Sensitivity of Ph 1 + CFU-GM to human recombinant interferon alpha and gamma alone and in combination

The in vitro effect of human recombinant interferon alpha (IFN) alone and in combination were studied on granulomonocytic colony forming units (CFU-GM) from the peripheral blood of 10 Ph 1+ chronic myeloid leukemia (CML) patients and from the marrow of 5 normal or non-leukemic subjects. alpha- and gamma-IFN alone determined a slight inhibition on colony growth with a preferential effect on "pure" macrophagic colonies. At maximum concentration (10(4) U/ml) leukemic colony inhibition was 46 +/- 34% for alpha IFN and 43 +/- 19% for gamma IFN. Culture growth with alpha + gamma IFN in combination were significantly inhibited (up to 96 +/- 4%) with a concentration-related effect. Similar results were obtained with normal CFU-GM. The synergism that was found in vitro is probably relevant for the in vivo therapeutic effects of these compounds in CML and suggest that the combination is worth testing in vivo.     

Sensitivity of chronic myeloid leukemia hemopoietic progenitors to PTT-119 in combination with human recombinant interferon alpha and gamma

PTT-119, a new synthetic alkylating compound, has shown a marked "in vitro" inhibitory effect on chronic myeloid leukemia (CML) granulo-monocytic precursors (CFU-GM) at doses greater than 5 micrograms/ml. Based on previous experiences of synergistic associations between alkylating drugs and biological modifiers, we tested the effects of low doses of PTT-119 (from 0.1 to 1 microgram/ml) in concert with alpha, gamma, or alpha + gamma interferons and compared to IFNs alone, in order to investigate an alternative choice for treatment of CML patients in chronic phase. Our results showed a significantly higher CFU-GM cloning inhibition after addition of 100 or 1,000 U/ml of alpha IFN to 0.1 microgram/ml PTT-119 (from 39.6% +/- 26.6 SD to 80.7% +/- 10 SD and 91.5% +/- 8 SD, respectively), while gamma IFN resulted in only a slight increase in colony growth inhibition when compared to the drug used alone. The association of alpha plus gamma IFN coupled with PTT-119 treatment did not significantly improve the results observed after exposure of leukemic progenitors to PTT-119 and alpha IFN alone. We conclude that a combined treatment with PTT-119 and IFN is probably worth testing both for purging methods before autologous bone marrow transplantation and for in vivo administration in chronic myeloid leukemia.     

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.     

Treatment of chronic myelogenous leukemia with interferons alpha and gamma

A 23-year-old male patient with Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML) was treated with both IFN alpha and IFN gamma. Normalization of leukocyte counts was reached after 3 months of treatment. Southern blot analysis failed to detect the neoplastic cell clone after 19 months of therapy. Cytogenetically, complete suppression of Ph positive cells in the patient's bone marrow and blood was observed after 20 months and 25 months, respectively. This response was achieved with doses of IFN alpha and IFN gamma which were considerably lower than the dosage of IFN used in single agent therapy of CML.     

Investigations on karyotype evolution in patients with chronic myeloid leukemia (CML)

In an attempt to relate karyotype evolution to clinical and hematological data serial chromosomal analyses were performed in 31 patients with chronic myeloid leukemia (CML), both in chronic and acute phases. Our results in Philadelphia chromosome (Ph1)-positive CML are in line with karyotype profiles described in the literature. In addition, we report on chromosomal findings in 4 cases of Ph1-negative disease, one presenting with an iso17q chromosome in the positive CML. The same chromosomal abnormality was observed in a small population of Ph1-negative cells present in one of two patients with mixed Ph1-positive/Ph1-negative CML. The first case of a female patient with the loss of a sex chromosome in Ph1-positive cells is reported. Two patients with unusually long and mild chronic phases despite the presence of trisomy 8 in their karyotypes are described. Our findings suggest that the order of appearance of additional chromosomal changes of CML is of prognostic significance for the progression and the clinical picture of the disease.     

