Molecular and cellular bases of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the overproduction of granulocytes, which leads to high white blood cell counts and splenomegaly in patients. Based on clinical symptoms and laboratory findings, CML is classified into three clinical phases, often starting with a chronic phase, progressing to an accelerated phase and ultimately ending in a terminal phase called blast crisis. Blast crisis phase of CML is clinically similar to an acute leukemia; in particular, B-cell acute lymphoblastic leukemia (B-ALL) is a severe form of acute leukemia in blast crisis, and there is no effective therapy for it yet. CML is induced by the BCR-ABL oncogene, whose gene product is a BCR-ABL tyrosine kinase. Currently, inhibition of BCR-ABL kinase activity by its kinase inhibitor such as imatinib mesylate (Gleevec) is a major therapeutic strategy for CML. However, the inability of BCR-ABL kinase inhibitors to completely kill leukemia stem cells (LSCs) indicates that these kinase inhibitors are unlikely to cure CML. In addition, drug resistance due to the development of BCR-ABL mutations occurs before and during treatment of CML with kinase inhibitors. A critical issue to resolve this problem is to fully understand the biology of LSCs, and to identify key genes that play significant roles in survival and self-renewal of LSCs. In this review, we will focus on LSCs in CML by summarizing and discussing available experimental results, including the original studies from our own laboratory.     

Detection of an antigen, associated with cells during the blast crisis of chronic myeloleukemia, in a cytotoxic test using xenogenic antibodies]

The immune antimyeloblast serum (AMS) was obtained from horses immunized with white blood cells from patients suffering from chronic myeloid leukemia (CML) at the blast crisis stage; the serum was completely absorbed with normal red blood cells and white blood cells (WBC). The absorbed antiserum remained cytotoxic to blast cells from 20 of 42 patients with CML at the blast crisis stage. AMS failed to react with the WBC from patients with CML in its chronic phase, and from patients with other types of leukemia Morphological studies indicated a possibility of identification of the antigen associated with myeloblasts from the blood of patients with CML blast crisis, by means to AMS.     

Acute leukemia in adults: assessment of remission induction with combination chemotherapy by clinical and cell-culture criteria.

Remission induction was assessed by clinical and cell-culture criteria for 65 patients with acute myelogenous leukemia (AML), 11 patients with chronic myelogenous leukemia (CML) in blast crisis and 19 patients with acute lymphoblastic leukemia (ALL). Cyclophosphamide, cytosine arabinoside and vincristine (CAV) therapy resulted in complete remission in 23 of 50 previously untreated patients with AML and in 3 of the 11 patients with CML. Fourteen patients with ALL responded to vincristine-prednisone induction therapy and two to induction therapy with CAV. The median duration of survival of the responding patients was 2.2 years, compared with 4 months for the patients who did not respond to treatment. Granulopoietic colony formation, assessed by assay of colony-forming units dependent on colony-stimulating activity in culture (CFU-C), was abnormal in 37 of 42 bone marrow aspirates from patients with AML before treatement. CFU-C concentration increased when leukocyte-conditioned medium (LCM) was added to the cultures; 13 cultures had normal or elevated CFU-C concentration with LCM. Marrow cells of patients with ALL or CML in blast crisis demonstrated a similar pattern. Serial studies of marrow CFU-C concentration of 31 patients with AML demonstrated a change to a normal pattern with successful remission induction. Results of this study suggest that administration of purified LCM to leukemic patients might increase granulocyte production from potential but unstimulated granulopoietic precursors. This therapy would lessen the probability of death from infection during remission induction.     

Tiazofurin: biological effects and clinical uses

Inosine 5'-phosphate dehydrogenase (IMPDH) activity is increased in all cancer cells. It is the rate-limiting enzyme of guanosine triphosphate (GTP) biosynthesis, and therefore, a sensitive target of chemotherapy. Tiazofurin selectively blocks IMPDH activity. Tiazofurin was found to have an antiproliferative effect on tumor cells in vitro and in the murine system. Based on these findings, Phase I trials were started elsewhere in patients with solid tumors, but were discontinued because of toxicity. In leukemic patients, we were able to demonstrate a good correlation between biochemical parameters (i.e., decline in IMPDH activity and GTP concentrations in blast cells) and clinical response. The most consistent responses to therapy were seen in patients with myeloid blast crisis of chronic myeloid leukemia. Severe toxicity was seen in the earlier patients in the study. However, better patient selection, limitation of treatment duration and earlier recognition and treatment of complications have now made it possible to administer tiazofurin without undue toxicity.     

