Synergy between imatinib and mycophenolic acid in inducing apoptosis in cell lines expressing Bcr-Abl

Bcr-Abl tyrosine kinase activity initiates a number of intracellular signaling cascades that result in leukemogenesis. Imatinib mesylate, a specific Bcr-Abl tyrosine kinase inhibitor, has been highly successful in the treatment of chronic myelogenous leukemia (CML). However, the emergence of imatinib resistance and the incomplete molecular response of a significant number of patients receiving this therapy have led to a search for combinations of drugs that will enhance the efficacy of imatinib. We have demonstrated that mycophenolic acid (MPA), a specific inosine monophosphate dehydrogenase (IMPDH) inhibitor that results in depletion of intracellular guanine nucleotides, is synergistic with imatinib in inducing apoptosis in Bcr-Abl-expressing cell lines. Studies of signaling pathways downstream of Bcr-Abl demonstrated that the addition of MPA to imatinib reduced the phosphorylation of both Stat5 and Lyn, a Src kinase family member. The phosphorylation of S6 ribosomal protein was also greatly reduced. These results demonstrate that inhibitors of guanine nucleotide biosynthesis may synergize with imatinib in reducing the levels of minimal residual disease in CML and lay the foundation for clinical trials in which IMPDH inhibitors are added to imatinib in patients who have suboptimal molecular responses to single agent therapy or who have progressive disease.     

Cell death provoked by loss of interleukin-3 signaling is independent of Bad, Bim, and PI3 kinase, but depends in part on Puma

Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.     

Inosine-5'-monophosphate dehydrogenase is required for mitogenic competence of transformed pancreatic beta cells

The relation of inosine-5'-monophosphate dehydrogenase (IMPDH; the rate-limiting enzyme in GTP synthesis) to mitogenesis was studied by enzymatic assay, immunoblots, and RT-PCR in several dissimilar transformed pancreatic ss-cell lines, using intact cells. Both of the two isoforms of IMPDH (constitutive type 1 and inducible type 2) were identified using RT-PCR in transformed beta cells or in intact islets. IMPDH 2 messenger RNA (mRNA) and IMPDH protein were both regulated reciprocally by changes in levels of their end-products. Flux through IMPDH was greatest in rapidly growing cells, due mostly to increased uptake of precursor. Glucose (but not 3-0-methylglucose, L-glucose, or fructose) further augmented substrate uptake and also increased IMPDH enzymatic activity after either 4 or 21 h of stimulation. Serum or ketoisocaproate also increased IMPDH activity (but not uptake). Two selective IMPDH inhibitors (mycophenolic acid and mizoribine) reduced IMPDH activity in all cell lines, and, with virtually identical concentration-response curves, inhibited DNA synthesis (assessed as bromodeoxyuridine incorporation) in response to glucose, serum, or ketoisocaproate. Inhibition of DNA synthesis was reversible, completely prevented by repletion of cellular guanine (but not adenine) nucleotides, and could not be attributed to toxic effects. Despite the fact that modulation of IMPDH expression by guanine nucleotides was readily detectable, glucose and/or serum failed to alter IMPDH mRNA or protein, indicating that their effects on IMPDH activity were largely at the enzyme level. Precursors of guanine nucleotides failed, by themselves, to induce mitogenesis. Thus, adequate IMPDH activity (and thereby, availability of GTP) is a critical requirement for beta-cell proliferation. Although it is unlikely that further increases in GTP can, by themselves, initiate DNA synthesis, such increments may be needed to sustain mitogenesis.     

Interleukin 3-dependent survival by the Akt protein kinase

Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.     

TEL/platelet-derived growth factor receptor beta activates phosphatidylinositol 3 (PI3) kinase and requires PI3 kinase to regulate the cell cycle

TEL/platelet-derived growth factor receptor beta (PDGF beta R) is the protein product of the t(5;12) translocation in chronic myelomonocytic leukemia. TEL/PDGF beta R transforms interleukin-3 (IL-3)-dependent Ba/F3 and 32D cells to IL-3 independence and induces a murine myeloproliferative disease in a bone marrow transplantation model of leukemogenesis. The fusion protein encodes a constitutively activated, cytoplasmic tyrosine kinase that activates multiple signal transduction pathways. To identify the signaling pathways that are necessary for transformation by TEL/PDGF beta R, transformed Ba/F3 and 32D cells were studied. TEL/PDGF beta R activates the kinase activity of phosphatidylinositol-3 (PI3) kinase and stimulates phosphorylation of its downstream substrates, including Akt and p70S6 kinase. Activation of this pathway requires the kinase activity of TEL/PDGF beta R and is inhibited by the PDGF beta R inhibitor, STI571. Furthermore, inhibition of PI3 kinase with the pharmacologic inhibitor, LY294002, inhibits growth of the transformed cells. Treated cells arrest in the G1 phase of the cell cycle within 16 hours but do not undergo apoptosis. To study the mechanism of cell cycle arrest by LY294002, the activity of the cdk4 complex, which regulates the transit of cells from the G1 to S phase in hematopoietic cells, was examined. Both STI571 and LY294002 lead to a decrease in the activity of cdk4 kinase activity and a decrease in expression of both Cyclin D2 and Cyclin E within several hours. These studies demonstrate the presence of a signaling pathway from TEL/PDGF beta R to PI3 kinase and subsequently to regulation of the cdk4 kinase complex. Activation of this pathway is necessary for transformation by TEL/PDGF beta R.     

Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. TPA (12-O-tetradecanoylphorbol 13- acetate) and interleukin-4 (IL-4) inhibit apoptosis of B-CLL lymphocytes ex vivo. We used specific inhibitors of protein kinase C (PKC), extracellular-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3-kinase) to study their involvement in TPA- and IL-4-induced survival of B-CLL lymphocytes. BisI, a specific inhibitor of PKC, induced apoptosis and inhibited the antiapoptotic activity of TPA and IL-4. B-CLL cells have a basal PKC activity that was increased by TPA but not by IL-4. TPA, but not IL-4, induced ERK activation. However, the inhibition of ERK activation did not affect the viability of B-CLL lymphocytes, demonstrating that this pathway is not involved in their survival. Inhibition of PI3-kinase by LY294002 induced apoptosis of B-CLL cells and inhibited the survival effect of IL-4 and TPA. In addition, Akt, a downstream effector of PI3-kinase activity, was phosphorylated by TPA and IL-4 in B-CLL cells, though PI3-kinase had no effect on PKC-dependent phosphorylation of Akt. Furthermore, the inhibition of PKC or PI3-kinase increased dexamethasone- and fludarabine-induced apoptosis ex vivo in the presence of survival factors. These results demonstrate that PKC and PI3-kinase are involved in the survival of B-CLL cells and suggest that inhibitors of these pathways could be combined with the drugs used in the treatment of B-CLL.     

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: Involvement of MEK upstream of Bad phosphorylation

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras–mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.     

Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells: possible involvement of STAT3 degradation by proteasome pathway in interleukin 6-dependent hematopoietic cells

The ubiquitin-proteasome pathway is responsible for selective degradation of short-lived cellular proteins and is critical for the regulation of many cellular processes. We previously showed that ubiquitin (Ub) secreted from hairy cell leukemia cells had inhibitory effects on clonogenic growth of normal hematopoietic progenitor cells. In this study, we examined the effects of exogenous Ub on the growth and survival of a series of human hematopoietic cells, including myeloid cell lines (HL-60 and U937), a B-cell line (Daudi), and T-cell lines (KT-3, MT-4, YTC-3, and MOLT-4). Exogenous Ub inhibited the growth of various hematopoietic cell lines tested, especially of KT-3 and HL-60 cells. The growth-suppressive effects of Ub on KT-3 and HL-60 cells were almost completely abrogated by the proteasome inhibitor PSI or MG132, suggesting the involvement of the proteasome pathway in this process. Furthermore, exogenous Ub evoked severe apoptosis of KT-3 and HL-60 cells through the activation of caspase-3. In interleukin-6 (IL-6)-dependent KT-3 cells, STAT3 was found to be conjugated by exogenous biotinylated Ub and to be degraded in a proteasome-dependent manner, whereas expression levels of STAT1, STAT5, or mitogen-activated protein kinase were not affected. Moreover, IL-6-induced the up-regulation of Bcl-2 and c-myc, and JunB was impaired in Ub-treated KT-3 cells, suggesting that the anti-apoptotic and mitogenic effects of IL-6 were disrupted by Ub. These results suggest that extracellular Ub was incorporated into hematopoietic cells and mediated their growth suppression and apoptosis through proteasome-dependent degradation of selective cellular proteins such as STAT3. (Blood. 2000;95:2577-2585)     

Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR

Recent evidence places the FRAP/mTOR kinase downstream of the phosphatidyl inositol 3-kinase/Akt-signaling pathway, which is up-regulated in multiple cancers because of loss of the PTEN tumor suppressor gene. We performed biological and biochemical studies to determine whether PTEN-deficient cancer cells are sensitive to pharmacologic inhibition of FRAP/mTOR by using the rapamycin derivative CCI-779. In vitro and in vivo studies of isogenic PTEN(+/+) and PTEN(-/-) mouse cells as well as human cancer cells with defined PTEN status showed that the growth of PTEN null cells was blocked preferentially by pharmacologic FRAP/mTOR inhibition. Enhanced tumor growth caused by constitutive activation of Akt in PTEN(+/+) cells also was reversed by CCI-779 treatment, indicating that FRAP/mTOR functions downstream of Akt in tumorigenesis. Loss of PTEN correlated with increased S6 kinase activity and phosphorylation of ribosomal S6 protein, providing evidence for activation of the FRAP/mTOR pathway in these cells. Differential sensitivity to CCI-779 was not explained by differences in biochemical blockade of the FRAP/mTOR pathway, because S6 phosphorylation was inhibited in sensitive and resistant cell lines. These results provide rationale for testing FRAP/mTOR inhibitors in PTEN null human cancers.     

