Pathogenesis Of Chronic Myelogenous Leukemia Research Articles

Identification of Alternative Splicing Events Regulated by the BCR-ABL Oncoprotein

Post-transcriptional modifications, including alternative splicing, are critical in determining the protein inventory of normal human cells. A number of malignancy-related and apoptosis-related genes have de-regulated alternative splicing due to mutation or deletion of genomic splice sites or through other, less well understood mechanisms. Chronic myelogenous leukemia (CML) is characterized by a translocation that produces the dominant acting BCR-ABL oncoprotein, which is responsible for genomic instability, resistance to apoptosis, cellular proliferation, differentiation abnormalities, and many other acquired cellular changes. It is likely that additional undiscovered properties of the BCR-ABL oncogene contribute to CML pathogenesis. Imatinib, an inhibitor of the BCR-ABL tyrosine kinase, induces a high rate of complete cytogenetic remission in CML. However, the ability of imatinib to eradicate the leukemia stem cell is unknown. Moreover, additional treatment targets will need to be identified to optimize therapies for imatinib-refractory patients. We used gene array analysis to investigate whether alternative splicing may be important in the biology and pathogenesis of CML. Two analyses were conducted, examining the splicing patterns of a total of 587 genes. 570 of the genes have been implicated in apoptosis, and 17 of the genes play a role in hematologic malignancies. In the first experiment, splicing profiles of all 587 genes (5832 independent splice events) were evaluated using a customized ExonHit array. Splicing profiles of three CML cell lines were compared to a well characterized primary thymus control tissue. Dye swaps confirmed experimental consistency. Bioconductor software was used to select events with greater than two-fold differential expression. We have identified 124 candidate splice forms in 91 genes that are differentially regulated in at least two of the three CML cell lines compared with control hematopoietic tissue. 88 of the 91 are implicated in apoptosis, while the remaining three are among the genes implicated in hematologic malignancies. The candidate genes identified by these means include WT1, RUNX1, PFDN5, and BRCA1. In a second experiment, the same 587 genes were examined in two CML cell lines treated with imatinib and compared to untreated controls. Prior pilot experiments identified a dose of imatinib that inhibited BCR-ABL but did not cause apoptosis during the course of the experiment. Using the same experimental procedures and analysis, we identified 41 candidate splice forms in 41 genes that were differentially expressed in imatinib-treated CML cell lines. Among these candidate genes, 40 are from the group of apoptosis-related genes, and one is from the group of genes related to hematologic malignancies. Candidate genes include MAPK3, FES, DCC, and TGFB1. Combining the two experiments, a group of 122 genes and 163 splice events were identified as potential BCR-ABL directed, imatinib-sensitive candidates. We performed validation experiments for one of these, WT1. Imatinib treatment consistently altered the ratios of specific splice forms present in CML cells. These findings are consistent with BCR-ABL dependent splicing of WT1 in CML. Because WT1 isoforms have differential effects on the survival, proliferation, and differentiation of leukemia cells, regulation of WT1 splicing by BCRABL may play an important role in the pathogenesis of CML. Overall, our results suggest that the BCR-ABL oncoprotein may cause significant dysregulation of mRNA splicing as part of the process of neoplastic transformation. We are currently validating selected genes among the 122 candidates in primary CML samples, performing bioinformatics analysis of the specific tumor-specific splice site anatomy to gain insights into potential upstream regulators, and investigating apoptosis regulation by selected candidate splice regulated targets.

A Distinctive Pattern of Different Gene Expression in Chronic Myelogenous Leukemia Patients