The dimeric Mcm8–9 complex of Xenopus laevis likely has a conserved function for resistance to DNA damage

The success of Imatinib mesylate (STI571, Gleevec) in treating chronic myeloid leukemia (CML) is, to date, the crowning achievement of targeted molecular therapy in cancer. Nearly 90% of newly diagnosed patients treated with Imatinib in the chronic phase of the disease achieve a complete cytogenetic response. However, more than 95% of these patients retain detectable levels of BCR-ABL mRNA and patients discontinuing Imatinib therapy almost invariably relapse, demonstrating that an Imatinib insensitive population of leukemia-initiating cells (LICs) persists in nearly all patients. These findings underscore the need for treatments specifically targeting the leukemia-initiating population of CML cells. While mounting evidence suggests that the LIC in the chronic phase of CML is the BCR-ABL positive hematopoietic stem cell, several recent publications suggest that during CML blast crisis, a granulocyte-macrophage progenitor (GMP) population also acquires LIC properties through activation of the β-catenin pathway. Characterization of these cells and evaluation of their sensitivity to Imatinib is critical to our understanding and treatment of CML blast crisis.     

Lysis of fresh leukemic blasts by interferon-activated human natural killer cells

In a comprehensive study of 30 leukemia patients, it was found that a measurable fraction of fresh leukemic blasts from 8 of 8 adult patients with chronic myelogenous leukemia (CML) in blast crisis and 10 of 11 pediatric patients with childhood acute lymphocytic leukemia (ALL) were efficiently lysed by human peripheral blood natural killer (NK) cells as measured in 4-hour chromium release assays. The observed lysis of these fresh, noncultured, neoplastic blasts was mediated by a population of interferon-augmentable, FcR-positive, non-adherent large granular lymphoid cells from normal donors, which were also able to kill the 'standard' NK target K562. It was of further interest that all 8 of the patients with blast crisis CML exhibited myeloid type morphology. Furthermore, neoplastic lymphoblasts from 9 of 10 patients with NK-susceptible childhood ALL lacked easily detectable B or T cell markers and were of 'null' cell type. In marked contrast to the lytic susceptibility of fresh leukemic blasts from patients with ALL and CML in blast crisis, fresh neoplastic granulocytes from 5 patients with chronic phase CML (2 of which eventually progressed to myeloid type blast crisis), as well as leukemic blasts from 8 patients with acute myeloid leukemias (AML, AMMoL, and AMoL) were resistant to lysis as mediated by human NK cells from normal donors. The clinical implications of these findings are discussed.     

Investigation of therapy resistance mechanisms in myeloid leukemia by protein profiling of bone marrow extracellular fluid

Evaluation of: Pizzatti L, Panis C, Lemos G et al. Label-free MS(E) proteomic analysis of chronic myeloid leukemia bone marrow plasma: disclosing new insights from therapy resistance. Proteomics 12(17), 2618–2631 (2012).

In spite of the effective chronic myeloid leukemia (CML) therapy, a small percentage will fail on therapy and develop acute myeloid leukemia-like blast crisis. Understanding the underlying biology of therapy resistance is probably needed to develop better blood cancer therapy, and CML may be an ideal disease model for future therapy that targets resistance mechanisms. Cell-stromal interactions and dissection of the interstitial tissue fluid is a relatively new source for understanding the resistance mechanisms. Abdelhay’s team have compared the proteome of bone marrow plasma in CML imatinib (Gleevec) responders and nonresponders. We discuss their findings of dysregulated redox and Wnt signaling in imatinib resistance, illustrating how powerful proteomics may be in the discovery of new therapeutic mechanisms.     

A biological marker model for predicting disease transitions

For patients with chronic myelogenous leukemia (CML), the effect of elevated blood levels of adenosine deaminase (ADA) is studied as a marker for transitions from stable disease to blast crisis and then to death. Data in the form of snapshots over time, with day, state of disease, and ADA level, are analyzed for 55 patients. A simple three-state Markov model with one-way transition probabilities dependent on ADA is used to determine if the marker has a significant effect on the prediction of changes from stable disease to blast crisis.     