Resveratrol induces apoptosis in thyroid cancer cell lines via a MAPK- and p53-dependent mechanism

Two papillary thyroid carcinoma (PTC) and two follicular thyroid carcinoma (FTC) cell lines treated with resveratrol (RV), 1-10 microM, showed activation and nuclear translocation of MAPK (extracellular signal-regulated kinase 1/2). Cellular abundance of the oncogene suppressor protein p53, serine phosphorylation of p53, and abundance of c-fos, c-jun, and p21 mRNAs were also increased by RV. Inhibition of the MAPK pathway by either H-ras antisense transfection or PD 98059, an MAPK kinase inhibitor, blocked these RV-induced effects. Addition of pifithrin-alpha, a specific inhibitor of p53, or transfection of p53 antisense oligonucleotides caused decreased RV-induced p53 and p21 expression in PTC and FTC cells. Studies of nucleosome levels estimated by ELISA and of DNA fragmentation showed that RV induced apoptosis in both papillary and follicular thyroid cancer cell lines; these effects were inhibited by pifithrin-alpha and by p53 antisense oligonucleotide transfection. PD 98059 and H-ras antisense transfection also blocked induction of apoptosis by RV. Thus, RV acts via a Ras-MAPK kinase-MAPK signal transduction pathway to increase p53 expression, serine phosphorylation of p53, and p53-dependent apoptosis in PTC and FTC cell lines.     

Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1beta-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1beta-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca(2+) channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse betaTC3 cells. Ca(2+) influx, specifically via L-type but not T-type channels, contributed to IL-1beta activation of JNK. Activation of p38 and ERK in response to IL-1beta was also dependent on L-type Ca(2+) influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca(2+) ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin-secreting cells.     

TEL/PDGFbetaR fusion protein activates STAT1 and STAT5: a common mechanism for transformation by tyrosine kinase fusion proteins

OBJECTIVE: TEL/PDGFbetaR is a tyrosine kinase fusion protein associated with the pathogenesis of chronic myelomonocytic leukemia. The following experiments were undertaken to understand the mechanisms whereby TEL/PDGFbetaR transforms cells. MATERIALS AND METHODS: Activation of JAK and STAT proteins was studied in an interleukin 3 (IL-3)-dependent cell line, Ba/F3, transformed to IL-3 independence by TEL/PDGFbetaR. RESULTS: TEL/PDGFbetaR activates STAT1 and STAT5 in transformed Ba/F3 cells through a JAK-independent pathway. Activation of STAT proteins requires the kinase activity of TEL/PDGFbetaR. JAK1, JAK2, JAK3, and TYK2 are not phosphorylated by TEL/PDGFbetaR. However, TEL/PDGFbetaR can phosphorylate STAT5 in transiently transfected COS cells, suggesting that TEL/PDGFbetaR may itself be the kinase involved in tyrosine phosphorylation of STAT proteins. In contrast, native PDGFbetaR stimulated by PDGF ligand does not activate STAT proteins to a significant degree in this hematopoietic context. STAT1 and STAT5 also are activated by TEL/ABL and TEL/JAK2 fusion proteins associated with human leukemia. CONCLUSIONS: STAT activation may be a common mechanism of transformation by leukemogenic tyrosine kinase fusion proteins.     

Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases

Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 microM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.     

Antileukaemic effect of PI3K‐mTOR inhibitors in acute myeloid leukaemia‐gene expression profiles reveal CDC25B expression as determinate of pharmacological effect

Acute myeloid leukaemia (AML) is a heterogeneous malignancy. Intracellular signalling through the phosphatidylinositol 3‐kinase (PI3K)‐Akt‐mammalian target of rapamycin (mTOR) pathway is important for regulation of cellular growth and metabolism, and inhibitors of this pathway is considered for AML treatment. Primary human AML cells, derived from 96 consecutive adult patients, were examined. The effects of two mTOR inhibitors (rapamycin, temsirolimus) and two PI3K inhibitors (GDC‐0941, 3‐methyladenine) were studied, and we investigated cytokine‐dependent proliferation, regulation of apoptosis and global gene expression profiles. Only a subset of patients demonstrated strong antiproliferative effects of PI3K‐mTOR inhibitors. Unsupervised hierarchical clustering analysis identified two main clusters of patients; one subset showing weak or absent antiproliferative effects (59%) and another group showing a strong growth inhibition for all drugs and concentrations examined (41%). Global gene expression analyses showed that patients with AML cell resistance against PI3K‐mTOR inhibitors showed increased mRNA expression of the CDC25B gene that encodes the cell cycle regulator Cell Division Cycle 25B. The antileukaemic effect of PI3K‐Akt‐mTOR inhibition varies between patients, and resistance to these inhibitors is associated with the expression of the cell cycle regulator CDC25B, which is known to crosstalk with the PI3K‐Akt‐mTOR pathway and mediate rapamycin resistance in experimental models.