression and are believed to be the root cause of cancer relapse. It was initially hypothesized that only HSCs could undergo malignant transformation into LSCs due to their inherent ability to self-renew. However, the study of acute promyeloid leukemia (APML), a subset of AML, showed that committed progenitors might be capable of developing into LSCs. The APML-associated fusion gene PML/ retinoic acid receptor-α (RARA), resulting from the t(15, 17) balanced reciprocal translocation, was present in CD34-CD38+ cell population but not in CD34+CD38 cell population [11]. As APML only represents a unique subset of AML, further

#### **Figure 1.**

*Hierarchical organization of normal hematopoiesis. HSCs are categorized as long-term HSCs, which are highly self-renewing cells that last a lifespan, and short-term HSCs that self-renew for a limited period and differentiate into multipotent progenitor (MPP). MPP differentiates further into lineage-restricted progenitors including common lymphoid progenitor (CLP) and common myeloid progenitor (CMP) from which all mature blood cells arise. NK, natural killer.*

**99**

**Figure 2.**

*remission.*

*Self-Renewal Pathways in Acute Myeloid Leukemia Stem Cells*

studies looked at mixed-lineage leukemia (MLL) fusion proteins and isolated cell populations of committed progenitors with MLL fusion mutations to examine their leukemogenesis potential. By injecting sublethally irradiated mice with the aforementioned cell population, leukemogenesis was observed and thus the ectopic renewal of genes in committed progenitors associated with self-renewal is a definite possibility [12, 13]. The cancer stem cell model implies that eradication of LSCs is crucial for developing relapse-free therapies and to achieve a long-term remission in AML. However, eradication of LSCs remains a hefty challenge, whose difficulty lies in the therapeutic targeting of key oncogenic pathways driving LSC formation and

maintenance without affecting normal adult HSCs and hematopoiesis.

Chemotherapy remains the first line treatment for AML, which has been relatively unchanged since its inception more than four decades ago. The chemotherapy drugs often used for AML treatment are a combination of cytarabine and an anthracycline drug such as daunorubicin. Insufficient inhibition of quiescent LSCs may be the culprit behind the failure of chemotherapy for the treatment of AML. Similar to their normal HSC counterpart, most LSCs remain quiescent or in the G0 phase of the cell cycle [14, 15]. This proves challenging as chemotherapy only interferes with DNA replication via DNA polymerase inhibition (e.g. cytarabine) or with DNA restructuring via topoisomerase II inhibition (e.g. daunorubicin) to induce apoptotic cell death in actively replicating cells [16, 17]. It is noted that LSCs can retain chemoresistant adaptations present in HSCs such as expression of P-glycoprotein, an efflux pump that may export chemotherapeutic agents and is associated with multidrug resistance and poor disease outcomes [18]. Consequently, most chemotherapy-induced cell deaths occur within leukemic blasts rather than in LSCs. Thus, the residual disease within patients is often characterized by the subpopulation of LSCs which are often quiescent and resistant to conventional chemotherapy leading to relapse (**Figure 2A**). This highlights the need for stem cell-targeted therapies so that the subpopulation of LSCs can be

**3. Cause of chemotherapy failure: leukemia stem cells**

eliminated allowing for long-term remission (**Figure 2B**).

*Conventional chemotherapy versus targeted therapies for AML treatment. (A) Chemotherapy is unable to eradicate LSCs, leading to relapse. (B) Targeted therapy specifically kills LSCs, resulting in relapse-free* 

*DOI: http://dx.doi.org/10.5772/intechopen.94379*

*Self-Renewal Pathways in Acute Myeloid Leukemia Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.94379*

*Acute Leukemias*

**2.2 Leukemia stem cells**

division or differentiate into lineage-committed progenitors via asymmetric division [7], which ultimately give rise to all blood lineage cells (**Figure 1**). A tight balance between self-renewal and differentiation is critical for sustaining the functional integrity of hematopoiesis, which prevents HSC exhaustion or hematologic malignancies such as leukemia. HSCs preferentially reside in a hypoxic microenvironment within the bone marrow in which they are maintained in a quiescent state [8]. Quiescence is important to preserve the genetic integrity of HSCs during adult homeostasis as frequent DNA replication may incur oncogenic mutations. Therefore, dysregulation of key HSC properties is essential for leukemia initiation as they enable the accumulation of genetic lesions and promote malignant transformation.

