**3.3 LSC display higher self-renewal ability**

Hope et al. proved that LSCs have self-renewal ability, which is one of the most prominent features of CSCs [37]. The self-renewal ability of LSCs may be one of the key factors that promote the development and metastasis of leukemia, and the molecular regulation mechanism is very complicated. Bmi-1 is a member of the PcG (polycomb group) transcriptional repressor family and is an essential factor in maintaining HSC self-renewal. Raffel et al. showed that miR-126 overexpression renders AML cells more resistant to standard chemotherapy and that treatment of primary AML cells results in the enrichment of LSC-like cells with increased

miR-126 levels [38]. Moreover, leukemic cells with high miR-126 expression were selected in refractory patients after induction chemotherapy, thus correlating high miR-126 levels to LSCs and therapy resistance. miR-126 knockdown leads to the expansion of HSCs but impaired maintenance of LSCs, and its overexpression promotes LSC self-renewal, which is inhibited in HSCs [39, 40]. In addition, all genes and signaling pathways that contribute to HSC self-renewal may be involved in LSCs, such as Wnt, Notch, HOX, and Shh. Recent studies have revealed that the activation of the Shh signaling pathway in LSCs by upregulation of SMO is essential for LSC survival maintenance [41, 42].

### **3.4 The special microenvironment (niche)**

The receptors CXCR4 on the LSC membrane and CXCL12 in the bone marrow microenvironment are required for LSC to maintain dormancy, self-renewal, differentiation, growth, and homing. However, targeted therapy for the niche will enhance the expression of the drug pump MDR1, which induced LSC insensitive to therapy and failed to achieve the goal of reversing its resistance [43].

### **3.5 Multiple signaling pathway abnormalities**

Recent studies have demonstrated that abnormal activation of multiple signaling pathways is one of the key mechanisms of LSC multidrug resistance, such as Sonic Hedgehog, Bmi-1, Nocth, and WNT. Among these pathways, the abnormality of Hedgehog (Hh) pathway is closely related to CSC resistance, such as increased endogenous synthesis of ligand protein Hh, loss of PCTH activity, inhibition of smoothened (SMO) signaling protein, mutation of SUFU, and overexpression of the transcription factor GLI1, thus regulating the downstream target gene and participating in the maintenance of stem cell proliferation, which are related to multiple hallmarks of tumor cell resistance [44, 45]. Studies have revealed that Hh signaling is abnormally activated in LSCs, GLI1 can induce endogenous BCL-2 expression, and the Hh pathway also up-regulates BCL-2 by activating PI3K/AKT, thus leading to apoptotic disorder and drug resistance of LSCs.