Introduction: Molecular chaperones have many functions, such as protecting other proteins against aggregation, assisting in folding of nascent proteins/refolding of damaged proteins and targeting severely damaged proteins to degradation. As one of the molecular chaperones, Hsp90 functions to facilitate the folding of newly synthesized and denatured client proteins, including mutated p53, Bcr-Abl, p185ErbB2 and Raf-1. The Bcr-Abl fusion gene encodes for the p210Bcr-Abl tyrosine kinase (TK) implicated in the pathogenesis of chronic myelogenous leukemia (CML). Studies in cultured cells have identified many signal transduction pathways activated by Bcr-Abl, including activation of the Ras, MAPK, JNK/SAPK, phosphatidylinositol-3 kinase, nuclear factor-B and STAT pathways. Imatinib mesylate (imatinib IM) is a tyrosine kinase inhibitor that competitively inhibits ATP binding in the kinase domains of both the Bcr-Abl and c-Abl kinases. It has been suggested that resistance to imatinib stems from Bcr-Abl gene amplification, leading to overexpression of Bcr-Abl protein or point mutations in the Bcr-Abl gene However, several groups suggested that there might be other forms of Bcr-Abl-independent imatinib resistance Recently, it has been reported that changes in histone deacetylase (HDAC) expression in leukemic cells could be involved in mechanisms for abnormal cellular proliferation that operate through chromatin-independent pathways and thereby could lead to acquired drug resistance of the cellsIn the present study, we evaluated in primary leukemic blasts, obtained from chronic myelogenous leukemia patients at onset, patients in blast crisis and patients which were imatinib-resistant The espression the sirtuin members family and HSP70, HSP90 i-NOS and bcl-2 was evaluated by Nortern blot and Western blot analysis.Material and Methods: Primary leukaemia blastsWe harvested primary blast rich mononuclear cells were obtained by gradient centrifugation on ficoll-hypaque of bone marrow and peripheral blood cells after obtaining appropriate informed consent.Northern blotTotal RNAs from control or treated cells were isolated using Tri Reagent Aliquots of RNA were electrophoresed and blotted onto nylon membranes, that hybridized to 32P-labelled probe.Western BlotCells were lysed and. then were centrifugated. Protein concentration was determined by the Bradford assay.. Equivalent amounts of protein loaded and electrophoresed and were transferred to nitrocellulose membranes, that were incubated with the different primary antibodies:,Result and Discussion:. In the present study, we evaluated a pattern of different gene expression by Northern Blot and Western Blot analysis in bone marrow and peripheral blood cells from 16 CML patients at onset, from 2 patients in blast crisis evolved under IM treatment, and 14 imatinib-resistant patients. Some RNAs were underexpressed in most or all samples tested and never overexpressed (eg SIRT2, SIRT3, SIRT4 and SIRT5), while others were overexpressed in the great majority of samples and rarely, if ever, underexpressed (eg SIRT1, SIRT7, HSP90, iNOS)Furthermore, we examined the level of heat-shock related proteins HSP90 and bcl-2 in 2 patients during treatment with IM. and one patient IM-resistant by western Blot analysis: HSP90 and BCl-2 increased one patient during treatment with IM, while both protein levels was very high in one one patient IM-resistant These results suggest that the difference of genes expression might contribute to patterns of clinical response.

The RALGEF Pathway Contributes to the Pathogenesis of Chronic Myelogenous Leukemia by BCR/ABL

Selected inhibitors of the BCR/ABL tyrosine kinase have shown a remarkable clinical activity in patients with chronic myelogenous leukemia (CML). However, these drugs do not completely eradicate leukemic cells and drug resistance emerges. Identification of additional contributors to the pathogenesis of CML remains to be important for developing strategies for overcoming resistance to BCR/ABL kinase inhibitors and for eradicating leukemic cells. We and others have previously shown that expression of BCR/ABL in mouse bone marrow cells by retroviral transduction and transplantation (BMT) efficiently induces a CML-like myeloproliferative disorder (MPD). Using this BMT model for CML, we have found that the Grb2 SH2 binding site Y177 of BCR/ABL, which is critical for BCR/ABL to activate the RAS pathway, is required for the induction of CML-like disease by BCR/ABL. We have further shown that oncogenic NRAS (NRASG12D) can rescue the defect of the Y177F mutant BCR/ABL in the induction of CML-like disease. To further elucidate the pathways downstream of NRAS responsible for this “rescue” effect, we utilized three well-characterized NRAS effector mutants, G12D/T35S (activating RAF but is impaired in binding PI3K and RALGEF), G12D/E37G (activating RALGEF but not RAF or PI3K), and G12D/Y40C (activating PI3K but not RAF or RALGEF). None of the three NRAS effector mutants could induce hematological malignancies by themselves, but all were capable of “rescuing” the CML defect of BCR/ABLY177F to some extent. Interestingly, the NRAS G12D/E37G effector mutant was the most efficient. We further examined the role of RALGEF pathway by introducing an activated RALGDSCAAX into our BCR/ABLY177F mouse model. RALGDS-CAAX was able to cooperate with BCR/ABLY177F to induce a CML-like MPD in nearly half of mice whereas RALGDSCAAX alone did not induce diseases in any mice. As a complement to this study, we also introduced dominant negative RAL A or RAL B in the background of BCR/ABL. This resulted in a significant delay in disease onset. Taken together, this data sheds light on the importance of the RALGEF pathway in BCR/ABL-mediated CML induction and supports the notion of inhibition of this pathway as a potential therapeutic strategy.

