Erlotinib antagonizes ABC transporters in acute myeloid leukemia

Erlotinib was originally developed as an epidermal growth factor receptor (EGFR)-specific inhibitor for the treatment of solid malignancies, yet also exerts significant EGFR-independent antileukemic effects in vitro and in vivo. The molecular mechanisms underlying the clinical antileukemic activity of erlotinib as a standalone agent have not yet been precisely elucidated. Conversely, in preclinical settings, erlotinib has been shown to inhibit the constitutive activation of SRC kinases and mTOR, as well as to synergize with the DNA methyltransferase inhibitor azacytidine (a reference therapeutic for a subset of leukemia patients) by promoting its intracellular accumulation. Here, we show that both erlotinib and gefitinib (another EGFR inhibitor) inhibit transmembrane transporters of the ATP-binding cassette (ABC) family, including P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP), also in acute myeloid leukemia (AML) cells that do not overexpress these pumps. Thus, inhibition of drug efflux by erlotinib and gefitinib selectively exacerbated (in a synergistic or additive fashion) the cytotoxic response of KG-1 cells to chemotherapeutic agents that are normally extruded by ABC transporters (e.g., doxorubicin and etoposide). Erlotinib limited drug export via ABC transporters by multiple mechanisms, including the downregulation of surface-exposed pumps and the modulation of their ATPase activity. The effects of erlotinib on drug efflux and its chemosensitization profile persisted in patient-derived CD34⁺ cells, suggesting that erlotinib might be particularly efficient in antagonizing leukemic (stem cell) subpopulations, irrespective of whether they exhibit or not increased drug efflux via ABC transporters.     

Erlotinib antagonizes ABC transporters in acute myeloid leukemia

Erlotinib was originally developed as an epidermal growth factor receptor (EGFR)-specific inhibitor for the treatment of solid malignancies, yet also exerts significant EGFR-independent antileukemic effects in vitro and in vivo. The molecular mechanisms underlying the clinical antileukemic activity of erlotinib as a standalone agent have not yet been precisely elucidated. Conversely, in preclinical settings, erlotinib has been shown to inhibit the constitutive activation of SRC kinases and mTOR, as well as to synergize with the DNA methyltransferase inhibitor azacytidine (a reference therapeutic for a subset of leukemia patients) by promoting its intracellular accumulation. Here, we show that both erlotinib and gefitinib (another EGFR inhibitor) inhibit transmembrane transporters of the ATP-binding cassette (ABC) family, including P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP), also in acute myeloid leukemia (AML) cells that do not overexpress these pumps. Thus, inhibition of drug efflux by erlotinib and gefitinib selectively exacerbated (in a synergistic or additive fashion) the cytotoxic response of KG-1 cells to chemotherapeutic agents that are normally extruded by ABC transporters (e.g., doxorubicin and etoposide). Erlotinib limited drug export via ABC transporters by multiple mechanisms, including the downregulation of surface-exposed pumps and the modulation of their ATPase activity. The effects of erlotinib on drug efflux and its chemosensitization profile persisted in patient-derived CD34⁺ cells, suggesting that erlotinib might be particularly efficient in antagonizing leukemic (stem cell) subpopulations, irrespective of whether they exhibit or not increased drug efflux via ABC transporters.     

Reelin activates SRC family tyrosine kinases in neurons

BACKGROUND: Reelin is a large signaling molecule that regulates the positioning of neurons in the mammalian brain. Transmission of the Reelin signal to migrating embryonic neurons requires binding to the very-low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor-2 (apoER2). This induces tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1), which interacts with a shared sequence motif in the cytoplasmic tails of both receptors. However, the kinases that mediate Dab1 tyrosine phosphorylation and the intracellular pathways that are triggered by this event remain unknown. RESULTS: We show that Reelin activates members of the Src family of non-receptor tyrosine kinases (SFKs) and that this activation is dependent on the Reelin receptors apoER2 and VLDLR and the adaptor protein Dab1. Dab1 is tyrosine phosphorylated by SFKs, and the kinases themselves can be further activated by phosphorylated Dab1. Increased Dab1 protein expression in fyn-deficient mice implies a response to impaired Reelin signaling that is also observed in mice lacking Reelin or its receptors. However, fyn deficiency alone does not compound the neuronal positioning defect of vldlr- or apoer2-deficient mice, and this finding suggests functional compensation by other SFKs. CONCLUSIONS: Our results show that Dab1 is a physiological substrate as well as an activator of SFKs in neurons. Based on genetic evidence gained from multiple strains of mutant mice with defects in Reelin signaling, we conclude that activation of SFKs is a normal part of the cellular Reelin response.     

