Haojian Zhang

Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia

Targeting of cancer stem cells is believed to be essential for curative therapy of cancers, but supporting evidence is limited. Few selective target genes in cancer stem cells have been identified. Here we identify the arachidonate 5-lipoxygenase (5-LO) gene (Alox5) as a critical regulator for leukemia stem cells (LSCs) in BCR-ABL–induced chronic myeloid leukemia (CML). In the absence of Alox5, BCR-ABL failed to induce CML in mice. This Alox5 deficiency caused impairment of the function of LSCs but not normal hematopoietic stem cells (HSCs) through affecting differentiation, cell division and survival of long-term LSCs (LT-LSCs), consequently causing a depletion of LSCs and a failure of CML development. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly by affecting LT-LSCs, and prolonged survival. These results demonstrate that a specific target gene can be found in cancer stem cells and its inhibition can completely inhibit the function of these stem cells.

HIF1α is required for survival maintenance of chronic myeloid leukemia stem cells

Hypoxia-inducible factor-1α (HIF1α), a master transcriptional regulator of the cellular and systemic hypoxia response, is essential for the maintenance of self-renewal capacity of normal HSCs. It is still unknown whether HIF1α has a role in survival regulation of leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). Using a mouse model of CML, here we report that HIF1α plays a crucial role in survival maintenance of LSCs. Deletion of HIF1α impairs the propagation of CML through impairing cell-cycle progression and inducing apoptosis of LSCs. Deletion of HIF1α results in elevated expression of p16(Ink4a) and p19(Arf) in LSCs, and knockdown of p16(Ink4a) and p19(Arf) rescues the defective colony-forming ability of HIF1α(-/-) LSCs. Compared with normal HSCs, LSCs appear to be more dependent on the HIF1α pathway. Together, these results demonstrate that HIF1α represents a critical pathway in LSCs and inhibition of the HIF1α pathway provides a therapeutic strategy for eradicating LSCs in CML.

PTEN is a tumor suppressor in CML stem cells and BCR-ABL–induced leukemias in mice

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is inactivated in many human cancers. However, it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome-positive leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. By using our mouse model of BCR-ABL-induced leukemias, we show that Pten is down-regulated by BCR-ABL in leukemia stem cells in CML and that PTEN deletion causes acceleration of CML development. In addition, overexpression of PTEN delays the development of CML and B-ALL and prolongs survival of leukemia mice. PTEN suppresses leukemia stem cells and induces cell-cycle arrest of leukemia cells. Moreover, PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL-induced leukemias and suggest a potential strategy for the treatment of Philadelphia chromosome-positive leukemia.

Activation, isolation, identification and in vitro proliferation of oval cells from adult rat livers

Abstract.  Oval cells, putative hepatic stem cells, could potentially provide a novel solution to the severe shortage of donor livers, because of their ability to proliferate and differentiate into functional hepatocytes. We have previously demonstrated that oval cells can be induced to differentiate into cells with morphologic, phenotypic, and functional characteristics of mature hepatocytes. In this study, we have established a new model combining ethionine treatment with partial hepatectomy to activate oval cells, then developed a procedure utilizing selective enzymatic digestion and density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population. We identified oval cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence of the presence of hepatic stem‐like cells in adult rat livers. Viewed by transmission electron microscopy, they were small cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles, including mitochondria and endoplasmic reticulum. Flow cytometric assay showed that these cells highly expressed OV‐6, cytokeratin‐19 (CK‐19) and albumin. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis displayed that the freshly isolated cells co‐expressed albumin, cytokeratin‐7 (CK‐7) and CK‐19 mRNA, indicating that they were essentially bipotential hepatic stem‐like cells. Furthermore, we set up a culture system containing growth factors and a fibroblast feeder layer, to provide nourishment to these cells. Thus, we were able to culture them in vitro for more than 3 months, with the number of cells doubling 100 times. Gene expressions of albumin, CK‐7 and CK‐19 in the cells derived from the expanding colonies at day 95 were confirmed by RT‐PCR analysis. These data suggested that the hepatic oval cells derived from adult rat livers possess a high potential to proliferate in vitro with a large increase in number, while maintaining the bipotential nature of hepatic stem cells.

