Leukemia, an effective model for chemical biology and target therapy¹

Introduction

It is increasingly clear that all biological processes, in principal, rely on chemical processes that are governed by the structure of the participating molecules and their interactions and, thus, the biological processes can be treated as chemical processes and studied in molecular detail. The Human Genome Program was a landmark event for life science and has uncovered a large number of novel genes and provided an unprecedented prospectus for target identification and the development of drugs. However, the function of these genes cannot be predicted merely from their structure, thereby demonstrating the need for the systematic collection of biological information in addition to the gene sequence to fully exploit the genomic data. As a result, the accumulation of genome sequence data has not resulted in the anticipated acceleration of novel therapeutic developments. We are now facing the exciting but daunting task of identifying and assigning functions and therapeutic potentials to all 30 000 human genes^[1]. To address the latter issue, a myriad of strategies have emerged to facilitate genomics-driven target identification and validation. The “functional genomics” technologies, such as genetic-based knock-out and knock-in, RNAi and ectopic expression of targeted genes, high-throughput cellular profiling of gene and protein expression, systematic analyses of protein complexes and bioinformatics, have played active roles in the past few years. Many of these techniques are used in limited classes of gene products, but none of the techniques has proven to be the generally useful strategy that was anticipated^[1,2].

Small molecules participate directly in many biological processes and function in diverse roles, including chemical triggers, inhibitors, stimulators and switches. In contrast, compared with genetic approaches, the effect of small molecules is generally fast and reversible because of the metabolism and excretion of the molecules, which enables transient study of the proteins. In addition, the effect of small molecules is tunable because a varying concentration can result in different degrees of phenotype expression^[3]. Therefore, small active molecules, providing a common link between the fields of chemistry and biology, have long had an important role in deciphering answers to fundamental biological questions, especially in the exploration of signaling networks for cellular activities. In the past decade, chemical biology, aimed at studying signaling networks for basic cellular activities using specific, active small molecules as probes, has developed rapidly. This rise in chemical biology greatly accelerates research on molecular mechanisms and target therapy of diseases, especially in malignant tumors. Complementary to genetics and genomics, chemical biology has become an effective tool to study the functions of proteins/genes and important cellular activities^[1,4,5]. The recent explosion of interest in chemical biology combined with genetic manipulation reflects a fascination with research at the interface of chemistry and biology, where chemical insights are brought to bear on exciting biological and biomedical problems^[6].

Leukemia, a heterogeneous group of hematopoietic malignancies that occurs worldwide, includes acute/chronic and myeloid/lymphocytic leukemias. Despite many important advances in understanding the biological, molecular and cytogenetic aspects of leukemia, as well as in the diagnosis and therapy of leukemia, the most people will die of their diseases^[7,8]. Obviously, it is imperative to understand the pathophysiological mechanisms of leukemia and to explore new therapeutic strategies. It has been widely understood that various kinds of leukemias present specific cytogenetic alterations, especially chromosome translocations, that generate abnormal oncogenic fusion proteins^[9]. These alterations disrupt the normal signaling and cause uncontrolled proliferation, blocked differentiation and/or damaged apoptosis. Because of these genetic and cellular features, and because of the ease of capture of leukemia samples and the relatively convenient evaluation of therapeutic effects, small molecule-based chemical biology has been widely used with leukemia and important progress has been achieved over the past two decades. Based on several examples, mainly from our own work, this review will outline the advances of chemical biology in understanding molecular mechanisms of cell differentiation and apoptosis induction and target therapy of leukemia.

All-trans retinoic acid-based chemical biology of leukemia

An important feature of acute myeloid leukemia (AML), which accounts for 75% of all acute leukemias, is the blockage of the differentiation of myeloid cells at different stages. Over the past 20 years, many studies have explored the differentiation-inducing agents for leukemic cells. A typical and successful example is the discovery of all-trans retinoic acid (ATRA) as a differentiation-inducing drug for acute promyelocytic leukemia (APL)^[10], a unique subtype of AML that is defined by blockage at the promyelocytic stage of myeloid cell maturation. Using cytogenetic analysis, it has been shown that over 95% of patients with APL carry specific chromosome translocation t(15,17), which leads to the fusion of the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor-alpha (RARα) gene on chromosome 17, and the generation of PML-RARα fusion protein^[11]. A series of in vitro and in vivo works, including those from transgenic mice^[12], have shown that this fusion protein plays a critical role in the pathogenesis of APL via interfering functions of wild-type PML, RARα and its heterodimeric partner retinoid X receptor (RXR), as well as obtaining its new functions, as widely reviewed^[13–15]. As widely confirmed, ATRA can induce complete clinical remission, although retinoid resistance and relapse frequently occur^[16]. Because ATRA can cleave the PML-RARα fusion protein and, more importantly, it can potently drive AML cells to undergo differentiation, ATRA as a probe has greatly promoted our understanding of signaling pathways of leukemogenesis and leukemic cell differentiation. Using chemical and biological studies based on ATRA and other differentiation-inducing agents, such as phorbol 12-myristate 13-acetate (PMA) and vitamin D₃, many differentiation and leukemogenesis-related molecules, such as CCAAT/enhancer binding protein (C/EBP), nuclear corepressors (N-CoR), Sin3A and histone deacetylase (HDAC), have been examined^[17,18]. Vice versa, knowledge of these differentiation-related molecules has also led to the development of new differentiation agents, such as HDAC inhibitor valproic acid^[19].

