TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Here we identified T cell immunoglobulin mucin-3 (TIM-3) as a surface molecule expressed on LSCs in most types of AML except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). TIM-3(+) but not TIM-3? AML cells reconstituted human AML in immunodeficient mice, suggesting that the TIM-3(+) population contains most, if not all, of functional LSCs. We established an anti-human TIM-3 mouse IgG2a antibody having complement-dependent and antibody-dependent cellular cytotoxic activities. This antibody did not harm reconstitution of normal human HSCs, but blocked engraftment of AML after xenotransplantation. Furthermore, when it is administered into mice grafted with human AML, this treatment dramatically diminished their leukemic burden and eliminated LSCs capable of reconstituting human AML in secondary recipients. These data suggest that TIM-3 is one of the promising targets to eradicate AML LSCs.     

Proteomic-based identification of Apg-2 as a therapeutic target for chronic myeloid leukemia

The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced “spontaneous” DNA damage and unfaithful repair in Philadelphia chromosome positive leukemia cells. Here, we investigated the changes of protein profile in H₂O₂-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially expressed and 13 proteins were identified with further MS analysis. Two essential proteins, Proto-oncogene tyrosine–protein kinase ABL1 (c-ABL) and Heat shock 70 kDa protein 4 (Apg-2), were confirmed by Western blot and showed consistent changes with proteomic results. Moreover, functional analysis demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL, BaF3-BCR/ABL^T315I). We also proved that Apg-2 inhibition aggravated H₂O₂ induced damage in BCR/ABL positive cells, and enhanced the sensitivity of BaF3-BCR/ABL^T315I to STI571. Taken together, the findings in this work provide us with some clues to a better understanding of the molecular mechanisms underlying BCR/ABL in the DNA damage/repair processes and demonstrated that Apg-2 would be a valid target for anti-leukemia drug development.     

Cancer stem cells as a therapeutic target in bladder cancer

Bladder cancer is one of the most prevalent genitourinary cancers responsible for about 150,000 deaths per year worldwide. Currently, several treatments, such as endoscopic and open surgery, appended by local or systemic immunotherapy, chemotherapy, and radiotherapy are used to treat this malignancy. However, the differences in treatment outcome among patients suffering from bladder cancer are considered as one of the important challenges. In recent years, cancer stem cells, representing a population of undifferentiated cells with stem-cell like properties, have been eyed as a major culprit for the high recurrence rate in superficial papillary bladder cancer. Cancer stem cells have been reported to be resistant to conventional treatments, such as chemotherapy, radiation, and immunotherapy, which induce selective pressure on tumoral populations resulting in selection and growth of the resistant cells. Therefore, targeting the therapeutic aspects of cancer stem cells in bladder cancer may be promising. In this study, we briefly discuss the biology of bladder cancer and then address the possible relationship between molecular biology of bladder cancer and cancer stem cells. Subsequently, the mechanisms of resistance applied by cancer stem cells against the conventional therapeutic tools, especially chemotherapy, are discussed. Moreover, by emphasizing the biomarkers described for cancer stem cells in bladder cancer, we have provided, described, and proposed targets on cancer stem cells for therapeutic interventions and, finally, reviewed some immunotargeting strategies against bladder cancer stem cells.     

Hypoxia selects bortezomib-resistant stem cells of chronic myeloid leukemia

We previously demonstrated that severe hypoxia inhibits growth of Chronic Myeloid Leukemia (CML) cells and selects stem cells where BCR/Abl(protein) is suppressed, although mRNA is not, so that hypoxia-selected stem cells, while remaining leukemic, are independent of BCR/Abl signaling and thereby refractory to Imatinib-mesylate. The main target of this study was to address the effects of the proteasome inhibitor Bortezomib (BZ) on the maintenance of stem or progenitor cells in hypoxic primary cultures (LC1), by determining the capacity of LC1 cells to repopulate normoxic secondary cultures (LC2) and the kinetics of this repopulation. Unselected K562 cells from day-2 hypoxic LC1 repopulated LC2 with rapid, progenitor-type kinetics; this repopulation was suppressed by BZ addition to LC1 at time 0, but completely resistant to day-1 BZ, indicating that progenitors require some time to adapt to stand hypoxia. K562 cells selected in hypoxic day-7 LC1 repopulated LC2 with stem-type kinetics, which was largely resistant to BZ added at either time 0 or day 1, indicating that hypoxia-selectable stem cells are BZ-resistant per se, i.e. before their selection. Furthermore, these cells were completely resistant to day-6 BZ, i.e. after selection. On the other hand, hypoxia-selected stem cells from CD34-positive cells of blast-crisis CML patients appeared completely resistant to either time-0 or day-1 BZ. To exploit in vitro the capacity of CML cells to adapt to hypoxia enabled to detect a subset of BZ-resistant leukemia stem cells, a finding of particular relevance in light of the fact that our experimental system mimics the physiologically hypoxic environment of bone marrow niches where leukemia stem cells most likely home and sustain minimal residual disease in vivo. This suggests the use of BZ as an enhanced strategy to control CML. in particular to prevent relapse of disease, to be considered with caution and to need further deepening.     

