Impaired mitochondrial oxidative metabolism in skeletal progenitor cells leads to musculoskeletal disintegration.

Although skeletal progenitors provide a reservoir for bone-forming osteoblasts, the major energy source for their osteogenesis remains unclear. Here, we demonstrate a requirement for mitochondrial oxidative phosphorylation in the osteogenic commitment and differentiation of skeletal progenitors. Deletion of Evolutionarily Conserved Signaling Intermediate in Toll pathways (ECSIT) in skeletal progenitors hinders bone formation and regeneration, resulting in skeletal deformity, defects in the bone marrow niche and spontaneous fractures followed by persistent nonunion. Upon skeletal fracture, Ecsit-deficient skeletal progenitors migrate to adjacent skeletal muscle causing muscle atrophy. These phenotypes are intrinsic to ECSIT function in skeletal progenitors, as little skeletal abnormalities were observed in mice lacking Ecsit in committed osteoprogenitors or mature osteoblasts. Mechanistically, Ecsit deletion in skeletal progenitors impairs mitochondrial complex assembly and mitochondrial oxidative phosphorylation and elevates glycolysis. ECSIT-associated skeletal phenotypes were reversed by in vivo reconstitution with wild-type ECSIT expression, but not a mutant displaying defective mitochondrial localization. Collectively, these findings identify mitochondrial oxidative phosphorylation as the prominent energy-driving force for osteogenesis of skeletal progenitors, governing musculoskeletal integrity.

Leukemia Stem Cells in 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 constitutively activated tyrosine kinase. Although BCR-ABL tyrosine kinase inhibitors (TKIs) are highly effective in treating CML at chronic phase, a number of patients develop drug resistance due to the inability of TKIs to kill leukemia stem cells (LSCs). Similar to other types of hematopoietic malignancies, LSCs in CML are believed to be a rare cell population responsible for leukemia initiation, disease progression, and drug resistance. Therefore, a full understanding of the biology of LSCs will help to develop novel therapeutic strategies for effective treatment of CML to possibly reach a cure. In recent years, a significant progress has been made in studying the biology of LSCs in both animal models and human patients at cellular and molecular levels, providing a basis for designing and testing potential molecular targets for eradicating LSCs in CML.

Targeting the Extracellular Signal-Regulated Kinase 5 Pathway to Suppress Human Chronic Myeloid Leukemia Stem Cells.

Tyrosine kinase inhibitors (TKi) are effective against chronic myeloid leukemia (CML), but their inefficacy on leukemia stem cells (LSCs) may lead to relapse. To identify new druggable targets alternative to BCR/ABL, we investigated the role of the MEK5/ERK5 pathway in LSC maintenance in low oxygen, a feature of bone marrow stem cell niches. We found that MEK5/ERK5 pathway inhibition reduced the growth of CML patient-derived cells and cell lines in vitro and the number of leukemic cells in vivo. Treatment in vitro of primary CML cells with MEK5/ERK5 inhibitors, but not TKi, strikingly reduced culture repopulation ability (CRA), serial colony formation ability, long-term culture-initiating cells (LTC-ICs), and CD26-expressing cells. Importantly, MEK5/ERK5 inhibition was effective on CML cells regardless of the presence or absence of imatinib, and did not reduce CRA or LTC-ICs of normal CD34+ cells. Thus, targeting MEK/ERK5 may represent an innovative therapeutic approach to suppress CML progenitor/stem cells.

Targeting chronic myeloid leukemia stem cells with the hypoxia-inducible factor inhibitor acriflavine.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell (HSC)-driven neoplasia characterized by expression of the constitutively active tyrosine kinase BCR/Abl. CML therapy based on tyrosine kinase inhibitors (TKIs) is highly effective in inducing remission but not in targeting leukemia stem cells (LSCs), which sustain minimal residual disease and are responsible for CML relapse following discontinuation of treatment. The identification of molecules capable of targeting LSCs appears therefore of primary importance to aim at CML eradication. LSCs home in bone marrow areas at low oxygen tension, where HSCs are physiologically hosted. This study addresses the effects of pharmacological inhibition of hypoxia-inducible factor-1 (HIF-1), a critical regulator of LSC survival, on the maintenance of CML stem cell potential. We found that the HIF-1 inhibitor acriflavine (ACF) decreased survival and growth of CML cells. These effects were paralleled by decreased expression of c-Myc and stemness-related genes. Using different in vitro stem cell assays, we showed that ACF, but not TKIs, targets the stem cell potential of CML cells, including primary cells explanted from 12 CML patients. Moreover, in a murine CML model, ACF decreased leukemia development and reduced LSC maintenance. Importantly, ACF exhibited significantly less-severe effects on non-CML hematopoietic cells in vitro and in vivo. Thus, we propose ACF, a US Food and Drug Administration (FDA)-approved drug for nononcological use in humans, as a novel therapeutic approach to prevent CML relapse and, in combination with TKIs, enhance induction of remission.