As an example, phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated protein that is localized in either the cell membrane or nucleus depending on its palmitoylated state. Increasing evidence has demonstrated the biological role of PLSCR1 in cell signaling, maturation and apopto-sis^[20]. ATRA can elevate PLSCR1 expression in ATRA-sensitive APL cells NB4 and HL60, but not in maturation-resistant NB4-LR1 cells. Furthermore, PMA, but not dimethyl sulfoxide (DMSO), sodium butyrate or vitamin-D₃-induced leukemic cell differentiation, is also accompanied by increased PLSCR1 protein^[20] . Based on the fact that ATRA and PMA can activate protein kinase Cdelta (PKCδ) by phosphoryla-tion, PKCδ-specific inhibitor rottlerin is shown to nearly eliminate ATRA-induced and PMA-induced PLSCR1 expression, whereas ectopic expression of a constitutively active form of PKCδ directly increases PLSCR1 expression^[21]. Thus, PKCδ is proposed to exert a critical role in ATRA-induced or PMA-induced PLSCR1 expression. Furthermore, interferon upregulates PLSCR1 by sequential activation of PKCδ, JNK and STAT1^[22]. In contrast, decreasing PLSCR1 expression with small interfering RNA (siRNA) inhibits ATRA/PMA-induced differentiation^[21], whereas inducible PLSCR1 expression induces growth arrest at the G₁ phase and granulocyte-like differentiation together with increased sensitivity to apoptosis induction^[23]. All these data support the anti-leukemia role of PLSCR1, although its exact molecular mechanisms need to be further investigated. In agreement with this notion, M2, M5a, and M5b type AML cells present lower levels of PLSCR1 than normal bone marrow cells, and the PLSCR1 mRNA level is associated with significantly longer overall survival^[24].

Arsenic trioxide-based chemical biology of leukemia

Following the successful practice of ATRA, several research groups in China, including ours, reported impressive clinical response rates in patients with APL who had received arsenic trioxide (As₂O₃), a common naturally occurring substance. Subsequently, clinical studies conducted in other nations confirmed earlier reports and As₂O₃, commercial name Trisenox (Cell Therapeutics, Seattle, WA, USA), was approved by the Food and Drug Administration for the treatment of relapsed/refractory APL^[25–32]. Later, single-agent As₂O₃-induced durable remission with minimal toxicity in the newly diagnosed APL was also reported^[33–35]. Following these successful experiences, the mechanisms of As₂O₃ action on APL cells and other cancer cells have been attracting wide interest and great progress has been made^[36–38]. In brief, As₂O₃ can rapidly modulate the subcellular localization of PML and PML-RARα proteins and degrade PML-RARα protein^[39–41]. In a cytological direction, As₂O₃ induces apoptosis in an impressive array of cancer cells in addition to APL^[42], in which various kinds of mechanisms have been documented^[43,44]. However, the clinical effectiveness of As₂O₃ appears to be restricted to APL, although clinical studies have shown that As₂O₃-based regimens are clinically active in patients with relapsed/refractory multiple myeloma (MM)^[45]. In contrast, in vivo observations in APL patients and animal models show that As₂O₃ can induce partial differentiation of APL cells^[27,41,46]. Hence, the in vitro differentiation-inducing effect of As₂O₃ was investigated in APL cells. Indeed, a relatively longer treatment of low-dose As₂O₃ (0.1–0.5 µmol/L) induces APL cells to undergo partial differentiation^[41,47]. However, it appears that it is not more significant than its in vivo activity. Thus, we speculated that some factors in the bone marrow (BM) microenvironment could modulate the in vivo activity of As₂O₃. Towards this end, we considered that oxygen concentration may be one such factor that impinges on the in vivo action of As₂O₃ based on the following facts: leukemic cells are cultured in vitro at ambient oxygen, whereas in vivo cells are physiologically exposed to much lower oxygen levels ranging from 16% in pulmonary alveoli to less than 6% in most peripheral organs of the body. Oxygen levels of BM in AML patients may be decreased as a result of the fast growth of leukemic cells, and are possibly further aggravated by the anemia that often accompanies newly diagnosed AML patients, although leukemic cells do not form a well-circumscribed “mass” in BM like they do in solid tumors. More importantly, angiogenesis, which increases blood supply and oxygen tension, is also vital in the pathogenesis of different hematologic malignancies and provides an independent predictor of outcome in adults with AML, while the anti-angiogenic effects of chemotherapeutic and other novel drugs for the treatment of leukemia, such as ATRA and As₂O₃, might contribute to their therapeutic potentials. We investigated whether low oxygen tension impacts on the action of As₂O₃ on APL cells. Unexpectedly, cobalt chloride (CoCl₂)/desfer-rioxamine (DFO)-mimicked hypoxia or moderate hypoxia (2% and 3% O₂) can directly trigger AML cells to undergo differentiation both in vitro and in vivo^[48–50]. The differentiation-inducing effect of As₂O₃ on APL cells is also enhanced by hypoxia mimetic agents^[51]. Using chemical interference, inducible expression and siRNA technology, it is confirmed that hypoxia-inducible factor (HIF)1α exerts a critical role in this event, possibly in a transcription activity independent manner^[49,52]. Furthermore, HIF-1α interacts with and increases the transcription activities of two known hematopoiesis transcription factors, AML1 and CCAAT/enhancer binding factor-alpha (C/EBPα)^[49,53-55]. A low concentration of Tiron, a non-toxic chelator used to alleviate acute metal overload, has recently been shown to be a potent inducer of cell differentiation in human promyelotic HL-60 leukemia cells via increased HIF-1α and C/EBPα^[56]. Intriguingly, HIF-1α is dysregulated in a murine APL model^[57]. Nguyen-Khac also recently reported that hypoxia accelerates the differentiation of normal CD34⁺ cells, whereas TEL-ARNT fusion protein, which results from t(1;12)(q21;p13), inhibits hypoxia-induced normal progenitor cell differentiation through the interaction of TEL-ARNT with HIF-1α ^[58,59].

Apoptosis induction-based chemical biology of leukemia

As an intrinsic cell death program, apoptosis plays a critical role in the tissue homeostasis of multicellular organisms, especially in organs, such as the hematopoietic system, where high rates of daily cell production are offset by rapid cell turnover. Indeed, the hematopoietic system provides numer-ous examples to show the importance of apoptotic cell death for achieving the control of homeostasis. Correspond-ingly, disorder of the apoptosis machinery would cause various diseases. For example, damaged apoptosis exerts an important role in the pathogenesis and resistance to chemotherapeutic drugs of cancers including leukemia^[60]. Therefore, to understand the core molecular mechanisms of the apoptosis machinery has become an important and interesting research direction.

Over the past years, much progress has been achieved in understanding the molecular mechanisms of initiation and regulation of apoptosis. In brief, apoptosis is mainly caused by the activation of intracellular aspartate-specific proteases, known as caspases, with over 10 members (eg, caspase-8, 9 and 3). The mechanisms of caspase activation by apoptosis inducers have been the center of much attention in recent years. To date, two pathways of caspase activation and, thus, apoptosis have been elucidated in great detail, that is, the mitochondria-centered intrinsic and death receptor-mediated extrinsic pathways. As for the former, multiple signals cause dysfunction of mitochondria, especially disrupted mitochondrial transmembrane potentials, which leads to the release of apoptogenic proteins, such as cytochrome c, second mitochondrial activator of caspase (Smac) and apoptosis-inducing factor (AIF), into the cytosol. Cytochrome c binds the apoptotic protease-activating factor 1 (Apaf1) and forms the apoptosome together with procaspase-9. In this multiprotein structure, caspase-9 is activated and then activated caspase-9 activates downstream effector caspases, mainly procaspase-3. In the extrinsic apoptotic pathway, death receptors such as Fas, containing a cytosolic death domain (DD), cluster in membranes when they bind to their ligands, which recruits caspase-binding adaptor proteins, such as the Fas-associating protein with death domain (FADD) that contains both a death domain (DD) and a death effector domain (DED). The DED of FADD binds DED-containing procaspases (eg caspase-8 and caspase-10), forming a death-inducing signaling complex (DISC) and resulting in downstream effector caspase-3. The activated caspase-3 cleaves numerous cellular target proteins, which in aggregate produce an apoptosis-characteristic morphology and biochemical alterations. In contrast, apoptosis machinery is also regulated by many proteins, including bcl-2 family members, inhibitors of caspases, oncoproteins and tumor-suppressing protein, and other signaling molecules. Thus, a delicate balance between pro-apoptotic and anti-apoptotic regulators of apoptosis pathways is essential to ensure the survival of long-lived cells and the proper turnover of short-lived cells in a variety of tissues.

During these great advances on apoptosis, chemical approaches with small-molecule apoptosis inducers and inhibitors as probes provided a huge help. In fact, a lot of information on cell apoptotic processes was contributed by chemical biological investigations^[61]. For example, wild p53 was found to actively mediate the apoptosis of the hematopoietic cells by depriving the cells of specific growth factors, such as IL-3, and p53 might be involved in the response of myeloid precursors to environmental cytokines for the maintenance of hematopoietic homeostasis^[62]. Recently, we reported that nanomolar concentrations of NSC606985, a rarely studied camptothecin analog, induces apoptosis in AML cells through the rapid proteolytic activation of PKCδ, followed by the loss of mitochondrial transmembrane potential and caspase-3 activation^[63]. Furthermore, the potential therapeutic effect of this agent on AML mice generated by syngenic grafts of leukemic blasts from transgenic mice with PML-RARα was also shown^[64]. Moreover, inducible expression of AML1-ETO, a fusion gene of the AML1 gene on chromosome 21 with the ETO (eight-twenty-one) gene on chromosome 8, endows leukemic cells with susceptibility to NSC606985 and other insult-induced apoptosis^[65]. Using subcellular and quantitative proteomic analyses, moreover, we found a set of unique deregulated proteins in NSC606985-induced apoptotic AML cell line NB4 cells, including 16 compartment-compartment translocated ones. These proteins contributed to multiple functional activities, such as DNA damage repair, chromosome assembly, mRNA processing, biosynthesis, modification and degradation of proteins (Figure 1)^[66]. These discoveries shed new insights for systematically understanding the mechanisms of camptothecin-induced apoptosis.