Treatment of Ph+ chronic myeloid leukemia by gamma interferon

The clinical, hematologic and cytogenetic effects of human recombinant gamma interferon (IFN) were investigated in 14 patients with Ph+ chronic myeloid leukemia (CML). Gamma-IFN was given at a daily dosage of 0.50 mg (= 10 x 10(6) U)/m2 from the 3rd week of treatment on, but the dosage had to be reduced to 0.25 mg/m2 in 10 cases and to 0.35 mg/m2 in 2 cases, because of the severity and persistence of side effects (mainly fever, fatigue, headache and pain). Only 2 patients tolerated the full dosage. The overall response rate was 64% (1 complete and 8 partial hematologic responses). Only patients in stable chronic phase responded. Two out of two patients in unstable chronic phase and two out of two patients in accelerated phase failed to respond. Eight out of nine responding patients remained in remission throughout the duration of treatment (30 to 35 weeks). No karyotypic conversion was detected. These data show that gamma IFN alone is effective in Ph+ CML, but that side effects can limit substantially the dosage and duration of treatment.     

Identification of a melanoma antigen, PRAME, as a BCR/ABL-inducible gene

In order to elucidate molecular events in BCR/ABL-induced transformation, we adopted a polymerase chain reaction (PCR)-based technique of differential display and compared mRNA expression in human factor-dependent cells, TF-1, with that in factor-independent cells, ID-1, which were established from TF-1 cells by transfection of BCR/ABL. Cloning and sequencing of a gene which was upregulated in ID-1 cells revealed that the gene was identical to a melanoma antigen, PRAME. Our present study demonstrated that PRAME was markedly expressed in primary leukemic cells with chronic myeloid leukemia (CML) in blastic crisis and Philadelphia (Ph)+-acute lymphoblastic leukemia (ALL), in which BCR/ABL played an important role as a pathogenic gene. Moreover, comparison of PRAME expression among CD34+ cells with CML in blastic, accelerated, and chronic phases revealed a higher expression in CML in advanced phases. Thus PRAME was considered to be a good candidate for a marker of Ph+-leukemic blast cells as well as a new target antigen of leukemic blast cells that cytotoxic T cells can recognize.     

Granulopoiesis in long-term culture by marrow from mice with busulfan-induced chronic latent aplasia

Mice given high-dose busulfan therapy develop a chronic latent marrow aplasia characterized by normal peripheral blood neutrophil numbers, hematocrits and marrow cellularity but reduced numbers of pluripotent hemopoietic stem cells (CFU-s) and granulocyte-monocyte progenitor cells (CFU-gm). To study the pathogenesis of this lesion, bone marrow was propagated in long-term marrow cultures (LTMC). Small amounts of normal marrow readily established and sustained long-term granulopoiesis in vitro. In contrast, inocula of marrow from busulfan-treated animals containing three to five times as many stem and progenitor cells failed to establish long-term granulopoiesis in vitro. These results suggest that high-dose busulfan therapy produces a qualitative defect in either the hemopoietic stem cells, the stromal-forming elements, or both, rendering them incapable of establishing long-term granulopoiesis in vitro. Furthermore, mixing experiments employing normal and busulfan-damaged marrow demonstrate that this qualitative defect is not due to the emergence of a suppressor cell population. LTMC can show types of marrow damage not detectable by other techniques currently available and represent a powerful tool for studying latent bone marrow failure.     

Stem cell and kinase activity-independent pathway in resistance of leukaemia to BCR-ABL kinase inhibitors

BCR-ABL tyrosine kinase inhibitors, such as imatinib (Gleevec) are highly effective in treating human Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) in chronic phase but not in terminal acute phase; acquired drug resistance caused mainly by the development of BCR-ABL kinase domain mutations prevents cure of the leukaemia. In addition, imatinib is ineffective in treating Ph+ B-cell acute lymphoblastic leukaemia (B-ALL) and CML blast crisis, even in the absence of the kinase domain mutations. This type of drug resistance that is unrelated to BCR-ABL kinase domain mutations is caused by the insensitivity of leukaemic stem cells to kinase inhibitors such as imatinib and dasatinib, and by activation of a newly-identified signalling pathway involving SRC kinases that are independent of BCR-ABL kinase activity for activation. This SRC pathway is essential for leukaemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Apart from BCR-ABL and SRC kinases, stem cell pathways must also be targeted for curative therapy of Ph+ leukaemia.     