Leukemia stem cells: the root of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by a chromosome translocation that generates the Bcr-Abl oncogene encoding a constitutive kinase activity. Despite remarkable success in controlling CML at chronic phase by Bcr-Abl tyrosine kinase inhibitors (TKIs), a significant proportion of CML patients treated with TKIs develop drug resistance due to the inability of TKIs to kill leukemia stem cells (LSCs) that are responsible for initiation, drug resistance, and relapse of CML. Therefore, there is an urgent need for more potent and safer therapies against leukemia stem cells for curing CML. A number of LSCassociated targets and corresponding signaling pathways, including CaMKII-γ, a critical molecular switch for co-activating multiple LSC-associated signaling pathways, have been identified over the past decades and various small inhibitors targeting LSC are also under development. Increasing evidence shows that leukemia stem cells are the root of CML and targeting LSC may offer a curable treatment option for CML patients. This review summarizes the molecular biology of LSC and itsassociated targets, and the potential clinical application in chronic myeloid leukemia.     

Mitochondrial metabolism in cancer stem cells: a therapeutic target for colon cancer

It has been proposed that the selective elimination of cancer stem cells (CSCs) using targeted therapy could greatly reduce tumor growth, recurrence, and metastasis. To develop effective therapeutic targets for CSC elimination, we aimed to define the properties of CSC mitochondria, and identify CSC-mitochondria-specific targets in colon cancer. We found that colon CSCs utilize mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP. We also found that forkhead box protein 1 (FOXM1)-induced peroxiredoxin 3 (PRDX3) maintains the mitochondrial function, and the FOXM1/PRDX3 mitochondrial pathway maintains survival of colon CSCs. Furthermore, FOXM1 induces CD133 (PROM1/prominin 1) expression, which maintains the stemness of colon CSCs. Together, our findings indicate that FOXM1, PRDX3, and CD133 are potential therapeutic targets for the elimination of CSCs in colon cancer. [BMB Reports 2015; 48(10): 539-540]     

Global dynamics of hematopoietic stem cells and differentiated cells in a chronic myeloid leukemia model

We consider a mathematical model describing evolution of normal and leukemic hematopoietic stem cells (HSC) and differentiated cells in bone marrow. We focus on chronic myeloid leukemia (CML), a cancer of blood cells resulting from a malignant transformation of hematopoietic stem cells. The dynamics are given by a system of ordinary differential equations for normal and leukemic cells. Homeostasis regulates the proliferation of normal HSC and leads the dynamics to an equilibrium. This mechanism is partially efficient for leukemic cells. We define homeostasis by a functional of either hematopoietic stem cells, differentiated cells or both cell lines. We determine the number of hematopoietic stem cells and differentiated cells at equilibrium. Conditions for regeneration of hematopoiesis and persistence of CML are obtained from the global asymptotic stability of equilibrium states. We prove that normal and leukemic cells can not coexist for a long time. Numerical simulations illustrate our analytical results. The study may be helpful in understanding the dynamics of normal and leukemic hematopoietic cells.     