Figure 1 Ideogram illustration of the proteins involved in NSC606985-induced NB4 cell apoptosis. With NSC606985, a camptothecin analog, as an apoptosis-inducing probe, we analyzed protein expression profiles of fractionated nuclei, mitochondria, raw endoplasmic reticula and cytosols of NSC606985-induced apoptotic AML cell line NB4 cells using two-dimensional electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry. A total of 90 unique deregulated proteins, including 16 compartment–compartment translocated proteins, were identified. These proteins contributed to multiple functional activities, such as DNA damage repair, chromosome assembly, mRNA processing, biosynthesis, modification and degradation of proteins. With their functional analyses, the possible roles of these deregulated proteins in NSC606985-induced apoptosis were demonstrated using an ideogram illustration. Upregulated proteins are marked in red and the downregulated proteins are marked in green. Modified proteins are marked with blue. All abbreviations are derived from the Swiss-Prot database.

In contrast, the application of chemical biology in understanding the core components of the apoptosis machinery at the molecular and structural levels aids the discovery of many new potential therapies for leukemia, as reviewed by Reed and Pellecchia^[67]. For instance, Bcl-2 family was recently raised up as the drug target for leukemia. The common aberrant expression of Bcl-2 in chronic lymphocytic leukemia (CLL) was proposed to confer CLL B-cell the unusual feature of resistance to undergoing apoptosis and dying properly, which is associated with leukemogenesis, poor response to chemotherapy and decreased overall survival of CLL. Consequently, selective apoptosis induction of malignant cells by interfering Bcl-2 is growing as a potential basic therapeutic strategy. In the past years, various apoptosis inducing and regulating drugs or lead compounds, including anti-apoptotic Bcl-2 or Bcl-X_L expression regulating drugs (such as synthetic retinoic acid and inhibitor of histone deacetylase) and drugs directly binding to Bcl-2 or other anti-apoptosis proteins (such as Gossypol isolated from cottonseed, HA14-1, BH3I-1, and BH3I-2, GX15-070) have been discovered.

AML cells are also characterized by constitutive and abnormal activation of the nuclear factor-kappaB (NF-κB) transcription factor. In addition, NF-κB also plays a crucial role in the pathogenesis, survival and resistance to apoptosis of HTLV-I-infected leukemic cells^[68], B-cell chronic lymphocytic leukemia (B-CLL)^[69], which is also supported by the fact that the PML can enhance the sensitivity of TNF-α induced apoptosis through the repression of the NF-κB survival pathway^[70], while transfection of a dominant-negative mutant NF-κB inhibitor represses p53-dependent apoptosis in acute lymphoblastic leukemia cells (ATL)^[71]. Taken together, NF-κB is a suitable target for the prevention and treatment of ATL^[68]. Interestingly, a specific pharmacological inhibitor of AS602868 could block NF-κB activation and lead to apoptosis of human primary AML cells. Moreover, AS602868 potentiated the apoptotic response induced by the current chemotherapeutic drugs doxorubicin, cytarabine and etoposide (VP16). In addition, NF-κB inhibition did not affect normal CD34⁺ hematopoietic precursors, suggesting that it could represent a new adjuvant strategy for AML treatment^[72]. At the same time, indole-3-carbinol, found in brassica species vegetables (such as cabbage, cauliflower and brussels spouts), can inhibit NF-κB and NF-κB-regulated gene expression and this mechanism may provide the molecular basis for its ability to suppress tumorigenesis and clinical application^[73].

Leukemogenic fusion proteins-based chemical biology

Different types of leukemia usually have specific chromosome translocations that cause the activation of onco-genes and, in particular, the formation of abnormal fusion genes, such as AML1-ETO^[74–76], a fusion protein generated by t(8,21) translocation that occurs in approximately 12% of all AML and 40% of M2-type AML. These fusion proteins play a pivotal role in leukemogenesis. Thus, using some small molecules to destroy them or to inhibit their activities may become an effective method to treat the corresponding type of leukemia, while these active small molecules would be more useful for understanding leukemogenic mechanisms of these fusion proteins. The destruction of PML-RARα by ATRA and As₂O₃ is a good model. Another typical example is the discovery of Bcr-Abl kinase inhibitor imatinib mesylate (also called Gleevec, STI571)^[77]. Bcr-Abl is a constitutively active, cytoplasmic tyrosine kinase that is produced by t(9;22) translocation in more than 95% of chronic myelogenous leukemia (CML) and about half of patients with adult-onset acute lymphoblastic leukemia (ALL). The central role of Bcr-Abl kinase in leukemogenesis promoted it as an ideal target for drug screen to treat CML. Hence, imatinib was discovered as a tyrosine kinase inhibitor and was introduced into the armamentarium of drugs for the treatment of CML and has since revolutionized its management^[77]. Imatinib has a high affinity for the ATP-binding site of Abl, and clinical trials have validated the promise of this molecular targeted therapy. In the more advanced phases of CML, imatinib was able to induce a major (complete or partial) cytogenetic response in 16%–60% of CML patients. In a phase III trial comparing imatinib with interferon-α plus cytosine arabinoside in newly diagnosed chronic phase CML, 85% of patients treated with imatinib attained a major cytogenetic response after a median follow-up of 19 months, compared to 22% in patients treated with the latter combination. A recent update has shown a further increase in major cytogenetic response of up to 92% of patients in the imatinib arm after a median follow-up of 54 months. In view of its high efficacy and low toxicity, imatinib has now replaced interferon-α as the frontline treatment for CML patients who are not eligible for allogeneic stem cell transplantation. Meanwhile, the application of imatinib as a probe greatly improved our understanding of Bcr-Abl-targeted signaling pathways, and has revealed a complex web of signals that promote cell division and survival^[78].

