Hematopoietic stem cell expansion and distinct myeloid developmental abnormalities in a murine model of the AML1-ETO translocation

The t(8;21)(q22;q22) translocation, which fuses the ETO gene on human chromosome 8 with the AML1 gene on chromosome 21 (AML1-ETO), is one of the most frequent cytogenetic abnormalities associated with acute myelogenous leukemia (AML). It is seen in approximately 12 to 15% of AML cases and is present in about 40% of AML cases with a French-American-British classified M2 phenotype. We have generated a murine model of the t(8;21) translocation by retroviral expression of AML1-ETO in purified hematopoietic stem cells (HSC). Animals reconstituted with AML1-ETO-expressing cells recapitulate the hematopoietic developmental abnormalities seen in the bone marrow of human patients with the t(8;21) translocation. Primitive myeloblasts were increased to approximately 10% of bone marrow by 10 months posttransplant. Consistent with this observation was a 50-fold increase in myeloid colony-forming cells in vitro. Accumulation of late-stage metamyelocytes was also observed in bone marrow along with an increase in immature eosinophilic myelocytes that showed abnormal basophilic granulation. HSC numbers in the bone marrow of 10-month-posttransplant animals were 29-fold greater than in transplant-matched control mice, suggesting that AML1-ETO expression overrides the normal genetic control of HSC pool size. In summary, AMLI-ETO-expressing animals recapitulate many (and perhaps all) of the developmental abnormalities seen in human patients with the t(8;21) translocation, although the animals do not develop leukemia or disseminated disease in peripheral tissues like the liver or spleen. This suggests that the principal contribution of AML1-ETO to acute myeloid leukemia is the inhibition of multiple developmental pathways.     

Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia.

The nature of null-cell acute lymphatic leukemia (ALL) was investigated with the aid of a thymic humoral factor (THF), bone marrow cells, and a local xenogeneic graft-versus-host reaction (GVHR). Lymphocytes obtained from the blood and bone marrow of six children with T-cell ALL, five with null-cell ALL, one with perinatal B-cell ALL, one with acute myelocytic leukemia, and one with erythroleukemia were tested for membrane surface markers (E, EAC, and SM Ig); functional activity of T cells was tested by a local GVHR. All of the specimens obtained at the initial presentation showed a lack of functional activity of the lymphocytes. Incubation of null cell and acute myelocytic leukemia (AML) bone marrow with THF led to the acquisition of the characteristics of functional, immunocompetent T cells. No such effect was seen when the bone marrow of T-cell ALL and peripheral blood lymphocytes of B-cell perinatal ALL were incubated with THF. This study demonstrates that the null cell in ALL bone marrow can be differentiated into a T cell whereas the stem cell in AML bone marrow constitutes a pluripotential undifferentiated cell which also can mature into a T cell.     

Antibody-producing human-human hybridomas. III. Derivation and characterization of two antibodies with specificity for human myeloid cells