Effect of Ras Inhibition in Hematopoiesis and BCR/ABL Leukemogenesis

Ras small GTPases are activated in many hematopoietic growth factor signaling and in hematological malignancies, but their role in hematopoiesis and leukemogenesis is not completely known. Here we examined the effect of Ras inhibition by a dominant negative mutant of Ras, N17 H-Ras, in adult hematopoiesis and in BCR/ABL leukemogenesis using the mouse bone marrow transduction and transplantation approach. We found that N17 H-Ras expression suppressed B- and T-lymphopoiesis and erythropoiesis. Interestingly, N17 H-Ras did not suppress myelopoiesis in the bone marrow, yet it greatly attenuated BCR/ABL-induced chronic myelogenous leukemia (CML)-like myeloproliferative disease. Most BCR/ABL + N17 H-Ras mice eventually developed pro-B lymphoblastic leukemia/lymphoma (B-ALL). These results suggest that Ras activation is essential for the development of lymphoid and erythroid cells but not myeloid cells and that Ras is a critical target of BCR/ABL in the pathogenesis of CML, but not B-ALL.

RETRACTED ARTICLE: Disruption of the Bcr-Abl/Hsp90 protein complex: a possible mechanism to inhibit Bcr-Abl-positive human leukemic blasts by novobiocin

The Bcr-Abl fusion gene encodes for the p210(Bcr-Abl) or p185(Bcr-Abl) tyrosine kinase (TK) implicated in the pathogenesis of chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia, respectively. Because Bcr-Abl TK is chaperoned by Hsp90 (90 kDa heat-shock protein), we investigated the effects of novobiocin (NB), an Hsp90 C-terminal inhibitor, on the viability of the Bcr-Abl-positive human leukemia cells HL-60/Bcr-Abl and K562, the expression of Bcr-Abl protein and the interaction between Hsp90 and Bcr-Abl TK. Present studies demonstrate that NB is a potent inhibitor of the growth of Bcr-Abl-positive human leukemia cells. NB induces cytosolic accumulation of cytochrome c and activation of caspase-9 and caspase-3, triggering apoptosis of HL-60/Bcr-Abl and K562 cells. Treatment of cell lines with NB disrupts Bcr-Abl /Hsp90 and Bcr-Abl /Hsp70 interactions, resulting in a decreased amount of intracellular Bcr-Abl protein levels. Co-treatment with the proteasome inhibitor N-acetyl leucyl-leucyl norlucinal increases NB-mediated accumulation of Bcr-Abl in the detergent-insoluble cellular fraction, which demonstrates that NB promotes proteasomal degradation of Bcr-Abl. Moreover, both imatinib-resistant K562/G01 and primary CML CD34(+) cells are sensitive to NB.

Experimental non-ATP-competitive therapies for chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell malignancy driven by the BCR-ABL fusion tyrosine kinase. The central role played by BCR-ABL1 in the pathogenesis of CML facilitated the development of the tyrosine kinase inhibitor (TKI) imatinib mesylate, the first actual targeted therapy in cancer history. Imatinib competes with ATP at the active site of BCR-ABL1 kinase. Despite outstanding clinical results, imatinib as well as other BCR-ABL1 TKIs have been associated with limited rates of complete molecular response and the development of mutations within the kinase domain of BCR-ABL1 that impairs TKI binding. To override such drawbacks, an array of novel non-ATP-competitive therapies with distinct mechanisms of action is undergoing preclinical, and in some cases, early clinical stages of development. This review focuses on the most promising among such therapeutics.

Array CGH Analysis at 60kb Resolution of CML Samples at Advanced Stage of Disease.