Imatinib is now frontline therapy for CML, but resistance is increasingly encountered, which has dampened the initial enthusiasm for this much heralded “magic bullet”. It has been shown that resistance to imatinib in CML occurs through the selection of tumor cells harboring Bcr-Abl kinase domain point mutations that interfere with drug binding. To date, at least 73 different point mutations leading to the substitution of 50 amino acid residues in the Abl kinase domain have been isolated from CML patients resistant to imatinib. Crystallographic studies revealed that imatinib binds to the ATP-binding site of Abl only when the activation loop of the kinase is closed and stabilizes the protein in this inactive conformation. Thus, most imatinib-resistant mutants should remain sensitive to inhibitors that bind Abl with less stringent conformational requirements. Based on this notion, an orally bioavailable Abl kinase inhibitor called Dasatinib (BMS-354825) has been examin-ed^[79], which presents a two-log increased potency relative to imatinib that retains activity against 14 of 15 imatinib-resistant Bcr-Abl mutants. Clinical trials of Dasatinib in imatinib-resistant and imatinib-intolerant CML and Ph chromosome-positive ALL are currently in progress^[80–82].

Conclusion

The rapid rise of chemical approaches, that is, studying signaling networks using basic cell activities with specific, active small molecules as probes, greatly accelerates research on the molecular mechanisms of disease and target therapy, especially in malignant tumors such as leukemia. With the application of chemical approaches combined with genetic manipulation, significant progress on the basic knowledge, diagnosis and therapy of leukemia has been achieved over the past few decades. In particular, discoveries of the clinical effectiveness of ATRA and As₂O₃ in the treatment of APL and of Bcr-Abl kinase inhibitor Imatinib and Dasatinib in the treatment of CML not only make target therapy of leukemia a reality, but also push mechanisms of leukemogenesis and leukemic cell activities forward. These successful practices are attracting wide interest on the application of a chemical approach in leukemia and other cancers.