Promoter methylation of p16 and EDNRB gene in leukemia patients in Taiwan

Both epigenetic and genetic alternations are involved in cancer formation. In this study, we have identified the methylation frequency of p16 and endothelin receptor type B (EDNRB) of 26 leukemia patients and 8 randomly selected normal blood donors in Taiwan. Promoter methylation of p16 was detected in 85% of acute lymphocytic leukemia (ALL), 83% in acute myeloid leukemia (AML) whereas no methylation was detected in chronic myeloid leukemia (CML) in blast crisis. Hypermethylation of EDNRB was observed in 92% of ALL, 75% AML and 100% in CML in blast crisis. No aberrant methylation of p16 and EDNRB was found in 8 normal blood donors. Taken together, aberrant methylation of p16 and EDNRB was highly prevalent in leukemia patients in Taiwan.     

慢性粒细胞性白血病急变的分子机制

慢性粒细胞性白血病(chronic myelogenous leukemia,CML)是源于造血干细胞伴有t(9;22)(q34;q11)染色体易位的恶性骨髓增生性疾病,其急变期与急性白血病相似,具有较强致死性。本文对CML急变分子机制有关的最新研究成果进行了综述,旨在深入理解CML急变的分子机制,并试图发现新的研究思路。     

In vitro culture studies of blood and marrow cells in chronic myeloid leukemia at different phases of the disease

The in vitro culture growth of peripheral blood (PB) and bone marrow (BM) cells were studied simultaneously from 100 adult patients with chronic myeloid leukemia at different phases. Sixty-five patients were investigated at initial diagnosis, 30 patients in control phase, and 41 patients in blast phase. In untreated chronic phase, the relative concentrations of granulocyte-macrophage progenitor cells (CFU-GM) in BM were not significantly different from those of normal controls, but there was generally a marked increase in circulating CFU-GM. The 6 Ph1-negative patients did not show different growth characteristics. We were unable to correlate the CFU-GM number to any of the hematologic parameters as well as to the response to busulfan therapy. Pretreated patients with excessive cluster formation did not necessarily indicate impending blast crisis. In hematologic remission, the numbers of CFU-GM in both BM and PB were well within the ranges of normal controls. Culture results in blast phase revealed a spectrum of abnormal growth. In myeloid crisis, 14/29 BM and 12/29 PB samples showed increased colony and cluster formations which were composed predominantly of immature cells with variable degeneration. Marrow cells in lymphoid crisis produced low numbers of both colonies and clusters in 5 out of 8 patients, while blood cells from 8 out of 10 patients formed large amount of colonies of normal morphology. This study indicates that the in vitro CFU-GM assay may have diagnostic utility in differentiating lymphoid crisis from myeloid crisis.     

Ultrastructural characterization of de novo and secondary leukemias

Cells from 95 patients with acute leukemia were studied by cytochemistry, light microscopy, and transmission electron microscopy (TEM), and were classified according to the French-American-British (FAB) guidelines. This group included 63 patients with acute nonlymphocytic leukemia (ANLL) de novo, 18 with acute lymphocytic leukemia (ALL), and 14 with ANLL as a second malignancy. In addition, 13 cases of chronic myelocytic leukemia in blast crisis were studied. Ultrastructural examination resulted in reclassification of 6 cases of ANLL de novo; two of these were reclassified from M2 (myeloblastic leukemia with maturation) to M3 variant (microgranular variant of hypergranular promyelocytic leukemia). The classification of the cases of CML in blast crisis was identical by light microscopy and TEM. IN 1 case of myeloblastic crisis, however, basophilic granules were demonstrated by TEM but were not appreciated by light microscopy. Classification of the cases of secondary leukemia was possible by light microscopy and cytochemistry in all 14 cases, but was often difficult since the cytochemical reactions were usually less intense than in de novo ANLL. This was particularly true in those cases classified as M1, and in such cases, TEM was required to confirm the diagnosis.     

Interferon alfa-2c in chronic myelogenous leukemia (CML): hematologic, cytogenetic and molecular-genetic response of patients with chronic phase CML previously resistant to therapy with interferon gamma

