Parthenolide and DMAPT induce cell death in primitive CML cells through reactive oxygen species.

Tyrosine kinase inhibitors (TKI) have become a first-line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34+ lin- ) cells. We found that both agents induced cell death in CML, while having little effect on the equivalent normal hematopoietic cells. PTL and DMAPT caused an increase in reactive oxygen species (ROS) levels and inhibited NF-κB activation. PTL and DMAPT inhibited cell proliferation and induced cell cycle arrest in G0 and G2 phases. Furthermore, we found cell cycle inhibition to correlate with down-regulation of cyclin D1 and cyclin A. In summary, our study shows that PTL and DMAPT have a strong inhibitory effect on CML cells. Given that cell cycle arrest was not dependent on ROS induction, we speculate that this effect could be a direct consequence of NF-κB inhibition and if this mechanism was to be evaded, PTL and DMAPT induced cell death would be potentiated.

Global gene expression profiles of hematopoietic stem and progenitor cells from patients with chronic myeloid leukemia: the effect of in vitro culture with or without imatinib.

In this study, we determined the gene expression profiles of bone marrow-derived cell fractions, obtained from normal subjects and Chronic Myeloid Leukemia (CML) patients, that were highly enriched for hematopoietic stem (HSCs) and progenitor (HPCs) cells. Our results indicate that the profiles of CML HSCs and HPCs were closer to that of normal progenitors, whereas normal HSCs showed the most different expression profile of all. We found that the expression profiles of HSCs and HPCs from CML marrow were closer to each other than those of HSCs and HPCs from normal marrow. The major biologic processes dysregulated in CML cells included DNA repair, cell cycle, chromosome condensation, cell adhesion, and the immune response. We also determined the genomic changes in both normal and CML progenitor cells under culture conditions, and found that several genes involved in cell cycle, steroid biosynthesis, and chromosome segregation were upregulated, whereas genes involved in transcription regulation and apoptosis were downregulated. Interestingly, these changes were the same, regardless of the addition of Imatinib (IM) to the culture. Finally, we identified three genes-PIEZO2, RXFP1, and MAMDC2- that are preferentially expressed by CML primitive cells and that encode for cell membrane proteins; thus, they could be used as biomarkers for CML stem cells.

Casiopeina III-Ea, a copper-containing small molecule, inhibits the in vitro growth of primitive hematopoietic cells from chronic myeloid leukemia.

Several novel compounds have been developed for the treatment of different types of leukemia. In the present study, we have assessed the in vitro effects of Casiopeina III-Ea, a copper-containing small molecule, on cells from patients with Chronic Myeloid Leukemia (CML). We included primary CD34+ Lineage-negative (Lin-) cells selected from CML bone marrow, as well as the K562 and MEG01 cell lines. Bone marrow cells obtained from normal individuals - both total mononuclear cells as well as CD34+ Lin- cells- were used as controls. IC50 corresponded to 0.5µM for K562 cells, 0.63µM for MEG01 cells, 0.38µM for CML CD34+ lin- cells, and 1.0µM for normal CD34+ lin- cells. Proliferation and expansion were also inhibited to significantly higher extents in cultures of CML cells as compared to their normal counterparts. All these effects seemed to occur via a bcr-abl transcription-independent mechanism that involved a delay in cell division, an increase in cell death, generation of Reactive Oxygen Species and changes in cell cycle. Our results demonstrate that Casiopeina III-Ea possesses strong antileukemic activity in vitro, and warrant further preclinical (animal) studies to assess such effects in vivo.

The rs61764370 Functional Variant in the KRAS Oncogene is Associated with Chronic Myeloid Leukemia Risk in Women.

BACKGROUND: Chronic myeloid leukemia (CML) is one of the most frequent hematopoietic malignancies in the elderly population; however, knowledge is limited regarding the genetic factors associated with increased risk for CML. Polymorphisms affecting microRNA (miRNA) biogenesis or mRNA:miRNA interactions are important risk factors in the development of different types of cancer. Thus, we carried out a case-control study to test the association with CML susceptibility of gene variants located in the miRNA machinery genes AGO1 (rs636832) and GEMIN4 (rs2740348), as well as in the miRNA binding sites of the genes BRCA1 (rs799917) and KRAS (rs61764370). MATERIALS AND METHODS: We determined the genotype of 781 Mexican-Mestizo individuals (469 healthy subjects and 312 CML cases) for the four polymorphisms using TaqMan probes to test the association with CML susceptibility. RESULTS: We found a borderline association of the minor homozygote genotype of the KRAS_rs61764370 polymorphism with an increased risk for CML susceptibility (P = 0.06). After gender stratification, this association was significant only for women (odds ratio [OR] = 13.41, P = 0.04). The distribution of the allelic and genotypic frequencies of the four studied SNPs was neither associated with advanced phases of CML nor treatment response. CONCLUSIONS: To the best of our knowledge, this study is the first to show a significant association of the KRAS_rs61764370 SNP with CML. To further determine such an association of with CML susceptibility, our results must be replicated in different ethnic groups.

CDKIs p18(INK4c) and p57(Kip2) are involved in quiescence of CML leukemic stem cells after treatment with TKI.

Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease. In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34(+)CD38(-)lin(-) LSC and HSC. Our results demonstrate that cellular location of p18(INK4c) and p57(Kip2) seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18(INK4c) and p57(Kip2) nuclear location. The differences in p18(INK4c)and p57(Kip2)activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.

Current patient management of chronic myeloid leukemia in Latin America: a study by the Latin American Leukemia Net (LALNET).

BACKGROUND: Treatment recommendations have been developed for management of patients with chronic myeloid leukemia (CML). METHODS: A 30-item multiple-choice questionnaire was administered to 435 hematologists and oncohematologists in 16 Latin American countries. Physicians self-reported their diagnostic, therapeutic, and disease management strategies. RESULTS: Imatinib is available as initial therapy to 92% of physicians, and 42% of physicians have access to both second-generation tyrosine kinase inhibitors. Standard-dose imatinib is the preferred initial therapy for most patients, but 20% would manage a young patient initially with an allogeneic stem cell transplant from a sibling donor, and 10% would only offer hydroxyurea to an elderly patient. Seventy-two percent of responders perform routine cytogenetic analysis for monitoring patients on therapy, and 59% routinely use quantitative polymerase chain reaction. For patients who fail imatinib therapy, 61% would increase the dose of imatinib before considering change to a second-generation tyrosine kinase inhibitor, except for patients aged 60 years, for whom a switch to a second-generation tyrosine kinase inhibitor was the preferred choice. CONCLUSIONS: The answers to this survey provide insight into the management of patients with CML in Latin America. Some deviations from current recommendations were identified. Understanding the treatment patterns of patients with CML in broad population studies is important to identify needs and improve patient care.

[Chronic myeloid leukemia in the 21st century: biology and treatment]. / La leucemia mieloide crónica en el siglo XXI: biología y tratamiento.

Chronic Myeloid Leukaemia (CML) is a clonal disease, originated at the level of Hematopoietic Stem Cells (HSC) and characterized by the presence of the Philadelphia (Ph) chromosome and its oncogenic product p210(BcrAbl). Such a protein has been shown to be essential for malignant transformation, since it is capable of altering cell adhesion, proliferation and apoptosis. Historically, CML has been treated by using different approaches: arsenic (in the early days), a variety of chemical agents (busulfan, hydroxyurea, cytarabine), cytokines (IFN-alpha, IFNalpha-PEG), hematopoietic cell transplant (HCT), and more recently drugs generated by design (imatinib, nilotinib, dasatinib). All these molecules exert specific effects on HSC and lead to a variety of clinical and biological responses. In this article, we present an overview about hematopoiesis in CML and its implications in the treatment of this disease.

Functional integrity in vitro of hematopoietic progenitor cells from patients with chronic myeloid leukemia that have achieved hematological remission after different therapeutic procedures.

In this study, we have assessed the in vitro growth of hematopoietic progenitor cells (HPC) from chronic myeloid leukemia (CML) patients that have recovered after different treatments. Bone marrow cells were obtained from 33 CML patients, including patients at diagnosis, before treatment (n=12), and patients that have achieved hematological remission (and in most cases a major cytogenetic response) after different therapeutic procedures (n=21), including patients treated with Interferon-alpha (IFN; n=5), imatinib mesylate (IMATINIB; n=8) and patients that received an allogeneic hematopoietic cell transplant (HCT; n=8). Marrow cells were enriched for CD34(+) cells and cultured in a serum- and stroma-free liquid culture system, supplemented with a combination of 8 recombinant cytokines. Normal samples were studied as controls. HPC from CML patients before therapy showed deficient proliferation and expansion potentials in culture (140-fold increase in nucleated cell number and 1.3-fold increase in colony-forming cell number) as compared to normal progenitors (1200-fold increase in nucleated cell number and 25-fold increase in colony-forming cell number). In contrast, HPC from patients treated with IMATINIB showed growth potentials similar to those of normal progenitors. Progenitors from patients after HCT also showed significant proliferation and expansion capacities. Interestingly, progenitors from IFN-treated patients showed proliferation and expansion kinetics similar to those of cells from untreated patients. These results indicate that, although treatment of CML patients with IFN, IMATINIB or HCT resulted in complete hematological remission (and a major cytogenetic response), only patients treated with IMATINIB and, to a lesser extent, with HCT showed a full hematopoietic recovery, as determined by the in vitro growth of HPC in our culture system.

HLA CW3 and HLA CW4 have a protective effect on acquisition of chronic myeloid leukemia on Mexican patients.

INTRODUCTION: Chronic myeloid leukemia (CML) is characterized by a chromosomal translocation t(9; 22) resulting in the chimeric ber-abl oncogene that encode for the p210 protein which has an increased tyrosine kinase activity. The fusion part of this protein contains a novel aminoacid sequence. If peptides derived from this leukemia-specific part of p210 are expressed in the context of HLA molecules on malignant cells this may elicit immunologically specific responses. Recent studies using synthetic peptides identical to the bcr-abl fusion region revealed that breakpoint specific peptides are capable of binding to the class I molecules HLA-A3, -A11 and -B8. It has been shown that individuals expressing HLA-A3 or HLA-B8 have a diminished risk for development of CML in Caucasoid populations. Other authors have reported a statistically significant increase in the frequency of Cw3 and Cw4 antigens in Causcasoids and European CML patients. These data suggested that Cw3 and Cw4 may be markers for CML susceptibility on these populations. OBJECTIVE: To asses a possible susceptibility effect of these HLA molecules we studied 63 CML Mexican Mestizo patients and 746 healthy subjects for the distribution of HLA class I and class II molecules. RESULTS: The gene products that showed statistically significant differences between CML patients and controls were DR14, DR3, Cw3 and Cw4. DR14 and DR3 were significantly increased in the patients group with respect to the controls group (DR14 antigen frequency: 3.17% vs. 0.29%; p = 0.03; DR3 20.63% vs. 8.33%; p = 0.0001). Cw3 and Cw4 were significantly decreased in the patient group (Cw3 26.9% vs. 49.11%; p = 0.03; Cw4 antigen frequency 23.8% vs. 86.28%; p = 0.0000001). The relative risk for DR14 was 10.48 (95% CI 1.52-79.29) and for DR3 was 3.96 (95% CI 2.05-7.71). The relative risk for Cw3 was 0.38 (95% CI 0.08-1.79) and for Cw4 was 0.11 (95% CI 0.048-0.81). CONCLUSION: These results suggest that the development of CML is apparently associated with HLA phenotypes specific to each population, and indicate that Cw3 and Cw4 expression may result in a protective effect on the CML acquisition on Mexican Mestizo population probably by bcr-abl breakpoint peptides presentation through these HLA molecules.

Kinetics of hematopoiesis in bone marrow cultures from patients with chronic myeloid leukemia: effect of recombinant cytokines in dexter-type long-term cultures.

Chronic myeloid leukemia (CML) is a hematological neoplasia that results from the transformation of a hematopoietic stem cell. It is characterized by the expansion of the myeloid lineage, which results in the accumulation of mature and immature granulocytes in peripheral blood and bone marrow. However, when CML marrow cells are cultured in Dexter-type long-term cultures (LTMC) hematopoiesis is defective and can be sustained for only a few weeks. One possible explanation for the deficient growth of hematopoietic cells in CML LTMC is that some factors that act as key regulators of hematopoiesis are absent in this experimental system. Thus, we tested this hypothesis by adding recombinant cytokines to these cultures. As a first approach, we added recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), rhGranulocyte-CSF (rhG-CSF) and rhErythropoietin (rhEPO); each factor was added individually once a week. Addition of rhGM-CSF and rhG-CSF resulted in a significant increase in the levels of nucleated cells and myeloid progenitors; the highest effects were seen in the presence of rhGM-CSF. Interestingly, such a cytokine also induced a significant decrease in the levels of erythroid progenitors. Recombinant hEPO had no significant effects on nucleated cells or myeloid progenitors, however, it induced a significant, although transient, increase in the levels of erythroid cells. The above results indicate that the hematopoietic regulators used here (rhGM-CSF, rhG-CSF and rhEPO) are capable of stimulating the growth of hematopoietic cells in LTMC from CML patients. Thus, this study demonstrates that it is, indeed, possible to manipulate CML LTMC by the addition of recombinant cytokines; this observation may be of particular relevance, since this in vitro experimental system has already been used as a method for purging of leukemic cells in autologous transplant settings. By using specific recombinant hematopoietic modulators it might be possible to make LTMC a more efficient system for such a clinical purpose.

[Analysis of Bcr-abl type transcript and its relationship with platelet count in Mexican patients with chronic myeloid leukemia]. / Análisis del tipo de transcrito bcr-abl y su relación con la cuenta plaquetaria en pacientes mexicanos con leucemia mieloide crónica.

PROBLEM: It has been suggested that type of chimeric mRNA is associated with differences in the clinical and hematologic characteristics of chronic myeloid leukemia (CML). However, prognostic value of type of chimeric mRNA bcr-xabl (b3a2 or b2a2) is still controversial. METHODS: We analyzed 97 cases of Philadelphia-positive CML to determine mRNA type by reverse-polymerase chain reaction (RT-PCR) and its relationship with clinical features. RESULTS: We detected b3a2 bcr-abl transcripts in 27 (28%) cases, b2a2 in 57 (59%) cases, and 13 (13%) with both mRNA transcripts b3a2/b2a2. These frequencies were the total reverse of other reports. Age, sex, hemoglobin, and white-cell counts showed no significant difference for those with either b3a2 or b2a2 bcr-abl transcripts. However, platelet counts of b3a2 patients were significantly higher than those of b2a2 patients (743.3 vs 477.3 x 109/L; p = 0.01). In addition, in the subgroup of patients whose white-cell count at diagnosis was < 100 x 10(9)/L, those with b3a2 transcript had a significantly higher platelet count (679.1 vs. 352.2 x 10(9)/L; p = 0.001). CONCLUSIONS: We observed reversed frequency of bcr-abl transcripts in this population, but agreement with other Latin-American reports. In addition, our data suggested that there is different CML biological behavior in our population and that there is a subpopulation of CML patients in whom b3a2 is associated witH higher thrombopoietic activity.