Arsenic trioxide represses constitutive activation of NF-kappaB and COX-2 expression in human acute myeloid leukemia, HL-60

It has been proposed that eukaryotic nuclear factor nuclear factor kappa-B (NF-kappaB) and cyclooxygenase-2 (COX-2) are implicated in the pathogenesis of many human diseases including cancer. Arsenic has been widely used in medicine in Oriental countries. Recent studies have shown that arsenic trioxide (As(2)O(3)) could induce in vitro growth inhibition and apoptosis of malignant lymphocytes, and myeloma cells. However, the molecular mechanisms by which As(2)O(3) initiates cellular signaling toward cell death are still unclear. In the present study, the effects of As(2)O(3) on NF-kappaB and COX-2 expression in HL-60 cells were investigated. As(2)O(3) suppressed DNA-binding activity of NF-kappaB composed of p65/p50 heterodimer through preventing the degradation of IkappaB-alpha and the nuclear translocation of p65 subsequently as well as interrupting the binding of NF-kappaB with their consensus sequences. Inhibitory effect of As(2)O(3) on NF-kappaB DNA activity was dependent upon intracellular glutathione (GSH) and H(2)O(2) level, but not superoxide anion. Futhermore, we found that As(2)O(3) also downregulated the expression of COX-2, which has NF-kappaB binding site on its promoter through repressing the NF-kappaB DNA-binding activity.     

SRC family kinases and receptors: analysis of three activation mechanisms by dynamic systems modeling

Src family kinases (SFKs) interact with a number of cellular receptors. They participate in diverse signaling pathways and cellular functions. Most of the receptors involved in SFK signaling are characterized by similar modes of regulation. This computational study discusses a general kinetic model of SFK-receptor interaction. The analysis of the model reveals three major ways of SFK activation: release of inhibition by C-terminal Src kinase, weakening of the inhibitory intramolecular phosphotyrosine-SH2 interaction, and amplification of a stimulating kinase activity. The SFK model was then extended to simulate interaction with growth factor and T-cell receptors. The modular SFK signaling system was shown to adapt to the requirements of specific signaling contexts and yield qualitatively different responses in the different simulated environments. The model also provides a systematic overview of the major interactions between SFKs and various cellular signaling systems and identifies their common properties.     

Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.     

Inactivation of SRC family tyrosine kinases by reactive oxygen species in vivo

Reactive oxygen species, including H2O2, O2*- and OH* are constantly produced in the human body and are involved in the development of cardiovascular diseases. Emerging evidence suggests that reactive oxygen species, besides their deleterious effects at high concentrations, may be protective. However, the mechanism underlying the protective effects of reactive oxygen species is not clear. Here, we reported a novel finding that H2O2 at low to moderate concentrations (50-250 microM) markedly inactivated Src family tyrosine kinases temporally and spatially in vivo but not in vitro. We further showed that Src family kinases localized to focal adhesions and the plasma membrane were rapidly and permanently inactivated by H2O2, which resulted from a profound reduction in phosphorylation of the conserved tyrosine residue at the activation loop. Interestingly, the cytoplasmic Src family kinases were activated gradually by H2O2, which partially compensated for the loss of total activities of Src family kinases but not their functions. Finally, H2O2 rendered endothelial cells resistant to growth factors and cytokines and protected the cells from inflammatory activation. Because Src family kinases play key roles in cell signaling, the rapid inactivation of Src family kinases by H2O2 may represent a novel mechanism for the protective effects of reactive oxygen species.     

