Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor.

Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.

cDNA microarray analysis of invasive and tumorigenic phenotypes in a breast cancer model.

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets.

c-Cbl associates directly with the C-terminal tail of the receptor for the macrophage colony-stimulating factor, c-Fms, and down-modulates this receptor but not the viral oncogene v-Fms.

The receptor for the macrophage colony-stimulating factor (CSF-1, also termed M-CSF), the tyrosine kinase c-Fms, was originally determined to be the oncogene product of the McDonough strain of feline sarcoma virus, v-Fms. The structural difference between c-Fms and v-Fms amounts to only five point mutations in the extracellular domain, two mutations in the cytoplasmic domain, and the replacement of 50 amino acids by 14 unrelated amino acids at the C-terminal tail. Here, we have identified c-Cbl as the direct binding partner for c-Fms. c-Cbl binds to phosphotyrosine residue 977 at the C-terminal end of feline c-Fms, which is absent in v-Fms. The replacement of the C-terminal end of v-Fms by the corresponding part of c-Fms (vc-Fms) restored the binding potential. As a result, vc-Fms reduced the transforming potency of v-Fms. The overexpression of Cbl did not influence the v-Fms-transformed phenotype, although c-Cbl forms a complex with v-Fms indirectly. In contrast, the expression of Cbl drastically reduced the vc-Fms-transformed phenotype and the activation of Erk and enhanced Fms ubiquitination via phosphotyrosine residue 977. Furthermore, the replacement of tyrosine 977 into phenylalanine in feline c-Fms and vc-Fms reduced the Cbl-dependent ubiquitination. These data suggest that an indirect association of c-Cbl via multimeric complex induced a different signaling pathway from the pathway induced by c-Cbl direct interaction.

Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma.

Functional identification and molecular cloning of a rat gene mediating growth inhibition and programmed cell death in normal and transformed rat cell lines.

We wished to identify DNA sequences conferring suppression of proliferation and transformed phenotypes. Thus, we have transfected DNA from normal rat cells, covalently linked to neo DNA coding for neomycin resistance into a tumorigenic, HRAS transformed rat cell line. Phenotypic revertants were selected after the first cycle of transfection by enrichment procedures that served to eliminate transformed cells. The revertant clones continued to express the HRAS oncogene, but exhibited a lower tumorigenicity, loss of anchorage-independent proliferation, flat morphology, and retardation of growth in monolayer culture. The reverted phenotype could be transferred in a second cycle of transfection into the HRAS transformed rat cells. Neo DNA ligated to genomic donor DNA was used as a tagging sequence to molecularly clone the transferred DNA sequence in a recombinant phage. Fragments of the cloned DNA detect a 2.5 kb transcript in parental cells and revertants. Thus, the recombinant phage harbors a putative growth inhibitory gene, designated trg, that is expressed at a higher level in rat embryo fibroblasts and in the REF52 cell line. Introduction of recombinant phage DNA into established 208F and Rat-2 cells and into HRAS-, v-fgr-, v-fms- and v-raf-transformed rat cell lines resulted in inhibition of growth and induction of programmed cell death.

Epidermal growth factor (EGF) modulation of feline sarcoma virus fms tyrosine kinase activity, internalization, degradation, and transforming potential in an EGF receptor/v-fms chimera.

The feline sarcoma virus oncogene v-fms has significantly contributed to the dissection of peptide growth factor action since it encodes the transmembrane tyrosine kinase gp140v-fms, a transforming version of colony-stimulating factor 1 receptor, a member of the growth factor receptor tyrosine kinase family. In this study, the functional significance of structural differences between distinct tyrosine kinase types, in particular between cellular receptors and viral transforming proteins of distinct structural types, has been further investigated, and their functional compatibility has been addressed. For this purpose, major functional domains of three structurally distinct tyrosine kinases were combined into two chimeric receptors. The cytoplasmic gp140v-fms kinase domain and the kinase domain of Rous sarcoma virus pp60v-src were each fused to the extracellular ligand-binding domain of the epidermal growth factor (EGF) receptor to create chimeras EFR and ESR, respectively, which were studied upon stable expression in NIH 3T3 fibroblasts. Both chimeras were faithfully synthesized and routed to the cell surface, where they displayed EGF-specific, low-affinity ligand-binding domains in contrast to the high- and low-affinity EGF-binding sites of normal EGF receptors. While the EFR kinase was EGF controlled for autophosphorylation and substrate phosphorylation in vitro, in vivo, and in digitonin-treated cells, the ESR kinase was not responsive to EGF. While ESR appeared to recycle to the cell surface upon endocytosis, EGF induced efficient EFR internalization and degradation, and phorbol esters stimulated protein kinase C-mediated downmodulation of EFR. Despite its ligand-inducible kinase activity, EFR was partly EGF independent in mediating mitogenesis and cell transformation, while ESR appeared biologically inactive.

Reassessment of the v-fms sequence: threonine phosphorylation of the COOH-terminal domain.

The v-fms oncogene product of the McDonough strain of feline sarcoma virus is a member of the receptor tyrosine kinase family. Its cellular counterpart, the c-fms product, is the receptor for colony-stimulating factor 1 (CSF-1) of macrophages. We have reanalyzed the v-fms gene by direct sequencing of a biologically active clone. An additional A nucleotide was detected in position 2810 of the published v-fms sequence. The frameshift changed the COOH-terminal sequence of the v-fms protein from -R-937-G-P-P-L-COOH to -Q-937-R-T-P-P-V-A-R-COOH. Antibodies against a synthetic peptide representing this new sequence precipitated the v-fms proteins from transformed NRK cells as well as from feline sarcoma virus (McDonough)-infected feline fibroblasts. We show by tryptic peptide mapping that threonine 939 present in the new sequence is phosphorylated by a yet unknown serine/threonine kinase in vivo. In chicken fibroblasts expressing the v-fms gene, this phosphorylation clearly depended on the addition of exogenous CSF-1. