KIT oncoprotein interactions in gastrointestinal stromal tumors: therapeutic relevance.

Most gastrointestinal stromal tumors (GISTs) express oncogenic and constitutively active forms of the KIT or platelet-derived growth factor receptor alpha (PDGFRA) receptor tyrosine kinase proteins, and these kinase oncoproteins serve as targets for effective therapies. Given that mutant KIT oncoproteins serve crucial transforming roles in GISTs, we evaluated interactions with the KIT oncoproteins and determined signaling pathways that are dependent on KIT oncogenic activation in GISTs. Tyrosine-phosphorylated KIT oncoproteins interacted with PDGFRA, PDGFRB, phosphatidylinositol 3-kinase (PI3-K) and PKCtheta in GIST cells, and these interactions were abolished by KIT inhibition with imatinib or PKC412 or KIT RNAi. Notably, tyrosine-phosphorylated PDGFRA was prominent in frozen GIST tumors expressing KIT oncoproteins, suggesting that KIT-mediated PDGFRA phosphorylation is an efficient and biologically consequential mechanism in GISTs. Activated signaling intermediates were identified by immunoaffinity purification of tyrosine-phosphorylated proteins in GIST cells before and after treatment with KIT inhibitors, and these analyses show that GRB2, SHC, CBL and MAPK activation are largely KIT dependent in GISTs, whereas PI3-K, STAT1 and STAT3 activation are partially KIT dependent. In addition, we found that phosphorylation of several tyrosine kinase proteins - including JAK1 and EPHA4 - did not depend on KIT activation. Likewise, paxillin activation was independent of the KIT oncogenic signal. These studies identify signaling pathways that can provide both KIT-dependent and KIT-independent therapeutic synergies in GIST, and thereby highlight clinical strategies that might consolidate GIST therapeutic response to KIT/PDGFRA inhibition.

The multikinase inhibitor midostaurin (PKC412A) lacks activity in metastatic melanoma: a phase IIA clinical and biologic study.

Midostaurin (PKC412A), N-benzoyl-staurosporine, potently inhibits protein kinase C alpha (PKCalpha), VEGFR2, KIT, PDGFR and FLT3 tyrosine kinases. In mice, midostaurin slows growth and delays lung metastasis of melanoma cell lines. We aimed to test midostaurin's safety, efficacy and biologic activity in a Phase IIA clinical trial in patients with metastatic melanoma. Seventeen patients with advanced metastatic melanoma received midostaurin 75 mg p.o. t.i.d., unless toxicity or disease progression supervened. Patient safety was assessed weekly, and tumour response was assessed clinically or by CT. Tumour biopsies and plasma samples obtained at entry and after 4 weeks were analysed for midostaurin concentration, PKC activity and multidrug resistance. No tumour responses were seen. Two (12%) patients had stable disease for 50 and 85 days, with minor response in one. The median overall survival was 43 days. Seven (41%) discontinued treatment with potential toxicity, including nausea, vomiting, diarrhoea and/or fatigue. One patient had >50% reduction in PKC activity. Tumour biopsies showed two PKC isoforms relatively insensitive to midostaurin, out of three patients tested. No modulation of multidrug resistance was demonstrated. At this dose schedule, midostaurin did not show clinical or biologic activity against metastatic melanoma. This negative trial reinforces the importance of correlating biologic and clinical responses in early clinical trials of targeted therapies.

Specific phospholipids enhance the activity of beta-lactam antibiotics against Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1 became considerably more sensitive to the action of ampicillin when grown in the presence of certain phospholipids. Only phospholipids capable of forming lipid bilayers or micelles proved to be capable of enhancing ampicillin activity. Of the phospholipids tested, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate, also called monopalmitoylphosphatidic acid (MPPA), was the best enhancer. In the absence of MPPA, the MIC and MBC of ampicillin for P. aeruginosa PAO1 were 1 and 2 g/L, respectively. In the presence of MPPA, the MIC and MBC were 20 and 40 mg/L, respectively. MPPA was shown to enhance ampicillin activity by binding both Ca(2+) and Mg(2+), suggesting that the mechanism of enhancement is similar to that previously reported for Ca(2+) and Mg(2+) chelators. Surprisingly, MPPA by itself slowed the growth of four mucoid multiply antibiotic-resistant strains of P. aeruginosa recently isolated from the sputum of cystic fibrosis patients, and enhanced their sensitivity to piperacillin. It also increased the sensitivity of two ceftazidime-resistant P. aeruginosa cystic fibrosis strains to ceftazidime.

Targeted suppression of cytokine production in monocytes but not in T lymphocytes by a tetravalent guanylhydrazone (CNI-1493).

Image analysis was used to study the cytokine-inhibitory effect of the nitric oxide inhibitor tetravalent guanylhydrazone (CNI-1493) in individual immunocytochemically stained human peripheral blood mononuclear cells (PBMC). CNI-1493 inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-1beta, IL-6, and IL-8 production whether or not LPS stimulation was enhanced by interferon (IFN)-gamma priming. Addition of TNF-alpha to CNI-1493-exposed LPS-stimulated cells partially restored the incidence of IL-1alpha-, IL-1beta-, and IL-8-producing cells. TNF-alpha production induced by costimulation by ligation of CD3 and CD28 was inhibited by CNI-1493 in monocytes but not in T lymphocytes. The prevalence of IL-2-, IFN-gamma-, and TNF-beta-producing T cells was not reduced by CNI-1493. Phorbol ester and ionomycin activation also resulted in a CNI-1493 -induced inhibition of TNF-alpha in monocytes but resistant production of TNF-alpha, IL-2, and IFN-gamma by T cells. Thus, CNI-1493 preferentially inhibited synthesis of proinflammatory cytokines in monocytes.

Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response.

The local production of proinflammatory cytokines mediates the host response to inflammation, infection, and injury, whereas an overexpression of these mediators can injure or kill the host. Recently, we identified a class of multivalent guanylhydrazone compounds that are effective inhibitors of proinflammatory cytokine synthesis in monocytes/macrophages. The structure of one such cationic molecule suggested a molecular mimicry with spermine, a ubiquitous endogenous biogenic amine that increases significantly at sites of inflammation and infection. Here, we addressed the hypothesis that spermine might counterregulate the innate immune response by downregulating the synthesis of potentially injurious cytokines. When spermine was added to cultures of human peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), it effectively inhibited the synthesis of the proinflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, MIP-1alpha, and MIP-1beta. The inhibition of cytokine synthesis was specific and reversible, with significant inhibition of TNF synthesis occurring even when spermine was added after LPS. The mechanism of spermine-mediated cytokine suppression was posttranscriptional and independent of polyamine oxidase activity. Local administration of spermine in vivo protected mice against the development of acute footpad inflammation induced by carrageenan. These results identify a distinct molecular counterregulatory role for spermine in downregulating the monocyte proinflammatory cytokine response.

CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.

Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation.

Suppression of proinflammatory cytokines in monocytes by a tetravalent guanylhydrazone.

An overproduction of proinflammatory cytokines by activated macrophages/monocytes mediates the injurious sequelae of inflammation, septic shock, tissue injury, and cachexia. We recently synthesized a tetravalent guanylhydrazone compound (CNI-1493) that inhibits cytokine-inducible arginine transport and nitric oxide (NO) production in macrophages, and protects mice against lethal endotoxemia and carrageenan-induced inflammation. During these investigations we noticed that CNI-1493 effectively prevented lipopolysaccharide (LPS)-induced NO production, even when added in concentrations 10-fold less than required to competitively inhibit L-arginine uptake, suggesting that the suppressive effects of this guanylhydrazone compound might extend to other LPS-induced responses. Here, we report that CNI-1493 suppressed the LPS-stimulated production of proinflammatory cytokines (tumor necrosis factor [TNF], interleukins 1beta and 6, macrophage inflammatory proteins 1alpha and 1beta) from human peripheral blood mononuclear cells. Cytokine suppression was specific, in that CNI-1493 did not inhibit either the constitutive synthesis of transforming growth factor beta or the upregulation of major histocompatibility complex class II by interferon gamma (IFN-gamma). In contrast to the macrophage suppressive actions of dexamethasone, which are overridden in the presence of IFN-gamma, CNI-1493 retained its suppressive effects even in the presence of IFN-gamma. The mechanism of cytokine-suppressive action by CNI-1493 was independent of extracellular L-arginine content and NO production and is not restricted to induction by LPS. As a selective inhibitor of macrophage activation that prevents TNF production, this tetravalent guanylhydrazone could be useful in the development of cytokine-suppressive agents for the treatment of diseases mediated by overproduction of cytokines.

Induction of transforming growth factor beta 1 and its receptors during all-trans-retinoic acid (RA) treatment of RA-responsive human neuroblastoma cell lines.