In spite of the universal presence of the BCR/ABL1 fusion gene, chronic myelogenous leukemia (CML) shows remarkable clinical and genetic diversity. The consequences of der(9)t(9;22) chromosome deletions, associated with poor survival, as well as the mechanism behind their formation remain unclear, as does our understanding of the molecular events driving the disease evolution. The presence of these deletions fuelled the expectations that cryptic genome-wide aberrations may be accountable for the disease progression. Following a comprehensive BAC aCGH analysis of 48 CML samples (Brazma et al., Genes, Chromosomes & Cancer, 2007 in press) we report high-resolution oligo-nucleotide array study of a further 30 CML accelerated/ blast phase samples. We were unable to confirm the high frequency of particular single BAC imbalances (CNVs), reported both by ourselves and others, possibly due to the manufacturer's array selection strategy. Never-the-less some of the CNVs and a wealth of new imbalances were obtained at 60kb resolution. It was possible to build a precise map of the amplicon affecting the sequences flanking the 3′ ABL1 breakpoint site, which include the LAMC3 and NUP214 genes. The presence of this amplicon was associated with therapy resistance. When assessed, at a resolution of 60 kb, the deletions of the regions flanking the ABL1/BCR breakpoint showed novel features:1.the genome loss affects preferentially both flanking sites as seen in 5 of the 6 ‘deleted' samples and2.the 120kb deletion identified is the smallest recorded so far.Most of the major cytogenetic features of the samples were confirmed and a number of cryptic genome imbalances were detected, from 120kb to 10Mb in size, involving regions rich in genes, some already implicated in the pathogenesis of CML. Finally, recurrent micro aberrations of several adjacent oligo-nucleotides affecting non-coding sequences were detected in as many as 2/3 of the samples.

Molecular diagnosis and monitoring in the clinical management of patients with chronic myelogenous leukemia treated with tyrosine kinase inhibitors

The well-established molecular pathogenesis of chronic myelogenous leukemia (CML) and its consequences for laboratory testing and clinical management illustrate a classic paradigm for the importance of molecular diagnostics in targeted drug therapy. The success of the tyrosine kinase inhibitor (TKI), imatinib, as the currently recommended first-line treatment of early chronic phase CML has both fueled the need for timely and reproducible molecular testing of the BCR-ABL1 fusion transcript in diagnosis and monitoring as well as necessitated the detection of kinase domain mutations that confer resistance to this agent. As, ongoing research continues to refine guidelines for monitoring residual disease in patients undergoing TKI therapy, an understanding of molecular technologies and their interpretation is critical. This review summarizes the molecular strategies that are currently employed in the initial diagnosis and subsequent management of CML patients maintained on TKI therapy.

The Interferon Regulatory Factor Family in Host Defense: Mechanism of Action

Transcription factors of the interferon regulatory factor (IRF) family commands the entire type I interferon (IFN) system from induction of IFNs to diverse IFN responses, thereby providing a principal basis for host resistance against pathogens. However, the family has various additional roles. Regulating the development of the immune system, IRFs shape the establishment and execution of innate and adaptive immunity. IRFs also regulate growth and differentiation of many cell types, thus playing a role in leukemia and other cancers. In addition, evidence indicates that IRFs confer antiviral mechanisms not directly ascribed to the IFN system. This review deals with the diverse roles of IRFs in host defense and discusses the molecular mechanisms by which they regulate target gene transcription.

THE DUAL SRC/ABL KINASE INHIBITOR SKI-606 EFFECTIVELY INHIBITS BCR-ABL KINASE ACTIVITY IN CHRONIC MYELOGENOUS LEUKEMIA PRIMITIVE PROGENITOR CELLS.

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell malignant disease that results from a translocation between chromosomes 9 and 22. The translocation generates the BCR/ABL gene, which has been shown to play a role in the pathogenesis of CML. Imatinib mesylate (IM), a Bcr-Abl kinase inhibitor, is highly effective in the treatment of CML. However CML stem cells appear to be relatively resistant to elimination by IM. Incomplete elimination of malignant progenitors may be related to incomplete Bcr-Abl kinase inhibition, persistent signaling through MAPK, or Bcr-Abl kinase mutations resulting in IM resistance. This phenomenon creates a need to develop new approaches that enhance elimination of the malignant progenitor cells. The dual Src/Abl kinase inhibitor SKI-606 has been reported to exert potent antiproliferative activity against CML cell lines in vitro and in xenograft models and is currently being investigated in phase 1/2 clinical trials. CD34+CD38− primitive progenitor and CD34+CD38+ committed progenitor cells from untreated CML patients were selected by flow cytometry and then cultured for 24 hours in growth factor-supplemented medium in a range of concentrations of SKI-606 (0-0.5 μM) and with 5 μM IM for comparison. The effect of SKI-606 on Bcr-Abl-kinase activity was assessed by Western blotting with anti-P-CrkL antibodies after overnight drug exposure of CML CD34+ cells. Importantly, 0.1 μM and 0.5 μM SKI-606 significantly suppressed phospho-CrkL levels, whereas a higher concentration of IM (5 μM) was needed to achieve a similar degree of suppression. Although treatment of CML CD34+ cells with IM was associated with increased p42/44 MAPK activity, a significant increase in MAPK activity was not observed when the same samples were treated with SKI-606. SKI-606 is therefore significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors. Unlike IM, SKI-606 treatment was not associated with a significant compensatory increase in p42/44 MAPK signaling, which could potentially be beneficial in targeting malignant stem cells. Further investigation into the effects of SKI-606 on CML stem cells as a single agent or in combination with other compounds is warranted.

Stromal cell-derived factor-1 chemokine gene variant in blood donors and chronic myelogenous leukemia patients.

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disorder that originates from a pluripotent stem cell expressing the bcr-abl oncogene. It is characterized by an abnormal release of the expanded, malignant stem cell clone from the bone marrow into the circulation. The stromal cell derived factor-1 (SDF-1) gene contains a common polymorphism, termed SDF1-3'A, in an evolutionarily conserved segment of the 3' untranslated region (UTR). In this work the SDF-1 genotypes of 25 patients (9-82 years old) who had been clinically and hematologically diagnosed with CML were compared with those of 60 healthy donors. In addition, the nature of bcr-abl hybrid mRNA and the association between demographic and hematological parameters were analyzed in cells from 12 CML patients (five women and seven men). All patients underwent blood collection during the chronic phase of disease after they received chemotherapy. b3a2 mRNA was detected in samples from eight of the CML patients and b2a2 mRNA was observed in four cases. An association between basophils and hemoglobin parameters was observed in that hemoglobin levels were higher in b2a2-expressing patients, and mean basophil levels were higher in patients expressing b3a2. Four of the CML patients (16%) were homozygous for 3'A allele. Of the patients who showed the presence of bcr-abl transcripts (N = 12), three presented the wt/wt genotype and nine were SDF1-3'A carriers. Three of the latter were homozygous for this mutation. It is possible that the bcr-abl fusion gene and the SDF1 genotype for 3'A allele have important implications for the pathogenesis of CML.

Regulation of Differentiation and Proliferation of Marrow Hematogenesis Stem Cells by Notch1 Signaling System from Patients with Aplastic Anemia and Chronic Myelogenous Leukemia.