Effects of the tyrosine kinase inhibitor imatinib mesylate (STI571) on bone marrow features in patients with chronic myelogenous leukemia

Preliminary data are available about bone marrow (BM) changes in patients with chronic myeloid leukemia (CML) who received the molecularly targeted and highly effective tyrosine kinase inhibitor Imatinib mesylate (STI571). This review is focused on a systematic assessment of BM features detectable at different stages of CML (stable, accelerated, blastic) following long-term (more than 10 months) treatment. By applying enzyme- and immunohistochemistry including monoclonal antibodies visualizing proliferating cell nuclear antigen (PCNA) and apoptosis (anti-apostatin), a more elaborate insight into alterations affecting hematopoiesis and the stroma compartment was gained. In patients with stable-phase CML therapy resulted in a significant reduction in cellularity, neutrophil granulopoiesis and number of megakaryocytes, accompanied by a retrieval of erythroid precursors. In patients with Imatinib as the only treatment morphometric analysis of CD61+ megakaryopoiesis was in keeping with a significant decrease in maturation defects implying a lesser amount of atypical micromegakaryocytes almost consistent with normalization. Moreover, a reduction of the initially enhanced (CD34+) microvessel density was detectable associated with a decrease in luminal distension. Regression of marked to moderate myelofibrosis was recognizable in about 70% of patients especially in the accelerated and blastic phases. The amount of myeloblasts, CD34+ progenitor cells and lysozyme-expressing immature myelomonocytic cells declined with treatment, but recurred in about 19% of patients that developed a leukemic relapse after 21+/-6 months of therapy. Data on proliferative activity and apoptosis in general supported in vitro findings concerning the inhibitory effect of this agent on growth associated with a tendency for stimulated apoptosis, at least in responding patients.     

Effect of cytosine arabinoside on the differentiation of granulocyte-monocyte progenitors (CFU-GM) and myeloid leukemia blasts (CFU-L) in vitro

The serum concentrations of Ara-C are in the range from 10(-6) to 10(-8) M in LD-Ara-C treated patients. The growth of CFU-GM from bone marrow of healthy volunteers was depressed depending on Ara-C-concentration applied in vitro. The growth of CFU-L from peripheral blood of two patients with AML (M 2) and one patient with CML in blast crisis was differently influenced by Ara-C-application in vitro. An elevated proportion of mature cells was observed in smears of cultured cells with Ara-C from two patients. The usefulness of Ara-C for a differentiation inducing therapy is discussed.     

Human long-term bone marrow cultures in aplastic anemia

Long-term bone marrow cultures (LTMC) were initiated with marrow from five normal subjects and eight patients with aplastic anemia (AA). Near confluent to confluent adherent layers developed in all cultures from normal subjects and AA patients. When present, the 'cobblestone' areas in LTMC from AA subjects were smaller than those observed in the LTMC from normal subjects. The decline in total and viable cell numbers in the LTMC was similar for both normal subjects and AA patients. Granulocyte-macrophage colony-forming units (CFU-gm) were present in nonadherent cells (NAC) from normal LTMC for a mean of 5.2 weeks. CFU-gm were present in the NAC of only two of the eight AA cultures for one week. The absent or small 'cobblestone' areas and the absence of CFU-gm production in AA-LTMC suggest a decrease in the reproductive potential of adherent hematopoietic stem cells, which may be the result of either an abnormal hematopoietic stem cell or an abnormal stromal microenvironment or both.     

Comparative proteome profiling and functional analysis of chronic myelogenous leukemia cell lines

The aim of the present study was the molecular profiling of different Ph+ chronic myelogenous leukemia (CML) cell lines (LAMA84, K562, and KCL22) by a proteomic approach. By employing two-dimensional gel electrophoresis combined with mass spectrometry analysis, we have identified 191 protein spots corresponding to 142 different proteins. Among these, 63% were cancer-related proteins and 74% were described for the first time in leukemia cells. Multivariate analysis highlighted significant differences in the global proteomic profile of the three CML cell lines. In particular, the detailed analysis of 35 differentially expressed proteins revealed that LAMA84 cells preferentially expressed proteins associated with an invasive behavior, while K562 and KCL22 cells preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, although representing the same pathological phenotype, show characteristics in their protein expression profile that suggest different phenotypic leukemia subclasses. These data contribute a new potential characterization of the CML phenotype and may help to understand interpatient variability in the progression of disease and in the efficacy of a treatment.     