References

1. Austin CP. The completed human genome: implications for chemical biology. Curr Opin Chem Biol 2003;7:511-5.
2. Friedman A, Perrimon N. Genome-wide high-throughput screens in functional genomics. Curr Opin Genet Dev 2004;14:470-6.
3. Doudna JA. Chemical biology at the crossroads of molecular structure and mechanism. Nat Chem Biol 2005;1:300-3.
4. Lipinski C, Hopkins A. Navigating chemical space for biology and medicine. Nature 2004;432:855-61.
5. Sawyer TK. Chemical biology and drug design: three-dimensional, dynamic, and mechanistic nature of two multidisciplinary fields. Chem Biol Drug Des 2006;67:196-200.
6. Lewis JD. The role of the chemical biology core facility at EMBL: a vision for a European roadmap. ACS Chem Biol 2007;2:21-3.
7. Appelbaum FR, Rosenblum D, Arceci RJ, Carroll WL, Breitfeld PP, Forman SJ, et al. End points to establish the efficacy of new agents in the treatment of acute leukemia. Blood 2007;109:1810-6.
8. Tallman MS, Gilliland DG, Rowe JM. Drug therapy for acute myeloid leukemia. Blood 2005;106:1154-63.
9. Scandura JM, Boccuni P, Cammenga J, Nimer SD. Transcription factor fusions in acute leukemia: variations on a theme. Oncogene 2002;21:3422-44.
10. Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 1988;72:567-72.
11. Cheng GX, Zhu XH, Men XQ, Wang L, Huang QH, Jin XL, et al. Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF-RARalpha and NPM-RARalpha. Proc Natl Acad Sci USA 1999;96:6318-23.
12. Kogan SC. Mouse models of acute promyelocytic leukemia. Curr Top Microbiol Immunol 2007;313:3-29.
13. Lutz PG, Moog-Lutz C, Cayre YE. Signaling revisited in acute promyelocytic leukemia. Leukemia 2002;16:1933-9.
14. Strudwick S, Borden KL. Finding a role for PML in APL pathogenesis: a critical assessment of potential PML activities. Leukemia 2002;16:1906-17.
15. Licht JD. Reconstructing a disease: what essential features of the retinoic acid receptor fusion oncoproteins generate acute promyelocytic leukemia? Cancer Cell 2006;9:73-4.
16. Chen GQ, Shen ZX, Wu F, Han JY, Miao JM, Zhong HJ, et al. Pharmacokinetics and efficacy of low-dose all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Leukemia 1996;10:825-8.
17. Kambhampati S, Verma A, Li Y, Parmar S, Sassano A, Platanias LC. Signalling pathways activated by all-trans-retinoic acid in acute promyelocytic leukemia cells. Leuk Lymphoma 2004;45:2175-85.
18. Zhao Q, Tao J, Zhu Q, Jia PM, Dou AX, Li X, et al. Rapid induction of cAMP/PKA pathway during retinoic acid-induced acute promyelocytic leukemia cell differentiation. Leukemia 2004;18:285-92.
19. Deubzer H, Busche B, Ronndahl G, Eikel D, Michaelis M, Cinatl J, et al. Novel valproic acid derivatives with potent differentiation-inducing activity in myeloid leukemia cells. Leuk Res 2006;30:1167-75.
20. Huang Y, Zhao Q, Chen GQ. Phospholipid scramblase 1. Sheng Li Xue Bao 2006;58:501-10.
21. Zhao KW, Li X, Zhao Q, Huang Y, Li D, Peng ZG, et al. Protein kinase Cdelta mediates retinoic acid and phorbol myristate acetate-induced phospholipid scramblase 1 gene expression: its role in leukemic cell differentiation. Blood 2004;104:3731-8.
22. Zhao KW, Li D, Zhao Q, Huang Y, Silverman RH, Sims PJ, et al. Interferon-alpha-induced expression of phospholipid scramblase 1 through STAT1 requires the sequential activation of protein kinase Cdelta and JNK. J Biol Chem 2005;280:42707-14.
23. Huang Y, Zhao Q, Zhou CX, Gu ZM, Li D, Xu HZ, et al. Antileukemic roles of human phospholipid scramblase 1 gene, evidence from inducible PLSCR1-expressing leukemic cells. Oncogene 2006;25:6618-27.
24. Yokoyama A, Yamashita T, Shiozawa E, Nagasawa A, Okabe-Kado J, Nakamaki T, et al. MmTRA1b/phospholipid scramblase 1 gene expression is a new prognostic factor for acute myelogenous leukemia. Leuk Res 2004;28:149-57.
25. Dombret H, Fenaux P, Soignet SL, Tallman MS. Established practice in the treatment of patients with acute promyleocytic leukemia and the introduction of arsenic trioxide as a novel therapy. Semin Hematol 2002;39:8-13.
26. Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qiu QY, et al. Use of arsenic trioxide (As₂O₃) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood 1997;89:3354-60.
27. Soignet SL, Maslak P, Wang ZG, Jhanwar S, Calleja E, Dardashti LJ, et al. Complete remission after treatment of acute promyelo-cytic leukemia with arsenic trioxide. N Engl J Med 1998;339:1341-8.
28. Sanz MA, Fenaux P, Lo Coco F. Arsenic trioxide in the treatment of acute promyelocytic leukemia. A review of current evidence. Haematologica 2005;90:1231-5.
29. Raffoux E, Rousselot P, Poupon J, Daniel MT, Cassinat B, Delarue R, et al. Combined treatment with arsenic trioxide and all-trans-retinoic acid in patients with relapsed acute promyelocytic leukemia. J Clin Oncol 2003;21:2326-34.
30. Lazo G, Kantarjian H, Estey E, Thomas D, O’Brien S, Cortes J. Use of arsenic trioxide (As₂O₃) in the treatment of patients with acute promyelocytic leukemia: the M. D. Anderson experience. Cancer 2003;97:2218-24.
31. Lu DP, Qiu JY, Jiang B, Wang Q, Liu KY, Liu YR, et al. Tetra-arsenic tetra-sulfide for the treatment of acute promyelocytic leukemia: a pilot report. Blood 2002;99:3136-43.