The discovery of the first cancer stem cell in AML two decades ago has led to a paradigm shift in our understanding of cancer cell biology and the way cancer can be treated and cured [9]. Cancer stem cells have been subsequently identified in a variety of tumors. Similar to normal HSCs, LSCs are a subpopulation of leukemic cells, which reside at the apex of a malignant hierarchy and possess the ability to self-renew and to differentiate into non-LSC bulk blasts [10]. LSCs with unlimited self-renewal capacity and chemoresistance are responsible for disease initiation and progression and are believed to be the root cause of cancer relapse. It was initially hypothesized that only HSCs could undergo malignant transformation into LSCs due to their inherent ability to self-renew. However, the study of acute promyeloid leukemia (APML), a subset of AML, showed that committed progenitors might be capable of developing into LSCs. The APML-associated fusion gene PML/ retinoic acid receptor-α (RARA), resulting from the t(15, 17) balanced reciprocal translocation, was present in CD34-CD38+ cell population but not in CD34+CD38 cell population [11]. As APML only represents a unique subset of AML, further

*Hierarchical organization of normal hematopoiesis. HSCs are categorized as long-term HSCs, which are highly self-renewing cells that last a lifespan, and short-term HSCs that self-renew for a limited period and differentiate into multipotent progenitor (MPP). MPP differentiates further into lineage-restricted progenitors including common lymphoid progenitor (CLP) and common myeloid progenitor (CMP) from which all mature* 

**98**

**Figure 1.**

*blood cells arise. NK, natural killer.*

studies looked at mixed-lineage leukemia (MLL) fusion proteins and isolated cell populations of committed progenitors with MLL fusion mutations to examine their leukemogenesis potential. By injecting sublethally irradiated mice with the aforementioned cell population, leukemogenesis was observed and thus the ectopic renewal of genes in committed progenitors associated with self-renewal is a definite possibility [12, 13]. The cancer stem cell model implies that eradication of LSCs is crucial for developing relapse-free therapies and to achieve a long-term remission in AML. However, eradication of LSCs remains a hefty challenge, whose difficulty lies in the therapeutic targeting of key oncogenic pathways driving LSC formation and maintenance without affecting normal adult HSCs and hematopoiesis.

#### **3. Cause of chemotherapy failure: leukemia stem cells**

Chemotherapy remains the first line treatment for AML, which has been relatively unchanged since its inception more than four decades ago. The chemotherapy drugs often used for AML treatment are a combination of cytarabine and an anthracycline drug such as daunorubicin. Insufficient inhibition of quiescent LSCs may be the culprit behind the failure of chemotherapy for the treatment of AML. Similar to their normal HSC counterpart, most LSCs remain quiescent or in the G0 phase of the cell cycle [14, 15]. This proves challenging as chemotherapy only interferes with DNA replication via DNA polymerase inhibition (e.g. cytarabine) or with DNA restructuring via topoisomerase II inhibition (e.g. daunorubicin) to induce apoptotic cell death in actively replicating cells [16, 17]. It is noted that LSCs can retain chemoresistant adaptations present in HSCs such as expression of P-glycoprotein, an efflux pump that may export chemotherapeutic agents and is associated with multidrug resistance and poor disease outcomes [18]. Consequently, most chemotherapy-induced cell deaths occur within leukemic blasts rather than in LSCs. Thus, the residual disease within patients is often characterized by the subpopulation of LSCs which are often quiescent and resistant to conventional chemotherapy leading to relapse (**Figure 2A**). This highlights the need for stem cell-targeted therapies so that the subpopulation of LSCs can be eliminated allowing for long-term remission (**Figure 2B**).

#### **Figure 2.**

*Conventional chemotherapy versus targeted therapies for AML treatment. (A) Chemotherapy is unable to eradicate LSCs, leading to relapse. (B) Targeted therapy specifically kills LSCs, resulting in relapse-free remission.*