Peripheral blood mononuclear cells from a patient with acute myeloid leukemia (AML) and spleen cells from a patient with chronic myeloid leukemia (CML) were fused with HAT-sensitive human B lymphoma cells (RH-L4) in attempts to generate human monoclonal antibodies (Mab) against antigens with high specificity for myeloid leukemia cells. Forty-seven of 246 hybridomas secreted Ig that bound to AML cell surface constituents, as determined by FACS analysis of viable cells that were FITC-stained with the human Mab as the first-step reagent and FITC-conjugated rabbit anti-human Ig as second-step. Two of the 47 human Mab (one from each patient and designated AML-19 and CML-20, respectively) bound to both autologous and allogeneic myeloid leukemia cells. No significant binding was observed to cell surface constituents on human bone marrow cells, granulocytes, lymphocytes, erythrocytes, thymocytes, monocytes, lymphoblastic leukemia cells, fibroblasts, malignant B and T lymphocytic cell lines, and murine bone marrow cells. Both human Mab were IgG and were cytotoxic to myeloid leukemia cells in the presence of complement. About 70% of peripheral blood cell samples from 46 AML patients contained AML-19- and CML-20-positive cells, but the reactivity pattern had no correlation to the morphologic FAB classification of the samples. The promyelocytic HL60 cell line and the K562 cell line reacted with the two antibodies. Dot blot analysis of binding of AML-19 and CML-20 to cellular extracts immobilized on nitrocellulose paper showed that both human Mab in this assay also reacted with normal bone marrow cells. This was supported by microscopic immunofluorescence because both human Mab stained intracytoplasmatic structures in normal bone marrow cells, but both intracytoplasmatic and cell surface components stained in myeloid leukemia cells. Moreover, immunoblotting demonstrated that both human Mab in leukemia cells reacted with two cellular proteins with Mr approximately 14,500 and 18,000, and in normal bone marrow cells with a molecule with Mr approximately 20,000. Immunoprecipitation of cell membrane molecules with both the AML-19 and CML-20 antibody precipitated from leukemic cells only the molecule with Mr approximately 18,000 and no components from normal bone marrow cells. It is concluded that myeloid leukemogenesis may result in generation of cell surface expression of either new or abnormally processed molecules that are immunogenic in the autochthonous host. These molecules may also be useful as markers in diagnosis of myeloid leukemia.     

Monoclonal antibodies to carbohydrate antigens in autologous bone marrow transplantation

Normal and malignant myeloid cells express a highly immunogenic oligosaccharide, lacto-n-fucopentaose-III (LNF-III), that has been identified by numerous monoclonal antibodies (MoAb). We have been interested in the use of a particular monoclonal antibody to LNF-III, PM-81, in the treatment of patients with acute myelogenous leukemia using the antibody to treat bone marrow in vitro. Following in vitro treatment of bone marrow with PM-81 and another MoAb, AML-2-23, the remaining cells are used as an autograft in a patient treated with high-dose chemotherapy and radiotherapy. In order to enhance the ability of the MoAb to lyse leukemic cells in the remission bone marrow, we have explored the effect of neuraminidase treatment on leukemia cells. In this paper we describe that myeloid leukemia cells expressing low levels of LNF-III by immunofluorescence can be shown to have high levels of LNF-III after neuraminidase treatment. In addition, we show that normal bone marrow progenitor cells do not have cryptic LNF-III antigen, thus allowing the application of this finding to the clinical setting. Moreover, we have shown that leukemia colony-forming cells from one patient with acute myelogenous leukemia express cryptic LNF-III and that after exposure to neuraminidase there was an increased ability of PM-81 in the presence of complement to eliminate these colony forming cells. These data indicate that the LNF-III moiety is almost universally expressed on myeloid leukemia cells and their progenitors but not expressed on normal progenitors. Thus, it may be possible to enhance leukemia cell kill in vitro by neuraminidase treatment of bone marrow.     

Impact of genetic background and aging on mesenteric collateral growth capacity in Fischer 344, Brown Norway, and Fischer 344 x Brown Norway hybrid rats

Available studies indicate that both genetic background and aging influence collateral growth capacity, but it is not known how their combination affects collateral growth. We evaluated collateral growth induced by ileal artery ligation in Fischer 344 (F344), Brown Norway (BN), and the first generation hybrid of F344 x BN (F1) rats available for aging research from the National Institute on Aging. Collateral growth was determined by paired diameter measurements in anesthetized rats immediately and 7 days postligation. In 3-mo-old rats, significant collateral growth occurred only in BN (35% +/- 11%, P < 0.001). The endothelial cell number in arterial cross sections was also determined, since this precedes shear-mediated luminal expansion. When compared with the same animal controls, the intimal cell number was increased only in BN rats (92% +/- 21%, P < 0.001). The increase in intimal cell number and the degree of collateral luminal expansion in BN rats was not affected by age from 3 to 24 mo. Immunohistochemical studies demonstrated that intimal cell proliferation was much greater in the collaterals of BN than of F1 rats. The remarkable difference between these three strains of rats used in aging research and the lack of an age-related impairment in the BN rats are novel observations. These rat strains mimic clinical observations of interindividual variation in collateral growth capacity and the impact of age on arteriogenesis and should be useful models to investigate the molecular mechanisms responsible for such differences.     