32. Cohen MH, Hirschfeld S, Flamm Honig S, Ibrahim A, Johnson JR, O’Leary JJ, et al. Drug approval summaries: arsenic trioxide, tamoxifen citrate, anastrazole, paclitaxel, bexarotene. Oncologist 2001;6:4-11.
33. Mathews V, George B, Lakshmi KM, Viswabandya A, Bajel A, Balasubramanian P, et al. Single-agent arsenic trioxide in the treatment of newly diagnosed acute promyelocytic leukemia: durable remissions with minimal toxicity. Blood 2006;107:2627-32.
34. George B, Mathews V, Poonkuzhali B, Shaji RV, Srivastava A, Chandy M. Treatment of children with newly diagnosed acute promyelocytic leukemia with arsenic trioxide: a single center experience. Leukemia 2004;18:1587-90.
35. Mathews V, Balasubramanian P, Shaji RV, George B, Chandy M, Srivastava A. Arsenic trioxide in the treatment of newly diagnosed acute promyelocytic leukemia: a single center experience. Am J Hematol 2002;70:292-9.
36. Rojewski MT, Korper S, Schrezenmeier H. Arsenic trioxide therapy in acute promyelocytic leukemia and beyond: from bench to bedside. Leuk Lymphoma 2004;45:2387-401.
37. Tallman MS. Arsenic trioxide: its role in acute promyelocytic leukemia and potential in other hematologic malignancies. Blood Rev 2001;15:133-42.
38. Zhang TD, Chen GQ, Wang ZG, Wang ZY, Chen SJ, Chen Z. Arsenic trioxide, a therapeutic agent for APL. Oncogene 2001;20:7146-53.
39. Chen GQ, Zhu J, Shi XG, Ni JH, Zhong HJ, Si GY, et al. In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As₂O₃) in the treatment of acute promyelocytic leukemia: As₂O₃ induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood 1996;88:1052-61.
40. Zhu J, Koken MH, Quignon F, Chelbi-Alix MK, Degos L, Wang ZY, et al. Arsenic-induced PML targeting onto nuclear bodies: implications for the treatment of acute promyelocytic leukemia. Proc Natl Acad Sci USA 1997;94:3978-83.
41. Chen GQ, Shi XG, Tang W, Xiong SM, Zhu J, Cai X, et al. Use of arsenic trioxide (As₂O₃) in the treatment of acute promyelocytic leukemia (APL): I. As₂O₃ exerts dose-dependent dual effects on APL cells. Blood 1997;89:3345-53.
42. Miller WH Jr, Schipper HM, Lee JS, Singer J, Waxman S. Mechanisms of action of arsenic trioxide. Cancer Res 2002;62:3893-903.
43. Zhu XH, Shen YL, Jing YK, Cai X, Jia PM, Huang Y, et al. Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentra-tions. J Natl Cancer Inst 1999;91:772-8.
44. Chen GQ, Zhou L, Styblo M, Walton F, Jing Y, Weinberg R, et al. Methylated metabolites of arsenic trioxide are more potent than arsenic trioxide as apoptotic but not differentiation inducers in leukemia and lymphoma cells. Cancer Res 2003;63:1853-9.
45. Berenson JR, Yeh HS. Arsenic compounds in the treatment of multiple myeloma: a new role for a historical remedy. Clin Lymphoma Myeloma 2006;7:192-8.
46. Kinjo K, Kizaki M, Muto A, Fukuchi Y, Umezawa A, Yamato K, et al. Arsenic trioxide (As₂O₃)-induced apoptosis and differentiation in retinoic acid-resistant acute promyelocytic leukemia model in hGM-CSF-producing transgenic SCID mice. Leukemia 2000;14:431-8.
47. Cai X, Shen YL, Zhu Q, Jia PM, Yu Y, Zhou L, et al. Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia. Leukemia 2000;14:262-70.
48. Huang Y, Du KM, Xue ZH, Yan H, Li D, Liu W, et al. Cobalt chloride and low oxygen tension trigger differentiation of acute myeloid leukemic cells: possible mediation of hypoxia-inducible factor-1alpha. Leukemia 2003;17:2065-73.
49. Jiang Y, Xue ZH, Shen WZ, Du KM, Yan H, Yu Y, et al. Desferrioxamine induces leukemic cell differentiation potentially by hypoxia-inducible factor-1 alpha that augments transcriptional activity of CCAAT/enhancer-binding protein-alpha. Leukemia 2005;19:1239-47.
50. Liu W, Guo M, Xu YB, Li D, Zhou ZN, Wu YL, et al. Induction of tumor arrest and differentiation with prolonged survival by intermittent hypoxia in a mouse model of acute myeloid leukemia. Blood 2006;107:698-707.
51. Yan H, Peng ZG, Wu YL, Jiang Y, Yu Y, Huang Y, et al. Hypoxia-simulating agents and selective stimulation of arsenic trioxide-induced growth arrest and cell differentiation in acute promyelocytic leukemic cells. Haematologica 2005;90:1607-16.
52. Xue ZH, Jiang Y, Yu Y, Wang LS, Chen GQ, Zhao Q. Metavana-date suppresses desferrioxamine-induced leukemic cell differentiation with reduced hypoxia-inducible factor-1alpha protein. Biochem Biophys Res Commun 2005;332:1140-5.
53. Chen GQ, Peng ZG, Liu W, Song LP, Jiang Y, Huang Y, et al. Hypoxia inducible factor-1alpha and leukemic cell differentiation. Sheng Li Xue Bao 2006;58:5-13.
54. Song LP, Zhang J, Wu SF, Huang Y, Zhao Q, Cao JP, et al. Hypoxia-inducible factor-1alpha-induced differentiation of myeloid leukemic cells is its transcriptional activity independent. Oncogene 2007. [Epub ahead of print].
55. Peng ZG, Zhou MY, Huang Y, Qiu JH, Wang LS, Liao SH, et al. Acute myeloid leukemia-1 antagonizes transcriptional activity of hypoxia inducible factor-1alpha protein through direct protein-protein interaction. Oncogene 2007. in press.
56. Kim JS, Cho EW, Chung HW, Kim IG. Effects of Tiron, 4,5-dihydroxy-1,3-benzene disulfonic acid, on human promyelotic HL-60 leukemia cell differentiation and death. Toxicology 2006;223:36-45.
