Escape From Treatment; the Different Faces of Leukemic Stem Cells and Therapy Resistance in Acute Myeloid Leukemia

Abstract

Standard induction chemotherapy, consisting of an anthracycline and cytarabine, has been the first-line therapy for many years to treat acute myeloid leukemia (AML). Although this treatment induces complete remissions in the majority of patients, many face a relapse (adaptive resistance) or have refractory disease (primary resistance). Moreover, older patients are often unfit for cytotoxic-based treatment. AML relapse is due to the survival of therapy-resistant leukemia cells (minimal residual disease, MRD). Leukemia cells with stem cell features, named leukemic stem cells (LSCs), residing within MRD are thought to be at the origin of relapse initiation. It is increasingly recognized that leukemia "persisters" are caused by intra-leukemic heterogeneity and non-genetic factors leading to plasticity in therapy response. The BCL2 inhibitor venetoclax, combined with hypomethylating agents or low dose cytarabine, represents an important new therapy especially for older AML patients. However, often there is also a small population of AML cells refractory to venetoclax treatment. As AML MRD reflects the sum of therapy resistance mechanisms, the different faces of treatment "persisters" and LSCs might be exploited to reach an optimal therapy response and prevent the initiation of relapse. Here, we describe the different epigenetic, transcriptional, and metabolic states of therapy sensitive and resistant AML (stem) cell populations and LSCs, how these cell states are influenced by the microenvironment and affect treatment outcome of AML. Moreover, we discuss potential strategies to target dynamic treatment resistance and LSCs.

Keywords: acute myeloid leukemia; leukemic stem cells; minimal residual disease; plasticity; therapy resistance.

Figure 1

The role of minimal residual disease, therapy resistance and LSCs in AML relapse development. In normal hematopoiesis (green box), quiescent hematopoietic stem cells (HSCs) with self-renewal capacity give rise to multipotent progenitors (MPPs), which can differentiate towards lymphoid primed multipotent progenitors (LMPPs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-macrophage progenitors (GMPs) and megakaryocyte erythroid progenitors (MEP). These lineage-committed progenitors can produce terminally differentiated lymphoid, myeloid and erythroid blood cells. AML originates from the transformation of normal HSCs, MPPs or more committed progenitors, developing in leukemic stem cells (LSCs) that subsequently can give rise to full-blown leukemia. AML initiated from HSC and MPP highly express the transcription factor EVI-1. At AML diagnosis (blue box), a heterogeneous leukemia cell population with a variety of sensitivities to therapy exists. Moreover, LSCs and normal hematopoietic (stem) cells, responsible for reconstituting the normal healthy blood cells after therapy, co-reside in the patient’s bone marrow. While treatment with standard induction chemotherapy results in complete remission in the majority of AML patients, a population of (chemo)therapy-resistant cells (TRCs) (minimal residual disease) constituting AML cells with leukemia-initiating potential survive the treatment. LSCs with leukemia-initiating potential within MRD could initiate sooner or later a relapse. Instead of (chemo)therapeutic selection of pre-existing subpopulations of LSCs, AML cells might adaptively obtain a leukemia re-initiating cell (LRC)-phenotype upon exposure to treatment.