Studies on microRNAs that are correlated with the cancer stem cells in chronic myeloid leukemia

Vascular remodeling is characterized by the aggregation of vascular smooth muscle cells (VSMCs) in intima. Previous studies have demonstrated that dehydroepiandrosterone (DHEA), a steroid hormone, can reverse vascular remodeling. However, it is still far clear that whether and how DHEA participates in the modulation of VSMCs activation and vascular remodeling. VSMCs were obtained from the thoracic aorta of SD rats. Cell proliferation was evaluated by CCK-8 assay and BrdU assay. To measure VSMCs migration activity, a transwell chamber assay was performed. Quantitative real-time RT-PCR and western blot were used to explore the molecular mechanisms. ROS generation by VSMCs was measured by DCF fluorescence. NADPH oxidase activity and SOD activity were measured by the corresponding kits. NF-κB activity was detected by NF-κB luciferase reporter gene assay. A rat carotid artery balloon injury model was built to evaluate the neointimal formation, and plasma PGF2 was measured by ELISA. Our results showed that DHEA significantly inhibited VSMCs proliferation after angiotensin (Ang II) stimulation by down-regulation of NADPH oxidase activity and ERK1/2 phosphorylation. Ang II can increase IL-6 and MCP-1 expression, but DHEA reverses these changes via inhibiting p38-MAPK/NF-κB (p65) signaling pathway. DHEA has no significant effects on VSMCs phenotype transition, but can reduce the neointimal to media area ratio after balloon injury. DHEA can alleviate oxidative stress and inflammation in VSMCs via ERK1/2 and NF-κB signaling pathway, but has no effect on VSMCs phenotype transition. Furthermore, DHEA attenuates VSMCs activation and neointimal formation after carotid injury in vivo. Taken together, DHEA might be a promising treatment for vascular injury under pathological condition.     

Establishment of a novel mesenchymal stem cell-based regimen for chronic myeloid leukemia differentiation therapy

Chronic myeloid leukemia (CML) is characterized by the accumulation of malignant and immature white blood cells which spread to the peripheral blood and other tissues/organs. Despite the fact that current tyrosine kinase inhibitors (TKIs) are capable of achieving the complete remission by reducing the tumor burden, severe adverse effects often occur in CML patients treated with TKIs. The differentiation therapy exhibits therapeutic potential to improve cure rates in leukemia, as evidenced by the striking success of all-trans-retinoic acid in acute promyelocytic leukemia treatment. However, there is still a lack of efficient differentiation therapy strategy in CML. Here we showed that MPL, which encodes the thrombopoietin receptor driving the development of hematopoietic stem/progenitor cells, decreased along with the progression of CML. We first elucidated that MPL signaling blockade impeded the megakaryocytic differentiation and contributed to the progression of CML. While allogeneic human umbilical cord-derived mesenchymal stem cells (UC-MSCs) treatment efficiently promoted megakaryocytic lineage differentiation of CML cells through restoring the MPL expression and activating MPL signaling. UC-MSCs in combination with eltrombopag, a non-peptide MPL agonist, further activated JAK/STAT and MAPK signaling pathways through MPL and exerted a synergetic effect on enhancing CML cell differentiation. The established combinational treatment not only markedly reduced the CML burden but also significantly eliminated CML cells in a xenograft CML model. We provided a new molecular insight of thrombopoietin (TPO) and MPL signaling in MSCs-mediated megakaryocytic differentiation of CML cells. Furthermore, a novel anti-CML treatment regimen that uses the combination of UC-MSCs and eltrombopag shows therapeutic potential to overcome the differentiation blockade in CML.Subject terms: Chronic myeloid leukaemia, Mesenchymal stem cells     

Abnormal lipid synthesis as a therapeutic target for cancer stem cells

Cancer stem cells (CSCs) comprise a subpopulation of cancer cells with stem cell properties, which exhibit the characteristics of high tumorigenicity, self-renewal, and tumor initiation and are associated with the occurrence, metastasis, therapy resistance, and relapse of cancer. Compared with differentiated cells, CSCs have unique metabolic characteristics, and metabolic reprogramming contributes to the self-renewal and maintenance of stem cells. It has been reported that CSCs are highly dependent on lipid metabolism to maintain stemness and satisfy the requirements of biosynthesis and energy metabolism. In this review, we demonstrate that lipid anabolism alterations promote the survival of CSCs, including de novo lipogenesis, lipid desaturation, and cholesterol synthesis. In addition, we also emphasize the molecular mechanism underlying the relationship between lipid synthesis and stem cell survival, the signal trans-duction pathways involved, and the application prospect of lipid synthesis reprogramming in CSC therapy. It is demonstrated that the dependence on lipid synthesis makes targeting of lipid synthesis metabolism a promising therapeutic strategy for eliminating CSCs. Targeting key molecules in lipid synthesis will play an important role in anti-CSC therapy.     

