**4.2 Genetic pathways of LSCs**

### **4.2.1 Wnt/Catenin**

The Wnt/Catenin signaling has been implicated in the self-renewal of LSCs. Wnt proteins are a large family of glycoproteins that bind to Frizzled receptors and LRP5/6 coreceptors. By stabilizing the mediator β-catenin, they start a complex signaling cascade that plays a significant role in regulating cell proliferation and differentiation. Wnt cascade has appeared as a critical regulator of stem cells self-renewal. Comparing the expression of normal hematopoietic stem cells to that of AML leukemic stem cells, evidences show that the Wnt signaling pathway is aberrantly regulated in leukemic stem cells.

Hematopoietic Stem Cell in Acute Myeloid Leukemia Development 271

In addition to genes involved in the control of stem cell self-renewal, leukemic stem cells are expected to express, at high levels, genes involved in anti-apoptotic mechanisms. In this context, particular attention has been focused on the study of NF-kB. NF-kB plays a critical role in inflammation, anti-apoptotic responses, and carcinogenesis. High NF-kB expression was found in primitive AML blasts. In particular, the constitutive activation of NF-kB was observed in AML cell populations enriched in leukemic stem cells, but not in normal hematopoietic stem cells. According to these observations, it seemed clear that NF-kB could be a potential

BMI1 is a polycomb group protein which, together with Ring1 proteins, is part of PRC1 complex that has histone H2A-K119 ubiquitin E3 ligase activity. BMI1 has a role in HOX gene (HOXC13) silencing by H2A ubiquitylation (Cao et al, 2005). BMI1 is also known to be important in the regulation and maintenance of proliferative/self-renewal potential in both normal hematopoietic and leukemic stem cells (Park et al, 2003). Upon knockdown of BMI-1, cells lose their ability to engraft and reconstitute leukemia in mice (Bomken et al., 2010).

A key component for understanding the biological mechanisms for tumor heterogeneity is the ability to functionally assess the capacity for limitless proliferative capacity for segregated populations of tumor cells. Unfortunately, for hematologic malignancies, *in vitro* culture assays are not entirely effective as a means of functionally assessing self-renewal capacity. Thus, transplantation assays in which candidate populations are assessed for their ability to establish long-term serial engraftment of recipient animals is the gold standard for assigning limitless proliferative capability, i.e. self-renewal. For murine studies, the availability of syngeneic transplantation models has been responsible in large part for our in

Since the 70 s, there is ample evidence supporting the existence of a discrete compartment of slowly cycling leukemic cells that are resistant to standard chemotherapeutic agents. These cell populations were felt to represent the leukemic stem cells and though many observations were consistent with this hypothesis, there was no direct evidence that this was indeed the case. As the first direct evidence for the existence of cancer stem cells, the work of Lapidot et al. (1994) represented a milestone in the history of the leukemic stem cell model. This study identified an infrequent population of leukemic cells capable of recapitulating the human tumor in xenotransplants. A key finding was that the SCID mouse leukemia repopulating cell, SRC, possessed a phenotype that was similar to that of the normal

Acute myeloid leukemia (AML) is a clonal disorder characterized by arrest of differentiation in the myeloid lineage coupled with an accumulation of immature progenitors in the bone marrow, resulting in hematopoietic failure (Pollyea et al., 2011). AML is the most common

depth understanding of the normal murine hematopoietic hierarchy.

therapeutic target for attempting leukemia stem cell eradication (Guzman et al., 2001).

**4.2.3 NF-kB** 

**4.2.4 BMI1** 

**4.3 Xenotransplantation model of leukemia** 

hematopoietic stem cell (CD34+ and CD38−).

**4.4 Acute myeloid leukemia** 

Fig. 6. LSCs main surface markers. LSCs can be defined as CD34, CD382, HLA-DR2, CD902, CD117 and CD123. The expression of CD123 seems to be leukemic-specific. Another LSC specific antigen is C-type lectin-like molecule-1, CLL-1.

#### **4.2.2 PTEN (Akt/mTOR)**

PTEN is a phosphatase that negatively regulates signaling through the PI3K pathway, attenuating proliferation and survival signals. PTEN deficiency causes an initial expansion of normal hematopoietic stem cells due to their cycling, followed by their exhaustion. In contrast to this requirement for PTEN in the maintenance of hematopoietic stem cells, leukemic stem cells arise and expand in numbers following PTEN deletion. The observation that PTEN deletion had opposite effects on normal hematopoietic stem cells compared to leukemic stem cells raised possibility for therapeutic targeting of this pathway to eliminate only the leukemic stem cells, without affecting normal hematopoietic stem cells. Since PTEN deletion causes increased AKT and mTOR activation, it seems logical that mTOR targeting by pharmacological agents, such as rapamycin, could represent an interesting option for AML treatment (Testa, 2011).