Notch genes encode evolutionarily conserved transmembrane receptors that regulate cell fate determination. Notch activation promotes proliferation and inhibits differentiation of bone marrow stem cells. Our research was to study the differential expression of Notch1 on bone marrow mononuclear cells (BMMNC) from chronic aplastic anemia (AA) and chronic myelogenous leukemia (CML) patients. We demonstrated that Notch signaling is inactivated in AA patients and activated in CML patients. In CML patients, Notch1 furthers stimulation by its ligand Jagged1, resulting in a strong increase of tumor cell growth. Therefore, we suggest that inactivated Notch signaling plays a pivotal role in the pathogenesis of AA and that activated Notch signaling plays a key role in the pathogenesis of CML. We used the antibody to inactivate Notch1 signaling in mice, and then to detect the hematopoietic state of these mice.Methods: 5ml marrow were abstracted from chronic AA patient s, CML patients in chronic phase and normal people and the BMMNC was separated from the marrow. RNA was abstracted from these cells. Real Time Quantitative PCR was used to investigate the expression of Notch1 on BMMNCs in 30 AA patients, 20 normal controls and 30 CML patients, and the changes of their expression after chemotherapy. Notch1 protein of these BMMNC was also detected through Western Blots. We injected monoclonal anti-Notch1 and isotonic Na chloride as controls into the tail vein of mice, to detect the hematopoietic state through bone marrow slides and flow cytometry.Results: Real Time PCR: Expression of Notch1 on BMMNCs from AA patients was lower than that of controls (P<0.05). The expression of Notch1 on BMMNCs from CML patients was higher than that of controls(P<0.05). Notch1 on the BMMNCs from AA patients was expressed higher after chemotherapy (P<0.05). Notch1 on the BMMNCs from CML patients was expressed lower after chemotherapy(P<0.05). Western Blots: Notch1 receptors were highly expressed in BMMNCs of CML patient. BMMNCs of AA patients expressed low amounts of Notch1. Jagged1 induces Notch signaling in cultured cells; We performed RT-PCR analysis of mRNA expression of Hes-1, a member of the hairy enhancer of split family of transcriptional repressors that are direct transcriptional targets of activated Notch. The result was that the expression of Hes-1 of CML was much higher than AA. The growth rate of CML BMMNCs was also much higher than AA BMMNCs. Compared with controls, the hematopoietic state of mice treated with anti-Notch1 had an obvious low hematopoietic ability.Conclusions: The expression of Notch1 on BMMNCs from AA patients and normal controls was different, which may account for the occurrence of AA. The expression of Notch1 of AA patients was improved after effective chemotherapy, which may be one of the reason of recovery of hematopoietic function. The expression of Notch1 on BMMNCs from CML patients was much higher than that of controls, which decreased after chemotherapy and may be one of the factors accounting for occurrence of CML. BMMNCs cocultured with Jagged-1 expressing cell lines can enhance the growth rate, which indicate that Notch1 can improve proliferation of leukemic cells. The mice treated with anti-Notch1 had lower hematopoietic ability manifesting that Notch1 signaling is essential to maintain normal hematopoietic function.

IRF-4 Functions as a Myeloid Tumor Suppressor.

Interferon regulatory factor 4 (IRF-4, also know as pip, LSIRF, ICSAT and MUM1) is a transcription factor with well-characterized functions in B and T cell development and immune response regulation. Though its expression has been demonstrated in macrophages, its function in the myeloid system is not as well characterized. The closely related IRF family member IRF-8 (ICSBP) has more clearly defined functions in the myeloid system. IRF-8 is a critical regulator of myelopoiesis, and its loss has been implicated in the pathogenesis of chronic myelogenous leukemia (CML). IRF-8 deficient mice manifest a CML-like syndrome, and we have shown previously that forced expression of IRF-8 in a BCR/ABL-induced murine model of CML represses the resulting myeloproliferative disease and prolongs survival. In the B-cell system, we have found that IRF-4 and IRF-8 function redundantly at the pre-B-to-B transition point of development. In this study we investigate whether IRF-4 and IRF-8 may also have overlapping function in myeloid cells. Our results show that mice deficient in both IRF-4 and IRF-8 develop from a very early age a more aggressive CML-like disease than mice deficient in IRF-8 alone. Bone marrow progenitors from IRF-4/8 DKO mice have increased proliferation, GM-CSF stimulated granulocytic differentiation, and colony-forming capacity compared to progenitors from WT or single KO mice. These data suggest that IRF-4 plays a role in myeloid lineage development and may act to suppress proliferation and granulocytic differentiation of myeloid progenitor cells. We further examined if, like IRF-8, IRF-4 is able to suppress BCR/ABL mediated transformation. We used a murine stem cell virus vector to co-express BCR/ABL and either IRF-4 or IRF-8 in BM progenitor cells and assessed colony forming capacity of infected cultures as well as the ability of IRF-4 to suppress BCR/ABL induced CML-like disease in a murine model. Our results demonstrate that IRF-4 potently suppresses BCR/ABL induced colony formation and that forced expression of IRF-4 suppresses BCR/ABL-induced CML-like disease in mice even more potently than IRF-8. These results provide direct evidence that IRF-4 is an important myeloid tumor suppressor and may allow elucidation of new molecular pathways significant to the pathogenesis of human CML.