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.     

Quantitative cytology in leukemia research

A study was undertaken to determine the usefulness of flow cytometric analysis of bone marrow cells as an objective means for diagnosis, classification and prognosis in patients with leukemia. Abnormal DNA content as a marker of neoplastic disease was found in only 15% of 264 adult patients with acute leukemia (13% in AML, 26% in ALL/AUL). Alternative means of tumor cell detection in heterogeneous marrow samples include determination of nucleolar antigen density and double-stranded RNA content. Phenotypic characterization of leukemia subtypes can be afforded by RNA content analysis of acridine orange-stained cells, demonstrating significantly higher mean RNA content values in AML, compared to ALL/AUL. Cytokinetic parameters amenable to flow cytometric analysis include measurements of cell cycle compartment distribution by DNA content, of cycle traverse rate by BUdR-induced modification of fluorescence intensity of DNA specific dyes and of growth fraction employing the method of in situ DNA denaturation and subsequent acridine orange staining. Determination of cell cycle distribution and RNA content pretreatment and serially during remission induction in 82 patients demonstrated a significantly lower pretreatment biopsy S phase proportion in responding patients with AML compared to individuals failing treatment whereas an opposite trend was noted in patients with ALL/AUL. While of no prognostic impact pretreatment, serial determinations of the RNA content during the first chemotherapy induction course revealed significant differences between responding and failing patients with AML. Also, patients attaining remission demonstrated a rise in marrow biopsy S phase compartment size by day 10 to 14 of treatment, thus, predicting remission during marrow hypoplasia. We conclude that quantitative cytologic examination of marrow cells from patients with acute leukemia provides useful diagnostic and prognostic information that should aid in the stratification of patients with poor prognosis to receive new agents.     

Contribution of an aged microenvironment to aging-associated myeloproliferative disease

The molecular and cellular mechanisms of the age-associated increase in the incidence of acute myeloid leukemia (AML) remain poorly understood. Multiple studies support that the bone marrow (BM) microenvironment has an important influence on leukemia progression. Given that the BM niche itself undergoes extensive functional changes during lifetime, we hypothesized that one mechanism for the age-associated increase in leukemia incidence might be that an aged niche promotes leukemia progression. The most frequent genetic alteration in AML is the t(8;21) translocation, resulting in the expression of the AML1-ETO fusion protein. Expression of the fusion protein in hematopoietic cells results in mice in a myeloproliferative disorder. Testing the role of the age of the niche on leukemia progression, we performed both transplantation and in vitro co-culture experiments. Aged animals transplanted with AML1-ETO positive HSCs presented with a significant increase in the frequency of AML-ETO positive early progenitor cells in BM as well as an increased immature myeloid cell load in blood compared to young recipients. These findings suggest that an aged BM microenvironment allows a relative better expansion of pre-leukemic stem and immature myeloid cells and thus imply that the aged microenvironment plays a role in the elevated incidence of age-associated leukemia.     

Benzene, the exposome and future investigations of leukemia etiology

Benzene exposure is associated with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and probably lymphoma and childhood leukemia. Biological plausibility for a causal role of benzene in these diseases comes from its toxicity to hematopoietic stem cells (HSC) or progenitor cells, from which all leukemias and related disorders arise. The effect of this toxicity is manifest as lowered blood counts (hematotoxicity), even in individuals occupationally exposed to low levels of benzene. Benzene can induce AML/MDS via several well-characterized pathways associated with these diseases. Through its metabolites, benzene induces multiple alterations that likely contribute to the leukemogenic process, and appears to operate via multiple modes of action. To improve mechanistic understanding and for risk assessment purposes, it may be possible to measure several of the key events in these modes of action in an in vitro model of the bone marrow stem cell niche. Even though benzene is leukemogenic at relatively low occupational levels of exposure, it seems unlikely that it is a major cause of leukemia in the general population exposed to benzene in the ppb range. Other established non-genetic causes of AML, e.g. smoking, ionizing radiation and cancer chemotherapy, also only explain about 20% of AML incidence, leaving ～80% unexplained. The question arises as to how to find the causes of the majority of de novo AMLs that remain unexplained. We propose that we should attempt to characterize the 'exposome' of human leukemia by using unbiased laboratory-based methods to find the unknown 'environmental' factors that contribute to leukemia etiology.     