Alpha- and gamma-interferons have been shown to actively suppress hematopoiesis in patients in the chronic phase of chronic myelogenous leukemia in vitro and in vivo. Since both interferons act through different receptors on their hematopoietic target cells, they are expected to be capable of independently inhibiting abnormal blood cell development in patients with chronic myelogenous leukemia. We have utilized recombinant human interferon alfa-2c to treat 11 patients with Philadelphia chromosome positive chronic myelogenous leukemia in chronic phase, who were resistant to previous interferon gamma therapy. Ten of the patients were evaluable for hematologic, cytogenetic and molecular-genetic response following interferon alfa-2c therapy for 6 to 30 months. In 5 patients, IFN alfa-2c treatment failed due to lack of hematologic response. A complete hematologic or partial hematologic response was achieved in the remaining 5 patients. Three of these experienced cytogenetic improvement with reappearence of 100% diploid hematopoietic cells and disappearence of c-abl/bcr rearrangement in one patient. In two patients interferon alfa-2c did not prevent transformation of the disease into an accelerated state or blast crisis, respectively. We conclude that recombinant human interferon alfa-2c may also control hematopoiesis in interferon-gamma resistant chronic myelogenous leukemia patients, although the long-term response will need to be elucidated in further studies.     

Daunomycin, cytosin-arabinoside and VP-16 (DAV) for myeloid blast crisis of CML

Nine patients with myeloid blast crisis of Philadelphia chromosome-positive chronic myelocytic leukemia received 1-3 courses of intensive induction chemotherapy with DAT (daunomycin, cytosin-arabinoside and 6-thioguanin) or DAV (daunomycin, cytosin-arabinoside and VP-16). Eight patients responded with clearing of blasts from peripheral blood giving a response rate of 89%. However, bone marrow aplasia with less than 5% blasts was seen in only 2 patients. These 2 patients subsequently received an allogeneic bone marrow transplant and achieved complete remissions of 3 and 6 month duration. All patients died due to progression of blast crisis. Median survival of the group was 164 days. These results were compared to a historical control group of 31 patients with myeloid blast crisis treated with vincristine and prednisone. Despite a significantly better response rate with DAV or DAT (8 of 9 versus 9 of 31, p = 0.01) survival was not significantly different than that of the control group.     

Dual PPARα/γ Ligand TZD18 Either Alone or in Combination with Imatinib Inhibits Proliferation and Induces Apoptosis of Human CML Cell Lines

Despite progress in the treatment of early-stage chronic myeloid leukemia (CML), the accelerated and blastic phases of CML still remain a therapeutic challenge. Persistence of BCR-ABL-positive (bcr-abl+) cells or secondary resistance during imatinib therapy frequently occurs. In this study, we investigated the activity of a novel dual ligand specific for peroxisome proliferator-activated receptor α and γ (PPARα/γ) against CML blast crisis cell lines. Exposure of these cell lines (K562, KU812 and KCL22) to TZD18 resulted in a growth inhibition in a dose- and time-dependent manner. This effect may not be mediated through PPARγ and/or PPARα activation, since antagonists of PPARg and/or PPARa could not reverse this inhibition. Western blotting analysis showed that expression of the cyclin dependent kinase inhibitor (CDKI) p27kip1 was enhanced, whereas levels of cyclin E, cyclin D2 and cyclin dependent kinase 2 (CDK-2) were decreased when these cells were treated with TZD18. Most interestingly, TZD18 synergistically enhanced the antiproliferative and pro-apoptotic effects of imatinib. Overall, our findings strongly suggest that TZD18, either alone or in combination with imatinib may be beneficial for the treatment of CML in myeloid blast crisis.     

ATM-dependent DNA damage-response pathway as a determinant in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) begins with an indolent chronic phase, and subsequently progresses to an accelerated or blastic phase. Although several genes are known to be involved in the progression to blastic phase, molecular mechanisms for the evolution toward blast crisis have not been fully identified. Oncogenic stimuli enforce cell proliferation, which requires DNA replication. Unscheduled DNA replication enforced by oncogenic stimuli leads to double strand breaks on DNA. We found the DNA damage-response pathway is activated in bone marrow of chronic-phase CML patients possibly due to an enforced proliferation signal by BCR-ABL expression. Since ataxia telangiectasia mutated (ATM) is a central player of the DNA damage-response pathway, we studied whether loss of this pathway accelerates blast crisis. We crossed Atm-knockout mice with BCR-ABL transgenic mice to test this hypothesis. Interestingly, the loss of one of the Atm alleles was shown to be enough for the acceleration of the blast crisis, which is supported by the finding of increased genomic instability as assayed by breakage–fusion–bridge (BFB) cycle formation. In light of these findings, the DNA damage-response pathway plays a vital role for determination of susceptibility to blast crisis in CML.     

Influence of BCR/ABL fusion proteins on the course of Ph leukemias

The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.