TIM-3 as a therapeutic target for malignant stem cells in acute myelogenous leukemia

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Recent studies have shown that many AML LSC-specific surface antigens could be such candidates. T cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML, except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). In mouse models reconstituted with human AML LSCs or human hematopoietic stem cells, a human TIM-3 mouse IgG2a antibody with complement-dependent and antibody-dependent cellular cytotoxic activities eradicates AML LSCs in vivo but does not affect normal human hematopoiesis. Thus, TIM-3 is one of the promising targets to eradicate AML LSCs.     

Molecular mechanisms for survival regulation of chronic myeloid leukemia stem cells

Studies on chronic myeloid leukemia (CML) have served as a paradigm for cancer research and therapy. These studies involve the identifi cation of the fi rst cancer-associated chromosomal abnormality and the subsequent development of tyrosine kinase inhibitors (TKIs) that inhibit BCR-ABL kinase activity in CML. It becomes clear that leukemia stem cells (LSCs) in CML which are resistant to TKIs, and eradication of LSCs appears to be extremely diffi cult. Therefore, one of the major issues in current CML biology is to understand the biology of LSCs and to investigate why LSCs are insensitive to TKI monotherapy for developing curative therapeutic strategies. Studies from our group and others have revealed that CML LSCs form a hierarchy similar to that seen in normal hematopoiesis, in which a rare stem cell population with limitless selfrenewal potential gives rise to progenies that lack such potential. LSCs also possess biological features that are different from those of normal hematopoietic stem cells (HSCs) and are critical for their malignant characteristics. In this review, we summarize the latest progress in CML field, and attempt to understand the molecular mechanisms of survival regulation of LSCs.     

Chronic myeloid leukemia: proving ground for cancer stem cells

A central question in cancer biology is whether malignancy arises in self-renewing tissue stem cells that suffer oncogene activation or in differentiated cells that acquire properties of unremitting self-renewal? In two papers, Weissman and colleagues document both mechanisms: chronic leukemia arising by mutation affecting the hematopoietic stem cell (HSC) and acute leukemia evolving from committed granulocyte-macrophage progenitors that have acquired the self-renewal machinery of HSCs.     

Impaired immunomodulatory function of chronic myeloid leukemia cancer stem cells and the possible mechanism involved in it

Backgroud

Cancer stem cells (CSCs) are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Development of specific therapies targeted at CSCs holds hope for the improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. This is particularly true in chronic myeloid leukemia (CML).

Methods

In this study, we isolated fetal liver kinase-1-positive (Flk1⁺) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph⁺) patients with stem cells property. We examined their biological characteristics as well as immunological function and further detected the possible molecular mechanism involved in the leukemia genesis.

Results

We showed that CML patient-derived Flk1⁺CD31^?CD34^? MSCs had normal morphology, phenotype and karyotype but appeared impaired immunomodulatory function. The capacity of Flk1⁺CD31^?CD34^? MSCs from CML patients to inhibit T lymphocyte activation and proliferation was impaired in vitro. CML patient-derived MSCs have dampening immunomodulatory functions, suggesting that the dysregulation of hematopoiesis and immune response might originate from MSCs rather than HSCs. These Ph⁺ putative CML hemangioblast upregulated TGF-β1 and resultantly activated matrix metalloproteinase-9 (MMP-9) to enhance s-KitL and s-ICAM-1 secretion, which activated c-kit⁺ HSCs from the quiescent state to proliferative state. Further studies showed that phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was involved in CML pathogenesis.

Conclusions

Flk1⁺CD31^?CD34^? MSCs that express BCR/ABL leukemia oncogene are CSCs of CML and they play a critical role in the progression of CML through PI3K/Akt/NF-κB/MMP-9/s-ICAM-1/s-KitL signaling pathway beyond HSCs.     

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.     

Update on acute myeloid leukemia stem cells:New discoveries and therapeutic opportunities

The existence of cancer stem cells has been wellestablished in acute myeloid leukemia. Initial proof of the existence of leukemia stem cells(LSCs) was accomplished by functional studies in xenograft models making use of the key features shared with normal hematopoietic stem cells(HSCs) such as the capacity of self-renewal and the ability to initiate and sustain growth of progenitors in vivo. Significant progress has also been made in identifying the phenotype and signaling pathways specific for LSCs. Therapeutically, a multitude of drugs targeting LSCs are in different phases of preclinical and clinical development. This review focuses on recent discoveries which have advanced our understanding of LSC biology and provided rational targets for development of novel therapeutic agents. One of the major challenges is how to target the selfrenewal pathways of LSCs without affecting normal HSCs significantly therefore providing an acceptable therapeutic window. Important issues pertinent to the successful design and conduct of clinical trials evaluating drugs targeting LSCs will be discussed as well.     