EVI-1 modulates arsenic trioxide induced apoptosis through JNK signalling pathway in leukemia cells

High expression of the oncogene ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukemia patients. This study aimed to examine the effects of arsenic trioxide (ATO) on EVI-1 in acute myeloid leukemia (AML). Mononuclear cells were isolated from the bone marrow and peripheral blood of AML patients and healthy donors. EVI-1 expression in hematopoietic cells was evaluated by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). ATO down-regulated EVI-1 mRNA in zebrafish in vivo as well as in primary leukemia cells and THP-1 and K562 cells in vitro. Additionally, ATO treatment induced apoptosis, down-regulated both EVI-1 mRNA and oncoprotein expression, increased the expression of pro-apoptosis proteins, and decreased the expression of anti-apoptotic proteins in leukemia cells in vitro. EVI-1 expression in leukemia cells (THP-1 and K562) transduced with EVI-1 siRNA was significantly reduced. Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of leukemia cell apoptosis. ATO may downregulate EVI-1 mRNA and oncoprotein levels and block the inhibitory effects of EVI-1 on the JNK pathway, which activates the JNK apoptotic pathway, thereby leading to the apoptosis of EVI-1 in AML patients.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Adoptive transfer of PR1 cytotoxic T lymphocytes associated with reduced leukemia burden in a mouse acute myeloid leukemia xenograft model

Background aimsTumor antigen-specific cytotoxic T lymphocytes (CTL) have been used in the treatment of human cancer, including leukemia. Several studies have established PR1 peptide, an HLA-A2.1-restricted peptide derived from proteinase 3 (P3), as a human leukemia-associated antigen. PR1-specific CTL elicited in vitro from healthy donors have been shown to lyse P3-expressing AML cells from patients. We investigated whether PR1-CTL can be adoptively transferred into NOD/SCID mice to eliminate human leukemia cells.MethodsPR1-CTL were generated in bulk culture from peripheral blood mononuclear cells (PBMC) stimulated with autologous dendritic cells. Human acute myeloid leukemia (AML) patient samples were injected and engrafted in murine bone marrow at 2 weeks post-transfer.ResultsFollowing adoptive transfer, bone marrow aspirate from mice that received AML alone had 72–88% blasts in a hypercellular marrow, whereas mice that received AML plus PR1-CTL co-infusion had normal hematopoietic elements and only 3–18% blasts in a hypocellular marrow. The PR1-CTL persisted in the bone marrow and liver and maintained a CD45RA^? CD28⁺ effector phenotype.ConclusionsWe found that adoptive transfer of PR1-CTL generated in vitro is associated with reduced AML cells in NOD/SCID mice. PR1-CTL can migrate to the sites of disease and maintain their capacity to kill the AML cells. The surface phenotype of PR1-CTL was consistent with their trafficking pattern in both vascular and end-organ tissues.     

Survey of activated FLT3 signaling in leukemia

Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia (normal and amplification of FLT3) cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr) that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.     

Membrane-permeant, bioactivatable analogues of cGMP as inducers of cell death in IPC-81 leukemia cells

We report an improved single-step synthesis to generate the membrane-permeant acetoxymethyl esters (AM-esters) of cGMP and three cGMP-analogues. These bioactivatable compounds were found to induce cell death in rat IPC-81 cells, a model system for acute myelocytic leukemia, in micromolar doses, while the corresponding non-modified cGMP-analogues were inactive.     