Heat Shock Protein 90: A Potential Therapeutic Target in Leukemic Progenitor and Stem Cells Harboring Mutant BCR-ABL Resistant to Kinase Inhibitors

Development of drug resistance has become a major obstacle for tyrosine kinase inhibitors (TKIs) in the treatment of Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) and other cancers. The BCR-ABL-T315I mutant does not respond to clinically available TKIs, although some newly developed anti-BCR-ABL-T315I TKIs are now being tested in patients. TKIs transiently inhibit kinase activity of BCR-ABL, but do not reduce the level of the BCR-ABL protein. Elimination of mutant BCR-ABL protein would provide a new therapeutic strategy for treating Ph+ leukemia. We recently showed that inhibition of heat shock protein 90 (Hsp90) by a novel Hsp90 inhibitor, IPI-504, causes BCR-ABL protein degradation, decreased numbers of leukemia stem cells, and prolonged survival of mice with CML induced by BCR-ABL-T315I. Here we discuss further the mechanisms and effectiveness of Hsp90 inhibition in suppression of survival and proliferation of leukemic progenitor and stem cells in CML mice, and the potential of this anti-Hsp90 strategy in treating CML patients, including those who have developed resistance to TKIs.     

In vitro antitumor effect of LAK cells and alpha interferon in combination on tumor cell lines.

In vitro antitumor effects of LAK cells and alpha-2b-Interferon (IFN) either alone or in combination were evaluated on NK resistant (K562) and NK sensitive (Namalwa, Raji) cell lines. Tumor cells were incubated with LAK cells for 4, 8 and 24 hours at a LAK: tumor cell ratio of 1:1, 10:1, 100:1, or with IFN for 48 and 96 h at the concentrations of 100, 1000, 10,000, 100,000 IU/ml. A clonogenic assay was utilised to enumerate residual cells after in vitro treatment. A positive correlation was found between tumor cell killing and effector: target ratio, IFN of 100:1 incubated for 4 h, and 100 IU/ml of IFN incubated for 48 h were further chosen. A synergistic effect was found when IFN was incubated before LAK cells or contemporarily, but not when IFN was incubated after LAK cells. These findings demonstrate that an additive or a synergistic effect in vitro can be obtained by adding the two agents in different sequences and suggest that a potential utility of LAK cells and IFN in vivo should be tested in clinical trials.     

Inhibition of PI3K/mTOR Overcomes Nilotinib Resistance in BCR-ABL1 Positive Leukemia Cells through Translational Down-Regulation of MDM2

Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.     

Cytogenetic findings in chronic myelogenous leukemia

Cytogenetic findings in chronic myeloic leukemia are represented in a survey. More than 90 per cent of CML are characterized by Ph1 chromosomes, with more than 90 per cent of the cases being involved in a translocation (9; 22). Further, non-incidental aberrations are +Ph1, isochromosome (17q) and +8 which particularly develop at the acute stage. Isochromosome 17q is assumed to be a marker for a straightly impending development of a blast crisis. Ph1-negative CML is connected with a comparatively bad prognosis for the patient. Partial trisomy 9q+ is indicated here as a marker chromosome. For the patient concerned congenital chromosome defects, such as the Down-syndrome, represent a higher risk of being affected with leukemia.     

Suppressor T cell activation by human leukocyte interferon

Murine fibroblast interferon (IFN beta) activates murine suppressor T lymphocytes in vitro, which suppress plaque-forming cell responses by spleen cells. Suppression of human in vitro immune responses by IFN was investigated to determine whether human IFN also activates suppressor T cells. Human leukocyte IFN (IFN alpha) suppressed pokeweed mitogen-induced polyclonal immunoglobulin production by human peripheral blood mononuclear cells (PBMC) by 80 to 90% at doses of 200 to 350 U/ml. Responses by IFN alpha-treated PBMC were suppressed in a dose-dependent manner; control cultures had maximal responses on day 7. PBMC incubated with 10,000 U/ml of IFN alpha contained activated suppressor cells that decreased pokeweed mitogen-stimulated, polyclonal immunoglobulin production by autologous cells by 70 to 80%. Suppression mediated by these cells was prevented by catalase, ascorbic acid, and 2-mercaptoethanol (2-ME). In murine systems, these reagents interfere with expression of suppressor T cell activity by preventing activation of soluble immune response suppressor. Selection procedures with monoclonal antibodies identified the suppressor cell as an OKT8+ (suppressor/cytotoxic) T lymphocyte. Selected OKT8+ cells required less IFN alpha (1000 U/ml) for activation and were effective in smaller numbers than unfractionated activated PBMC. IFN alpha-activated suppressor cells also inhibited proliferation in mixed lymphocyte and mitogen-stimulated PBMC cultures; again, catalase and 2-ME blocked suppression. These results indicate that IFN alpha activates suppressor T cells in human PBMC cultures; the ability of catalase, 2-ME, and ascorbic acid to block suppression suggests that these suppressor T cells have certain similarities to IFN beta or to concanavalin A-activated murine suppressor T cells.