57. Yuan W, Payton JE, Holt MS, Link DC, Watson MA, DiPersio JF, Ley TJ. Commonly dysregulated genes in murine APL cells. Blood 2007;109:961-70.
58. Salomon-Nguyen F, Della-Valle V, Mauchauffe M, Busson-Le Coniat M, Ghysdael J, Berger R, et al. The t(1;12)(q21;p13) translocation of human acute myeloblastic leukemia results in a TEL-ARNT fusion. Proc Natl Acad Sci USA 2000;97:6757-62.
59. Nguyen-Khac F, Della Valle V, Lopez RG, Ravet E, Mauchauffe M, Friedman AD, et al. Functional analyses of the TEL-ARNT fusion protein underscores a role for oxygen tension in hematopoietic cellular differentiation. Oncogene 2006;25:4840-7.
60. Zhivotovsky B, Orrenius S. Carcinogenesis and apoptosis: paradigms and paradoxes. Carcinogenesis 2006;27:1939-45.
61. Huang Z. The chemical biology of apoptosis. Exploring protein-protein interactions and the life and death of cells with small molecules. Chem Biol 2002;9:1059-72.
62. Blandino G, Scardigli R, Rizzo MG, Crescenzi M, Soddu S, Sacchi A. Wild-type p53 modulates apoptosis of normal, IL-3 deprived, hematopoietic cells. Oncogene 1995;10:731-7.
63. Song MG, Gao SM, Du KM, Xu M, Yu Y, Zhou YH, et al. Nanomolar concentration of NSC606985, a camptothecin analog, induces leukemic-cell apoptosis through protein kinase Cdelta-dependent mechanisms. Blood 2005;105:3714-21.
64. Liu W, Zhu YS, Guo M, Yu Y, Chen GQ. Therapeutic efficacy of NSC606985, a novel camptothecin analog, in acute myeloid leukemic mice. Leukemia Res 2007. [Epub ahead of print].
65. Lu Y, Xu YB, Yuan TT, Song MG, Lubbert M, Fliegauf M, et al. Inducible expression of AML1-ETO fusion protein endows leukemic cells with susceptibility to extrinsic and intrinsic apoptosis. Leukemia 2006;20:987-93.
66. Yu Y, Wang LS, Shen SM, Xia L, Zhang L, Zhu YS, et al. Subcellular proteome analysis of camptothecin analog NSC606985-treated acute myeloid leukemic cells. J Proteome Res 2007. [Epub ahead of print].
67. Reed JC, Pellecchia M. Apoptosis-based therapies for hematologic malignancies. Blood 2005;106:408-18.
68. Mori N, Yamada Y, Ikeda S, Yamasaki Y, Tsukasaki K, Tanaka Y, et al. Bay 11-7082 inhibits transcription factor NF-kappaB and induces apoptosis of HTLV-I-infected T-cell lines and primary adult T-cell leukemia cells. Blood 2002;100:1828-34.
69. Munzert G, Kirchner D, Stobbe H, Bergmann L, Schmid RM, Dohner H, et al. Tumor necrosis factor receptor-associated factor 1 gene overexpression in B-cell chronic lymphocytic leukemia: analysis of NF-kappa B/Rel-regulated inhibitors of apoptosis. Blood 2002;100:3749-56.
70. Wu WS, Xu ZX, Hittelman WN, Salomoni P, Pandolfi PP, Chang KS. Promyelocytic leukemia protein sensitizes tumor necrosis factor alpha-induced apoptosis by inhibiting the NF-kappaB survival pathway. J Biol Chem 2003;278:12294-304.
71. Zhou M, Gu L, Zhu N, Woods WG, Findley HW. Transfection of a dominant-negative mutant NF-kB inhibitor (IkBm) represses p53-dependent apoptosis in acute lymphoblastic leukemia cells: interaction of IkBm and p53. Oncogene 2003;22:8137-44.
72. Frelin C, Imbert V, Griessinger E, Peyron AC, Rochet N, Philip P, et al. Targeting NF-kappaB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells. Blood 2005;105:804-11.
73. Takada Y, Andreeff M, Aggarwal BB. Indole-3-carbinol suppresses NF-kappaB and IkappaBalpha kinase activation, causing inhibition of expression of NF-kappaB-regulated antiapoptotic and metastatic gene products and enhancement of apoptosis in myeloid and leukemia cells. Blood 2005;106:641-9.
74. Li X, Xu YB, Wang Q, Lu Y, Zheng Y, Wang YC, et al. Leukemogenic AML1-ETO fusion protein upregulates expression of connexin 43: the role in AML 1-ETO-induced growth arrest in leukemic cells. J Cell Physiol 2006;208:594-601.
75. Zhang L, Wang LS, Xu Y, Xia L, Chen WL, Zheng Y, et al. Comparative proteomic analysis of human leukemic cells with and without inducible expression of leukemogenic AML1-ETO protein. Chin J Physiol 2006;49:182-91.
76. Gao FH, Wang Q, Wu YL, Li X, Zhao KW, Chen GQ. c-Jun N-terminal kinase mediates AML1-ETO protein-induced connexin-43 expression. Biochem Biophys Res Commun 2007;356:505-11.
77. Deininger M, Buchdunger E, Druker BJ. The development of imatinib as a therapeutic agent for chronic myeloid leukemia. Blood 2005;105:2640-53.
78. Kharas MG, Fruman DA. ABL oncogenes and phosphoinositide 3-kinase: mechanism of activation and downstream effectors. Cancer Res 2005;65:2047-53.
79. Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL. Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 2004;305:399-401.
80. Hochhaus A, Kantarjian HM, Baccarani M, Lipton JH, Apperley JF, Druker BJ, et al. Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy. Blood 2007;109:2303-9.
81. Guilhot F, Apperley J, Kim DW, Bullorsky EO, Baccarani M, Roboz GJ, et al. Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in accelerated phase. Blood 2007;109:4143-50.
82. Cortes J, Rousselot P, Kim DW, Ritchie E, Hamerschlak N, Coutre S, et al. Dasatinib induces complete hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in blast crisis. Blood 2006;109:3207-13.