All-Trans Retinoic Acid Activity in Acute Myeloid Leukemia: Role of Cytochrome P450 Enzyme Expression by the Microenvironment

Differentiation therapy with all-trans retinoic acid (atRA) has markedly improved outcome in acute promyelocytic leukemia (APL) but has had little clinical impact in other AML sub-types. Cell intrinsic mechanisms of resistance have been previously reported, yet the majority of AML blasts are sensitive to atRA in vitro. Even in APL, single agent atRA induces remission without cure. The microenvironment expression of cytochrome P450 (CYP)26, a retinoid-metabolizing enzyme was shown to determine normal hematopoietic stem cell fate. Accordingly, we hypothesized that the bone marrow (BM) microenvironment is responsible for difference between in vitro sensitivity and in vivo resistance of AML to atRA-induced differentiation. We observed that the pro-differentiation effects of atRA on APL and non-APL AML cells as well as on leukemia stem cells from clinical specimens were blocked by BM stroma. In addition, BM stroma produced a precipitous drop in atRA levels. Inhibition of CYP26 rescued atRA levels and AML cell sensitivity in the presence of stroma. Our data suggest that stromal CYP26 activity creates retinoid low sanctuaries in the BM that protect AML cells from systemic atRA therapy. Inhibition of CYP26 provides new opportunities to expand the clinical activity of atRA in both APL and non-APL AML.     

The peculiarities of cytogenetic changes in different types of myelodysplastic syndrome

A cytogenetic study of bone marrow aspirate from 32 patients with different types of myelodysplastic syndrome (MDS) has been carried out. The patients were from eight regions of Ukraine. Chromosome deletions prevailed in the spectrum of karyotype changes. The largest number of chromosome abnormalities was revealed in patients with a refractory anemia with an excess of blasts (66.6% of cases). Chromosomal changes that involved three or more chromosomes occurred among 27% of all karyotype changes examined by us. Transformation of myelodysplastic syndrome to acute myeloid leukemia (AML) was found in 5 patients (45.4% of the cases) among 11 patients with abnormal karyotypes. We propose that cytogenetic confirmation of increased apoptosis in the bone marrow from the myelodysplastic syndrome patients is a phenomenon of chromosome fragmentation. The risk of transformation of myelodysplastic syndrome to acute myeloid leukemia was measured with the use of a new international score system, IPSS.     

Human Vγ9Vδ2 T cells specifically recognize and kill acute myeloid leukemic blasts

Vγ9Vδ2 T cells are attractive candidates for antileukemic activity. The analysis of Vγ9Vδ2 T cells in newly diagnosed acute myeloid leukemia (AML) patients revealed that their absolute cell numbers were normal in the blood as well as in the bone marrow but showed a striking imbalance in the differentiation subsets, with preponderance of the effector memory population. This unusual phenotype was restored after removal of leukemic cells in patients, which reached complete remission after chemotherapy, suggesting that leukemic cells might be involved in the alteration of γδ T cell development in AML. Accordingly, coculture between AML cells and Vγ9Vδ2 T cells induced selection of effector cells. In accordance with their effector memory status, in vitro proliferation of Vγ9Vδ2 T cells was reduced compared with normal controls. Nevertheless, Vγ9Vδ2 T cells efficiently killed autologous AML blasts via the perforin/granzyme pathway. The ligands for DNAM-1 were expressed by AML cells. We showed that killing of AML blasts was TCR and DNAM-1 dependent. Using a xenotransplantation murine model, we showed that Vγ9Vδ2 T cells homed to the bone marrow in close proximity of engrafted leukemic cells and enhanced survival. These data demonstrate that Vγ9Vδ2 T cells are endowed with the ability to interact with and eradicate AML blasts both in vitro and in a mouse model. Collectively, our data revealed that Vγ9Vδ2 T cells have a potent antileukemic activity provided that optimal activation is achieved, such as with synthetic TCR agonists. This study enhances the interest of these cells for therapeutic purposes such as AML treatment.