The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells

A therapeutic strategy for treating cancer is to target and eradicate cancer stem cells (CSCs) without harming their normal stem cell counterparts. The success of this approach relies on the identification of molecular pathways that selectively regulate CSC function. Using BCR-ABL–induced chronic myeloid leukemia (CML) as a disease model for CSCs, we show that BCR-ABL downregulates the Blk gene (encoding B-lymphoid kinase) through c-Myc in leukemic stem cells (LSCs) in CML mice and that Blk functions as a tumor suppressor in LSCs but does not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Blk suppresses LSC function through a pathway involving an upstream regulator, Pax5, and a downstream effector, p27. Inhibition of this Blk pathway accelerates CML development, whereas increased activity of the Blk pathway delays CML development. Blk also suppresses the proliferation of human CML stem cells. Our results show the feasibility of selectively targeting LSCs, an approach that should be applicable to other cancers.

Scd1 Plays a Tumor-Suppressive Role in Survival of Leukemia Stem Cells and the Development of Chronic Myeloid Leukemia

ABSTRACT 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.

The existence of epithelial‐to‐mesenchymal cells with the ability to support hematopoiesis in human fetal liver

The fetal liver is the major hematopoietic organ during mid‐gestation, and it is also a source of stem cells that exist in a complex environment. In this study, we isolated a population of actively replicating cells with the characteristic of the epithelial‐to‐mesenchymal transition (EMT) from fetal liver. These cells were identified with the epithelial markers, including α‐fetoprotein (AFP), albumin (ALB), cytokeratins (CK) 7, and CK18, as well as the mesenchymal markers, such as α‐smooth muscle actin (ASMA), CD29, CD44, CD49, CD54, collagen I and osteopontin (OPN). Furthermore, they also expressed some hematopoiesis‐related genes. In addition, the cell population had the ability to retain hematopoietic stem cells (HSCs) in an undifferentiated state in vitro during cytokine‐stimulated proliferation. These results provide an insight about early human liver development and may also help to understand hematopoiesis in the fetal liver.

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 identification of the first 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 difficult. 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 self-renewal 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.

Functional Ramifications for the Loss of P-Selectin Expression on Hematopoietic and Leukemic Stem Cells

Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between blood cells and their microenvironment to preserve the homeostatic balance of long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and differentiated cells. Adhesion molecules like P-selectin (encoded by the Selp gene) are essential to hematopoiesis, and their dysregulation has been linked to leukemogenesis. Like HSCs, leukemic stem cells (LSCs) depend upon their microenvironments for survival and propagation. P-selectin plays a crucial role in Philadelphia chromosome -positive (Ph+) chronic myeloid leukemia (CML). In this paper, we show that cells deficient in P-selectin expression can repopulate the marrow more efficiently than wild type controls. This results from an increase in HSC self-renewal rather than alternative possibilities like increased homing velocity or cell cycle defects. We also show that P-selectin expression on LT-HSCs, but not ST-HSCs and MPPs, increases with aging. In the absence of P-selectin expression, mice at 6 months of age possess increased levels of short-term HSCs and multipotent progenitors. By 11 months of age, there is a shift towards increased levels of long-term HSCs. Recipients of BCR-ABL-transduced bone marrow cells from P-selectin-deficient donors develop a more aggressive CML, with increased percentages of LSCs and progenitors. Taken together, our data reveal that P-selectin expression on HSCs and LSCs has important functional ramifications for both hematopoiesis and leukemogenesis, which is most likely attributable to an intrinsic effect on stem cell self-renewal.

GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. Transformation of HSCs by the breakpoint cluster region-ABL tyrosine kinase (BCR-ABL) oncogene causes chronic myelogenous leukemia (CML). The E-twenty six (ets) transcription factor GA binding protein (GABP) is a tetrameric transcription factor complex that contains GABPα and GABPβ proteins. Deletion in bone marrow of Gabpa, the gene that encodes the DNA-binding component, caused cell cycle arrest in HSCs and profound loss of hematopoietic progenitor cells. Loss of Gabpα prevented development of CML, although mice continued to generate BCR-ABL–expressing Gabpα-null cells for months that were serially transplantable and contributed to all lineages in secondary recipients. A bioinformatic screen identified the serine-threonine kinase protein kinase D2 (PRKD2) as a potential effector of GABP in HSCs. Prkd2 expression was markedly reduced in Gabpα-null HSCs and progenitor cells. Reduced expression of PRKD2 or pharmacologic inhibition decreased cell cycling, and PRKD2 rescued growth of Gabpα-null BCR-ABL–expressing cells. Thus, GABP is required for HSC cell cycle entry and CML development through its control of PRKD2. This offers a potential therapeutic target in leukemia.