Deletion of Alox5 gene decreases osteogenic differentiation but increases adipogenic differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) have great potential in bone tissue engineering to repair large bone defects. Before their clinical application, investigations are needed to discover the genes and osteoconductive scaffolds that influence their differentiation toward an osteogenic lineage. Alox5 plays controversial and complex roles in the regulation of bone and fat metabolism. To detect the effect of Alox5 on osteogenic and adipogenic differentiation of iPSCs, both Alox5 knockout mouse iPSCs (Alox5-KO-iPSCs) and wild-type mouse iPSCs (Wild-iPSCs) were developed. The mRNA levels of many osteogenic markers in Alox5-KO-iPSCs were significantly reduced, while many adipogenic markers were enhanced. Furthermore, when implanted in rat cranial critical-sized defects with collagen/chitosan/hydroxyapatite scaffolds (CCHS), Alox5-KO-iPSCs produced significantly less new bone than Wild-iPSCs and both cell-scaffold groups had no tumor formation. There was a significant difference in the expression of Cox2 during the osteogenic and adipogenic differentiation between the two kinds of iPSCs in vitro. In conclusion, firstly, Alox5 knockout reduced the osteogenic but increased the adipogenic differentiation potential of mouse iPSCs. These disorders might be related to the change of Cox2 expression. Secondly, combined with iPSCs, CCHS can serve as a potential substrate to repair critical-sized bony defects. However, more studies are required to confirm the mechanisms through which Alox5 affects the osteogenic and adipogenic abilities of iPSCs in vivo and the effect of Cox2 inhibition in this system.     

Scd1 plays a tumor-suppressive role in survival of leukemia stem cells and the development of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is derived from a stem cell, and it is widely accepted that the existence of leukemia stem cells (LSCs) is one of the major reasons for the relapse of CML treated with kinase inhibitors. Key to eradicating LSCs is to identify genes that play a critical role in survival regulation of these stem cells. Using BCR-ABL-induced CML mouse model, here we show that expression of the stearoyl-CoA desaturase 1 (Scd1) gene is downregulated in LSCs and that Scd1 plays a tumor-suppressive role in LSCs with no effect on the function of normal hematopoietic stem cells. Deletion of Scd1 causes acceleration of CML development and conversely overexpression of Scd1 delays CML development. In addition, using genetic approaches, we show that Pten, p53, and Bcl2 are regulated by Scd1 in LSCs. Furthermore, we find that induction of Scd1 expression by a PPARγ agonist suppresses LSCs and delays CML development. Our results demonstrate a critical role for Scd1 in functional regulation of LSCs, providing a new anti-LSC strategy through enhancing Scd1 activity.     

Spred2 is involved in imatinib-induced cytotoxicity in chronic myeloid leukemia cells

Spreds, a recently established class of negative regulators of the Ras-ERK (extracellular signal-regulated kinase) pathway, are involved in hematogenesises, allergic disorders and tumourigenesis. However, their role in hematologic neoplasms is largely unknown. Possible effects of Spreds on other signal pathways closely related to Ras-ERK have been poorly investigated. In this study, we investigated the in vitro effects of Spred2 on chronic myeloid leukemia (CML) cells. In addition to inhibiting the well-established Ras-ERK cascade, adenovirus-mediated Spred2 over-expression inhibits constitutive and stem cell factor (SCF)-stimulated sphingosine kinase-1 (SPHK1) and Mcl-1 expression, as well as inhibiting proliferation and inducing apoptosis in CML cells. In K562 cells and primary CML cells, imatinib induces endogenous Spred2 expression. Spred2 silencing by stable RNA interference partly protects K562 cells against imatinib-induced apoptosis. Together, these data implicate Spred2 in imatinib-induced cytotoxicity in CML cells, possibly by inhibiting the Ras-ERK cascade and the pro-survival signaling molecules SPHK1 and Mcl-1. These findings reveal potential targets for selective therapy of CML.