State and role of SRC family kinases in replication of herpes simplex virus 1

An earlier report showed that infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) interacts with the SH3 domains of a recently discovered adaptor protein, CIN85. Here, we report the following. (i) ICP0 also interacts with other SH3 domain-containing proteins and, in particular, with nonneuronal members of the Src kinase family. (ii) HSV-1 infection enhanced the activating phosphorylation of Tyr416 of the members of the Src kinase family, modestly enhanced the kinase activity of Src, and posttranslationally modified at least one additional member of the Src kinase family by phosphorylation in a manner dependent on the viral gene products ICP0, unique short 3 (U(S)3), and unique long 13 (U(L)13). (iii) To define the roles of Src kinase family members, we examined the accumulation of viral proteins, DNA, and mRNA and virus yields from wild-type mouse embryo fibroblasts and sibling cells lacking Src, Fyn, and Yes (SYF-); a mutant cell line, +Src, in which Src was restored to SYF- cells; and the mutant cell line (CSK-) lacking the negative regulator Csk gene of the Src kinase family. Representative alpha, beta, and gamma2 proteins accumulated in the largest amounts in SYF- cells and the smallest amounts in +Src compared to wild-type cells. The CSK- cells yielded smaller amounts of the gamma2 protein and at least 10-fold less virus than wild-type cells. We conclude that HSV-1 proteins regulate the activities of Src family kinases to achieve optimal viral yields in the course of viral replication.     

Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases

Src family kinases (SFKs) are abundant in chromaffin cells that reside in the adrenal medulla and respond to cholinergic stimulation by secreting catecholamines. Our previous work indicated that SFKs regulate acetylcholine- or nicotine-induced secretion, but the site of modulatory action was unclear. Using whole cell recordings, we found that inhibition of SFK tyrosine kinase activity by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine) treatment or expression of a kinase-defective c-Src reduced the peak amplitude of nicotine-induced currents in chromaffin cells or in human embryonic kidney cells ectopically expressing functional neuronal alpha3beta4alpha5 acetylcholine receptors (AChRs). Conversely, the phosphotyrosine phosphatase inhibitor, sodium vanadate, or expression of mutationally activated c-Src resulted in enhanced current amplitudes. These results suggest that SFKs and putative phosphotyrosine phosphatases regulate the activity of AChRs by opposing actions. This proposed model was supported further by the findings that SFKs physically associate with the receptor and that the AChR is tyrosine-phosphorylated.     

Annular promyelocytes in acute myeloid leukemia

Granulocytes with ring-shaped nuclei (annular granulocytes; ring granulocytes) are normal bone marrow constituents in rodents. Studies in man have shown a small number of these cells in cases of myeloproliferative diseases. Myelocytes and metamyelocytes have also been described. Similar to rodents and some other animal species, the annular promyelocyte also exists in humans. The significance of these very rare cells in human haemopoiesis becomes an interesting question.     

Interleukin-17 in acute myeloid leukemia

There are several reports that angiogenesis plays important roles in hematological malignancies including acute myeloid leukemia (AML). Human interleukin-17 (IL-17) is a proinflammatory cytokine produced by activated CD4 T cells. IL-17 plays a potential role in T cell mediated angiogenesis. The role of IL-17 in pathologic angiogenesis has not been evaluated yet. The aim of the study was to determine plasma level of IL-17 in patients with AML. IL-17 levels were measured by ELISA in plasma samples taken from 68 adult patients with AML before chemotherapy was administered. In addition 20 out of 68 patients were reanalysed after achieving complete remission (CR). Ten samples from healthy volunteers were evaluated as the control. In this study we have demonstrated that serum level of IL-17 is not elevated in AML patients. These results suggest that angiogenesis in AML is not mediated by CD4 T cells. To our knowledge this is the first report about IL-17 serum level in acute leukemias. We are currently evaluating IL-17 levels in others haematological malignancies.     