Figure 2

The different processes involved in therapy resistance in AML (stem) cells. Plasticity and therapy resistance: Within the hypoxic BM niche, persistent AML (stem) cells might exist prior to drug treatment or might become resistant and obtain leukemia re-initiating potential upon exposure to a treatment, such as chemotherapy or venetoclax. The transcriptional signature of AML LSCs is associated with “stemness” and leukemia-initiating capacity. LSCs aberrantly express lymphoid and myeloid antigens, including CD123, CCL1, CD25, CD32, CD44, GPR56 and IL1RAP. These cell surface markers differ within and between individual AML patients and can change during the course of the disease. Different processes are involved to induce therapy resistance: Epigenetically regulated therapy resistance (yellow box): LSCs and therapy-resistant AML cells show modulated expression of components of the PRC2 complex (i.e. EZH1/2), upregulation of BET proteins (i.e. Brd4), and altered methylation profile caused by enhanced HDM activity (i.e. LSD1, KDM2, KDM4, and KDM6) and HMT activity (i.e. DOT1L and G9a). These differential epigenetic processes induce transcriptional deregulation of genes, like MEIS1, Myc and HOXA. Microenvironment and therapy resistance (green box): In response to hypoxia, HIF-1α signaling promotes expression of VEGF, CXCR4, and SCF. CXCR4 on AML cells interact with CXCL12, increasing stromal protective effects. VEGF expressing ECs protect VEGFR3-expressing AML cells from chemotherapy-induced apoptosis, due to increased BCL2/Bax ratios. LSCs express VLA-4, VLA-5 and LFA-1 on their cell surface, which interact with the stromal ligands VCAM-1, ICAM-1 and fibronectin to support attachment to stromal cells, promoting NF-κB signaling. SCF enhances anti-apoptotic and proliferative effects of fibronectin expressed on AML cells. Pro-inflammatory cytokines, including TNFα, influence cell adhesion, promoting LSC survival and chemotherapy resistance through modulation of NF-κB signaling. Several members of the TGFβ family suppress proliferation of AML cells and enhance chemotherapy resistance. Metabolism and therapy resistance (orange box): AML LSCs often lack the ability to enhance glycolysis and therefore switch from anaerobic glycolysis to mitochondria-mediated OXPHOS to generate ATP. Therapy-resistant AML cells show increased mitochondrial mass and high OXPHOS. In addition to glucose, FFA can by metabolized to drive the TCA cycle and OXPHOS. Adipocytes, the major MSC present in the BM, support survival of AML cells by stimulating FAO and OXPHOS through fatty acid transfer. Part of the LSC population expresses the fatty acid transporter CD36 to control uptake of FFA. CPT1, regulated by PPAR, controls FAO translocation by conjugating FFA with carnitine for translocation into the mitochondrial matrix. FABP4 is involved in the interaction of adipocytes with LSCs. Furthermore, LSCs are able to reduce ROS production generated by mitochondria in response to hypoxia, by activation of autophagy. Inhibition of BCL2 by venetoclax efficiently reduces LSC survival by impairing homeostasis and inhibiting OXPHOS. Integrated Stress Response induced resistance (blue box): In response to stress stimuli, such as ROS, the PERK-eIF2α ISR pathway is activated in LSCs. eIF2α is phosphorylated by GCN2 or PERK, reducing global protein synthesis while allowing translation of specific genes, including ATF4. Increased activity of the ISR pathway protects LSCs to enable restoration of homeostasis favoring survival. Signaling and therapy resistance (purple box): Upregulation of FOXM1 activates the Wnt/β-catenin signaling pathway by direct binding to β-catenin, inhibiting its degradation, preserving LSC quiescence and promoting LSC self-renewal. Overexpression of miR-126 repress multiple components of the PI3K/Akt pathway, resulting in the proliferation of LSCs, delayed G₀ exit of LSCs and enhances resistance to combination chemotherapy. AML, acute myeloid leukemia; ac, acetyl group; ATF4, activating transcription factor 4; ATP, adenosine tri-phosphate; BET, bromodomain and extra-terminal motif; Brd4, bromodomain-containing 4; BM, bone marrow; CCL1, C-type lectin-like molecule 1; CPT1, carnitine O-palmitoyltransferase 1; CXCR4, CXC chemokine receptor-type 4; CXCL12, CXC motif chemokine ligand 12; eIF2α, eukaryotic initiation factor 2α; EC, endothelial cells; EED, embryonic ectoderm development; EZH1/2, zeste homolog 1 or 2; FAO, fatty acid oxidation; FABP4, fatty acid binding protein 4; FFA, free fatty acids; GCN2, general control non-derepressible 2; HDM, histone demethylase; HIF-1α, hypoxia-inducing factor 1α; HMT, histone methyl transferase; HSC, hematopoietic stem cell; ICAM-1, intracellular adhesion molecule 1; IL3, interleukin 3; ISR, integrated stress response; KDM, histone lysine demethylase; LFA-1, lymphocyte function-associated antigen 1; LSD1, lysine-specific histone demethylase 1; LSCs, leukemic stem cells; me, methyl group; MSC, mesenchymal stromal cells; NF-κB, nuclear factor κB; P, phosphorylation; PPAR, peroxisome proliferator-activated receptor; PERK, protein kinase RNA-like ER kinase; PI3K, phosphatidylinositol 3-kinase; PRC2, polycomb complex 2; ROS, reactive oxygen species; SCF, stem cell factor; SUZ12, suppressor of zeste 12; TCA, tricarboxylic acid cycle; TGFβ, transforming growth factor β; tmTNFα, transmembrane tumor necrosis factor α; VCAM-1, vascular cell adhesion molecule 1; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VLA-4, very late antigen-4; VLA-5, very late antigen-5.