Alox5 Blockade Eradicates JAK2V617F-Induced Polycythemia Vera in Mice.

Myeloproliferative neoplasms such as polycythemia vera (PV), which are associated with the JAK mutation V617F, remain incurable despite progress in the use of JAK2 inhibitors for treatment of some of these diseases. In this study, we employed mice that undergo JAK2V617F-induced PV as a tool to explore new candidate targets for therapy. Our investigations focused on the lipid metabolic enzyme arachidonate 5-lipoxygenase (Alox5), which we found to be strongly upregulated by JAK2V617F in hematopoietic cells in vitro and in vivo Notably, genetic deletion of Alox5 or its inhibition in mice with a bioactive small-molecule inhibitor was sufficient to attenuate PV development. This therapeutic effect was associated with induction of a blockade in cell-cycle progression and also with apoptosis in PV cells. Genetic loss exerted an inhibitory effect on PV-initiating cells. Similarly, Alox5 inhibition was sufficient to suppress colony formation in human JAK2V617F-expressing CD34+ cells. Mechanistic investigations showed that Alox5 inhibition reduced AKT activation and decreased ß-catenin expression in JAK2V617F-expressing cells. Together, our results define Alox5 as a key genetic effector of JAK2V617F in driving PV, and they identify this enzyme as a candidate therapeutic target to treat this refractory myeloproliferative neoplasm. Cancer Res; 77(1); 164-74. ©2016 AACR.

Cell Cycle Analysis of CML Stem Cells Using Hoechst 33342 and Propidium Iodide.

Chronic myeloid leukemia (CML) is a myeloproliferative disease with an expansion of white blood cells. The current treatments for CML are shown not to be long-term effective because of CML stem cells' insensitivity to tyrosine kinase inhibitors. Therefore, studying more about CML stem cells is essential to understand the pathways of CML stem cell development and proliferation and finally lead to effective treatments to eliminate CML stem cells and eradicate CML. This chapter describes two methods to analyze cell cycle of CML stem cells. The rare population of CML stem cells can be identified by staining with cell surface markers, and then DNA-binding dyes Hoechst 33342 and propidium iodide (PI) are added to stain the DNA content which is changed when cells go through different phases of the cell cycle. Samples are run through the flow cytometer to be analyzed based on different absorbance and emission wavelengths of different florescent colors.

Arachidonate 15-lipoxygenase is required for chronic myeloid leukemia stem cell survival.

Cancer stem cells (CSCs) are responsible for the initiation and maintenance of some types of cancer, suggesting that inhibition of these cells may limit disease progression and relapse. Unfortunately, few CSC-specific genes have been identified. Here, we determined that the gene encoding arachidonate 15-lipoxygenase (Alox15/15-LO) is essential for the survival of leukemia stem cells (LSCs) in a murine model of BCR-ABL-induced chronic myeloid leukemia (CML). In the absence of Alox15, BCR-ABL was unable to induce CML in mice. Furthermore, Alox15 deletion impaired LSC function by affecting cell division and apoptosis, leading to an eventual depletion of LSCs. Moreover, chemical inhibition of 15-LO function impaired LSC function and attenuated CML in mice. The defective CML phenotype in Alox15-deficient animals was rescued by depleting the gene encoding P-selectin, which is upregulated in Alox15-deficient animals. Both deletion and overexpression of P-selectin affected the survival of LSCs. In human CML cell lines and CD34+ cells, knockdown of Alox15 or inhibition of 15-LO dramatically reduced survival. Loss of Alox15 altered expression of PTEN, PI3K/AKT, and the transcription factor ICSBP, which are known mediators of cancer pathogenesis. These results suggest that ALOX15 has potential as a therapeutic target for eradicating LSCs in CML.