Expression analysis of PCNA gene in chronic myelogenous leukemia—Combined application of siRNA silencing and expression arrays

Imatinib metylase is the first choice treatment for BCR/ABL positive chronic myelogenous leukemia (CML). However, as some CML patients develop resistance to imatinib therapy, there is a significant interest in development of alternative treatment strategies, such as identifying targets other than BCR/ABL that may participate in CML. Previously, we demonstrated strong PCNA up-regulation in CML patients. To further study its role in CML pathogenesis, we performed silencing of PCNA expression followed by array experiments. PCNA inhibition led to down-regulation of CDK1, CDK4, PLK1, ERK3, JNK1, STAT5, and several inhibitors of apoptosis (DAXX, Mdm2, survivin). The following genes were up-regulated: CDK inhibitors p21 and p19-INK4D, pro-apoptotic FAST kinase, fibronectin, etc . However, as PCNA affects cell growth in naturally proliferating cells as well as in cancerous cells, it seems to act a secondary role relating to proliferation activity of leukemic cells.

Hematopoietic growth factors, signaling and the chronic myeloproliferative disorders

The chronic myeloproliferative diseases (CMDs) are a group of conditions characterized by unregulated blood cell production, that due either to excessive numbers of erythrocytes, leukocytes or platelets, or their defective function cause symptoms and signs of fatigue, headache, ruddy cyanosis, hemorrhage, abdominal distension, and the complications of vascular thrombosis. In the late 19th century Vaquez provided the first description of polycythemia vera (PV) and Hueck defined idiopathic myelofibrosis (IMF). In 1920, di Guglielmo established criteria for patients with essential thrombocythemia (ET). In 1951, Dameshek argued that these disorders, along with chronic myelogenous leukemia (CML) display many similar clinical and laboratory features [Dameshek W. Some speculations on the myeloproliferative syndromes. Blood 1951;6:372–5], and grouped them. In 2002, the World Health Organization expanded the definition of CMDs to also include chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia/hypereosinophilic syndrome (CEL/HES) and systemic mast cell disorder (SMCD) [Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 2002;100:2292–302]. While the molecular pathogenesis of CML is well known [Melo JV, Deininger MW. Biology of chronic myelogenous leukemia-signaling pathways of initiation and transformation. Hematol Oncol Clin North Am 2004;18:545–68], and the causes of CEL/HES and SMCD have been identified in about half of all cases [Gotlib J, Cools J, Malone III JM, Schrier SL, Gilliland DG, Coutre SE. The FIP1L1–PDGFRalpha fusion tyrosine kinase in hypereosinophilic syndrome and chronic eosinophilic leukemia: implications for diagnosis, classification, and management. Blood 2004;103:2879–91; Valent P, Akin C, Sperr WR, Horny HP, Metcalfe DD. Mast cell proliferative disorders: current view on variants recognized by the World Health Organization. Hematol Oncol Clin North Am 2003;17:1227–41], until very recently the etiologies of the three classically defined CMDs, PV, IMF and ET, were poorly understood. Each of these disorders is characterized by excessive hematopoiesis, a process usually dependent on one or more hematopoietic growth factors (HGFs). This review will focus on how our knowledge of the molecular mechanisms by which HGFs are produced, bind cell surface receptors and transduce survival and proliferative signals have provided the platform on which the multiple origins of CMDs can be understood and novel therapeutic interventions designed.

Tratamento da recidiva da leucemia mielóide crônica após transplante de medula óssea alogênico utilizando mesilato de imatinibe: relato de três casos

Imatinib mesylate (MI), a selective tyrosine kinase inhibitor involved in the pathogenesis of chronic myelogenous leukemia (CML), has become the first-line treatment for this disease. Donor lymphocyte infusion (DLI) has been considered as the standard treatment for relapse after allogeneic bone marrow transplantation (BMT), even though it is frequently associated with graft versus host disease and myelosuppression. Because of the satisfactory results and tolerance of the treatment of CML, the authors used MI as an alternative therapy for DLI in patients that relapsed after BMT. They obtained cytogenetic remission in two cases, with, in one case, proven conversion to the donor chimera. The third case evolved with progression of the disease and a second BMT was required. Since this is a new alternative, this descriptive study suggests it should be considered as an alternative therapy for relapse after BMT.