The role of Src family kinases in egg activation

Solitary amoebae of Dictyostelium discoideum are frequently exposed to stressful conditions in nature, and their multicellular development is one response to environmental stress. Here we analyzed an aggregation stage abundant gene, krsA, homologous to human krs1 (kinase responsive to stress 1) to understand the mechanisms for the initiation of development and cell fate determination. The krsA- cells exhibited reduced viability under hyperosmotic conditions. They produced smaller aggregates on membrane filters and did not form aggregation streams on a plastic surface under submerged starvation conditions, but were normal in sexual development. During early asexual development, the expression of cAMP-related genes peaked earlier in the knockout mutants. Neither cAMP oscillation in starved cells nor an increase in the cAMP level following osmotic stress was observed in krsA-. The nuclear export signal, as well as the kinase domain, in KrsA was necessary for stream formation. These results strongly suggest that krsA is involved in cAMP relay, and that signaling pathways for multicellular development have evolved in unison with the stress response.     

The role,mechanism and potentially therapeutic application of microRNA-29 family in acute myeloid leukemia

Abnormal proliferation, apoptosis repression and differentiation blockage of hematopoietic stem/progenitor cells have been characterized to be the main reasons leading to acute myeloid leukemia (AML). Previous studies showed that miR-29a and miR-29b could function as tumor suppressors in leukemogenesis. However, a comprehensive investigation of the function and mechanism of miR-29 family in AML development and their potentiality in AML therapy still need to be elucidated. Herein, we reported that the family members, miR-29a, -29b and -29c, were commonly downregulated in peripheral blood mononuclear cells and bone marrow (BM) CD34+ cells derived from AML patients as compared with the healthy donors. Overexpression of each miR-29 member in THP1 and NB4 cells markedly inhibited cell proliferation and promoted cell apoptosis. AKT2 and CCND2 mRNAs were demonstrated to be targets of the miR-29 members, and the role of miR-29 family was attributed to the decrease of Akt2 and CCND2, two key signaling molecules. Significantly increased Akt2, CCND2 and c-Myc levels in the AML cases were detected, which were correlated with the decreased miR-29 expression in AML blasts. Furthermore, a feed-back loop comprising of c-Myc, miR-29 family and Akt2 were found in myeloid leukemogenesis. Reintroduction of each miR-29 member partially corrected abnormal cell proliferation and apoptosis repression and myeloid differentiation arrest in AML BM blasts. An intravenous injection of miR-29a, -29b and -29c in the AML model mice relieved leukemic symptoms significantly. Taken together, our finding revealed a pivotal role of miR-29 family in AML development and rescue of miR-29 family expression in AML patients could provide a new therapeutic strategy.     

Antiapoptotic microenvironment of acute myeloid leukemia

We showed previously that tumor-derived supernatant (TSN) from acute myeloid leukemia (AML) myeloblasts inhibits peripheral blood T cell activation and proliferation, rendering the T cells functionally incompetent. We show here that the AML TSN also significantly delays apoptosis of both resting and stimulated T cells, as judged by reduction in annexin V/propidium iodide staining. In addition, we show that this is not unique to T cells and that AML TSN inhibits apoptosis of peripheral B cells, neutrophils, and monocytes. Furthermore, it also enhances the survival of other AML myeloblasts with lower viability. Investigations into the mechanism demonstrate a reduction in the cleavage of procaspase-3, -8, and -9 and the caspase substrate, poly(ADP-ribose)polymerase (PARP). This may be due to Bcl-2, which is normally down-regulated in CD3/CD28-stimulated T cells, but is maintained in the presence of AML TSN. We conclude that AML cells generate an antiapoptotic microenvironment that favors the survival of malignant cells, but also inhibits apoptosis of other normal hemopoietic cells. Reversal of these immunosuppressive effects and restoration of normal immune responses in patients with AML would improve the success of immunotherapy protocols.     

Genetic profile of acute myeloid leukemia

Understanding genomic events and the cascade of their effects in cell function is crucial for identifying distinct subsets of acute myeloid leukemia and developing new therapeutic strategies. Conventional cytogenetics, fluorescence in situ hybridization investigations and molecular studies have provided much information over the past few years. This review will focus on major genomic mechanisms in acute myeloid luekemia and on the genes implicated in the pathogenesis of specific subtypes.