Recent work on a variety of normal and malignant cell lines has shown that induction and secretion of biologically active TGF-beta may occur after exposure to all-trans-retinoic acid (RA), coincident with decreased growth rate and/or differentiation. This study evaluates the expression and regulation of transforming growth factor beta (TGF-beta) and its receptors during RA-induced cell growth arrest and induction of differentiation in the RA-sensitive human neuroblastoma cell line SMS-KCNR and the RA-resistant neuroblastoma cell line SK-N-AS. RA treatment of SMS-KCNR cells results in a 40-fold increase in TGF-beta 1 mRNA after 4 days of RA, a dose-dependent increase in TGF-beta 1 secretion, an increase in types I (TBRI) and III (TBRIII) TGF-beta receptor proteins, and an increase in type II TGF-beta receptor (TBRII) mRNA coincident with RA-responsiveness of the cells. However, in the RA-resistant line SK-N-AS, TGF-beta 1 is constitutively secreted at levels that are unchanged after RA treatment, and although TBRI and TBRIII mRNA is expressed in untreated SK-N-AS cells, levels of TBRI and TBRIII protein and TBRII mRNA decrease after RA treatment. Thus, in RA-sensitive neuroblastoma cells, RA treatment may result in the induction of a negative autocrine TGF-beta 1 growth regulatory loop. These results suggest the hypothesis that: (a) induction of a TGF-beta 1 negative autocrine growth loop may be a necessary component for RA-responsiveness of neuroblastoma cells in vivo; and (b) the inability to induce or maintain this TGF-beta 1 negative autocrine growth loop may be a mechanism of RA resistance in neuroblastoma.

Expression of stem cell factor and c-kit in human neuroblastoma. The Children's Cancer Group.

During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Because melanocytes derive from neural crest cells, the role of SCF and c-kit was investigated in the neural crest-derived childhood tumor neuroblastoma. Using reverse transcription-polymerase chain reaction analysis, simultaneous expression of steady-state mRNA for the SCF ligand and its receptor c-kit was found in 14 of 14 (100%) human neuroblastoma cell lines and clones and in 8 of 18 (45%) human neuroblastoma tumor samples. Functional blockade of c-kit receptors in the cell lines SK-N-BE(2) and SH-SY5Y using the mouse monoclonal anti-c-kit antibody SR-1 resulted in a significant decrease in cellular growth rate when measured by either 3H-thymidine incorporation or clonogenicity. In addition, higher levels of c-kit mRNA expression were associated with parental neuroblastoma cell lines and subclones with a neuronal (N) differentiation phenotype, whereas lower levels of c-kit mRNA were associated with neuroblastoma cell line subclones having a schwannian/glial/melanocytic pattern of differentiation. However, the differentiation phenotype of neuroblastoma cell lines was not directly altered when c-kit expression was blocked using the SR-1 antibody. In summary, these data indicate that c-kit receptor expression may play a significant role in the growth regulation of the two neuroblastoma cell lines examined and suggest that c-kit may also play a similar role in neuroblastoma growth regulation in vivo. Simultaneous expression of SCF and c-kit mRNA in both neuroblastoma cell lines and tumors implies that c-kit may act as part of an autocrine growth loop in conjunction with endogenous production of SCF in this disease.

Neuropeptide Y expression in the developing adrenal gland and in childhood neuroblastoma tumors.

Neuropeptide Y (NPY) expression is limited to tissues of the central and peripheral nervous system. In the adrenal gland, NPY is found in a subset of cells of the adrenal medulla. Using in situ hybridization analysis, NPY mRNA expression was characterized during human fetal adrenal medullary development. We found a biphasic pattern of NPY mRNA expression during the development of the human adrenal medulla. NPY mRNA is detectable at the earliest evaluable time point (7.5 weeks of gestational age) through 18 weeks of gestational age, and is then not detectable until 8 months after birth. We also analyzed NPY mRNA expression in neuroblastoma tumors, which often arise in the adrenal medulla. Thirty-eight neuroblastoma tumors were analyzed for NPY mRNA expression using in situ hybridization. We found NPY mRNA expression in 30 of 38 tumors; 15 of 15 Stage IVS tumors from children under 1 year of age at diagnosis expressed NPY mRNA, whereas 0 of 4 Stage IV tumors from children less than 1 year of age at diagnosis expressed NPY mRNA. These data suggest that in children under 1 year of age at diagnosis, Stage IVS and Stage IV neuroblastoma may be marked by the presence or absence, respectively, of NPY mRNA expression. Moreover, since NPY is expressed for only a short period of time during embryogenesis, these tumors may arise from different neuroblast populations occurring during the course of adrenal medullary development.

Human neuroblastoma tumor cell lines correspond to the arrested differentiation of chromaffin adrenal medullary neuroblasts.

We have examined the hypothesis that nonhematopoietic malignancies may contain cells corresponding to those which occur during the differentiation of tissue precursors. Neuroblastoma, an embryonal tumor of the adrenal medulla, was studied because of its well described ability to differentiate both in vivo and in vitro. We examined the expression of four genes during development of the human adrenal medulla: tyrosine hydroxylase, chromagranin A, pG2, and beta-2-microglobulin. The sequential expression of these genes by adrenal neuroblasts marks successive stages during maturation of the chromaffin lineage. We also observed a population of neuroblasts during adrenal medullary development that did not express any of these four genes, suggestive of adrenal medullary cells differentiating along nonchromaffin lineage(s). We then evaluated 27 neuroblastoma cell lines for the expression of these genes and found that 24 expressed chromaffin markers, with 19 of these mimicking the pattern of gene expression found during development. Three cell lines did not express tyrosine hydroxylase, chromogranin A, or pG2, consistent with either a very undifferentiated neural crest cell or maturation along a nonchromaffin lineage. These data indicate that neuroblastoma tumor cells correspond to adrenal neuroblasts arrested during morphogenesis of the adrenal medulla and raise the possibility that malignant transformation of cells at different stages of tissue maturation may contribute to the diversity that characterizes tumors of solid tissues.

Neuropeptide Y expression distinguishes malignant from benign pheochromocytoma.

No reliable gross or microscopic features distinguish benign from malignant pheochromocytomas. The diagnosis of malignant pheochromocytoma is based solely on the presence of regional or distant metastases. This study evaluated the expression of neuropeptide Y messenger RNA (mRNA) in nine benign and 11 malignant pheochromocytomas and has found that neuropeptide Y mRNA was expressed in all nine benign tumors but in only four of 11 malignant tumors (P = .0084). These data suggest that the determination of neuropeptide Y expression in the evaluation of patients with pheochromocytoma may have prognostic significance.

Analysis of nerve growth factor receptor expression in human neuroblastoma and neuroepithelioma cell lines.

A series of 22 neuroepithelioma and neuroblastoma cell lines were screened for expression of nerve growth factor receptor (NGFR) by flow cytometry, Western blotting, and Northern blotting. All 5 neuroepithelioma cell lines expressed cell surface NGFR, with 30-69% of cells NGFR positive, but the 17 neuroblastoma cell lines tested had a smaller percentage of cell surface NGFR-positive cells (0-21%) and 10 lines were completely lacking cell surface NGFR. SY5Y, a variant line with a neuronal phenotype derived from neuroblastoma line SKNSH, expressed much more NGFR than SHEP, a variant line with an epithelial-like phenotype also derived from SKNSH. By Western blotting, the Mr approximately 69,000 NGFR band was detected for all four neuroepithelioma cell lines tested but was visible for only 8 of 15 neuroblastoma cell lines tested. The band was most intense for neuroepithelioma cell lines SKNMC and TC32. For these two lines, a Mr approximately 56,000 and a Mr approximately 60,000 band were also detected. By Northern blotting, all three neuroepithelioma cell lines tested were positive for the 3.8 kilobase NGFR mRNA, but only 8 of 15 neuroblastoma cell lines were positive. Neuroepithelioma cell line TC32 and neuroblastoma cell line GICAN had the strongest expression of NGFR mRNA. These results demonstrate that NGFR is a biological marker for neuroepithelioma and that NGFR expression is heterogeneous for neuroblastoma cell lines. This series of neural cell lines differing in NGFR expression will be useful for future studies of regulation of NGFR expression and neuronal differentiation.

Basic molecular biology for the pediatric hematologist/oncologist.

Recent advances in molecular biology have permitted a broader understanding of the pathogenesis of human cancer. Furthermore, the elucidation of pathogenetic mechanisms underlying the malignant transformation of normal cells has begun to suggest new approaches for the diagnosis, prognosis, and therapy of cancers. To familiarize the practicing hematologist/oncologist with the implications of current advances in the molecular biology of cancer, this article will review the basic concepts of molecular biology, and will summarize salient aspects of the most frequently used techniques. Also, several biologic principles which have lead to a further understanding of oncogenesis will be discussed in detail.

Chromogranin A expression in normal and malignant human tissues.

We used a recombinant cDNA probe for human chromogranin A to measure the expression of mRNA encoded by this gene in a variety of normal human tissues and tumor specimens using Northern blot and in situ hybridization analysis. With few exceptions, the expression of chromogranin A mRNA appears to be restricted to normal tissues and tumors of neuroendocrine lineage. However, we have detected mRNA expression of this gene in 1 of 14 cell lines and 2 of 13 tumor specimens of colon adenocarcinoma. The finding of chromogranin A expression in some colon carcinomas suggests that a previously unrecognized subgroup of these tumors has neuroendocrine features. The detection of this subgroup demonstrates the potential for improving tumor classification through the use of techniques and reagents developed by recombinant DNA technology.

Detection of N-myc gene expression in neuroblastoma tumors by in situ hybridization.

The presence of N-myc DNA amplification in human neuroblastoma tumors has been shown to be an independent prognostic factor predicting rapid progression of disease. Southern blot analysis has been used previously to detect N-myc amplification in these tumors. The authors report an analysis of N-myc gene expression by in situ hybridization in 28 human neuroblastoma tumors previously studied by Southern blot analysis. In the LA-N-5 human neuroblastoma cell line known to be amplified for N-myc, reaction conditions favoring RNA-RNA hybridization yielded an optimal signal. Using these hybridization conditions, in situ hybridization analysis of N-myc expression in 28 human neuroblastoma tissues correlated perfectly with N-myc DNA amplification in these tumors as detected by Southern blot analysis. In particular, there were no tumors in which N-myc RNA expression was found by in situ hybridization analysis in the absence of DNA amplification detectable by Southern blot, nor were there tumors that had DNA amplification in the absence of RNA expression. Heterogeneity of N-myc RNA expression was observed both among cells in any given tumor area, as well as within different areas of a single tumor. N-myc expression by in situ hybridization analysis was not observed in those tumors with more neuronally differentiated, ganglioneuroma histology. It is concluded that in situ hybridization of tissue sections is as effective as Southern blot analysis of tumor cell DNA in identifying human neuroblastoma tumors in which the N-myc gene is of prognostic significance.

Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription.

Narcotic withdrawal syndrome in young adults after the therapeutic use of opiates.

Five young adults with malignant neoplasms developed symptoms of narcotic withdrawal six to 48 hours after the abrupt discontinuation of narcotic therapy that had been administered for six to 21 days. The symptoms, which included agitation, irritability, muscular jerks, abdominal pain, diarrhea, burning sensations, "gooseflesh," and itching, rapidly resolved when narcotic therapy was reinstituted. Four patients were then successfully weaned from opiates without recurrence of their withdrawal symptoms; the fifth patient continues to receive methadone hydrochloride treatment for continuing pain.

In vitro and in vivo chemotherapy screening of the divalent cation chelator 1,10-orthophenanthroline.

1,10-Orthophenanthroline (OP) is a divalent cation chelating agent with known cytotoxicity to human normal and malignant T-lymphocytes. To determine whether OP might be a useful anticancer agent with specific T cell toxicity, OP's effect on cell growth was determined on colony-forming cells. The assay used supported growth of both malignant lymphoid and normal myeloid colony-forming cells (CFU-C) and thus a direct comparison of OP's antilymphoid and antimyeloid toxicity was obtained. The malignant lymphoid cells tested were established from patients at relapse and were resistant to conventional chemotherapeutic agents in vitro. While OP was found to be toxic to all cells tested, some selective kill of malignant cells over CFU-C occurred. OP's cytotoxicity was time-dependent and a three-log enhanced kill occurred when the drug exposure time was increased from 1 to 24 h. When test cells were continously exposed to OP, the ID50 was less than 1 micrograms/ml for malignant lymphoid cells and the sensitivity index (SI = x ID50 CFU-C divided by x ID50 cell line) ranged from 1.5 to 3.0. The National Cancer Institute currently screens new compounds for antitumor activity by determining whether the test drug is toxic to a mouse lymphocytic leukemia cell line (P388). While the mouse P388 cells were sensitive to OP in vitro, no effect was seen when OP was administered in vivo, even when schedules designed to take advantage of OP's time-dependent toxicity were used. Since malignant cells were sensitive to OP (ID50 less than 1 micrograms/ml), and some selectivity over CFU-C occurred (SI greater than 1), OP may be a useful agent for control of leukemic cell growth in vitro. However, since OP did not control the growth of P388 cells in vivo, additional studies designed to enhance the therapeutic index of OP in vivo are needed.