Chronic Myelogenous Leukemia: From Molecular Biology to Clinical Aspects and Novel Targeted Therapies

The critical causative event in chronic myelogenous leukemia (CML) is the fusion of the head of the bcr gene with the body of the abl gene, named bcr/abl gene. This chimeric BCR/ABL molecule transforms primary myeloid cells to leukemic cells and induces a CML-like disease in mice. The mouse CML model expressing the BCR/ABL molecule has provided important new insights into the molecular pathophysiology of CML and has directly answered many questions regarding this disease. Furthermore, numerous clinical studies have demonstrated a correlation between leukemic clinical features and the position of the breakpoint in the BCR gene of the chimeric BCR/ABL gene. Understanding of the molecular pathogenesis of CML has led to the development of several novel therapies. The BCR/ABL molecule is unique oncogeneiety, having ABL tyrosine kinase activity, making it an ideal target for drug development. Subsequent clinical studies now realize the hypothesis that selective inhibition of the abl tyrosine kinase activity using imatinib mesylate might be useful for the treatment of CML. This article reviews the history of BCR/ABL molecular biology, including the CML model mouse, clinical molecular studies and the recent findings of imatinib mesylate and more potent tyrosine kinase inhibitors developed for the treatment of CML.

Multiple roles of Rap1 in hematopoietic cells: complementary versus antagonistic functions

AbstractSmall G proteins serve as critical control points in signal transduction, integrating a wide range of stimuli to dictate discrete cellular outcomes. The outcomes of small G-protein signaling can both potentiate and antagonize one another. Studies in hematopoietic cells have uncovered multiple functions for the small G protein, Rap1 (Ras-proximate-1). Because Rap1 can regulate cell proliferation, differentiation, and adhesion through distinct mechanisms, it serves as a paradigm for the need for tight cellular control of small G-protein function. Rap1 has received recent attention for its role in enhancing integrin-dependent signals. This action of Rap1 augments a variety of processes that characterize hematopoietic-cell function, including aggregation, migration, extravasation, and homing to target tissues. Rap1 may also regulate cellular differentiation and proliferation via pathways that are distinct from those mediating adhesion, and involve regulation of the mitogen-activated protein (MAP) kinase or ERK (extracellular signal-regulated kinase) cascade. These actions of Rap1 occur in selected cell types to enhance or diminish ERK signaling, depending on the expression pattern of the MAP kinase kinase kinases of the Raf family: Raf-1 and B-Raf. This review will examine the functions of Rap1 in hematopoietic cells, and focus on 3 cellular scenarios where the multiple actions of Rap1 function have been proposed. Recent studies implicating Rap1 in the maturation of megakaryocytes, the pathogenesis of chronic myelogenous leukemia (CML), and activation of peripheral T cells will receive particular attention.

P210BCR-ABL inhibits SDF-1 Chemotactic Response via Alteration of CXCR4 Signaling and Down-regulation of CXCR4 Expression

It has been shown that p210(BCR-ABL) significantly impairs CXCR4 signaling. We report here that the migratory response to SDF-1 was profoundly altered in blast crisis, whereas chronic-phase CD34(+) cells migrated normally to this chemokine. This migratory defect was associated with a low CXCR4 membrane expression. In vitro STI-571 treatment of CD34(+) cells from patients in blast crisis markedly increased the CXCR4 transcript and CXCR4 membrane expression. Because p210(BCR-ABL) frequently increases with disease progression, we determined the effects of high and low p210(BCR-ABL) expression on CXCR4 protein in the granulocyte macrophage colony-stimulating factor-dependent human cell line MO7e. p210(BCR-ABL) expression distinctly alters CXCR4 protein through two different mechanisms depending on its expression level. At low expression, a signaling defect was detected with no modification of CXCR4 expression. However, higher p210(BCR-ABL) expression induced a marked down-regulation of CXCR4 that is related to its decreased transcription. The effect of p210(BCR-ABL) required its tyrosine kinase activity. Collectively, these data indicate that p210(BCR-ABL) could affect CXCR4 by more than one mechanism and suggest that down-regulation of CXCR4 may have important implications in chronic myelogenous leukemia pathogenesis.

On the Molecular Origins of the Chronic Myeloproliferative Disorders: It All Makes Sense

On the Molecular Origins of the Chronic Myeloproliferative Disorders: It All Makes Sense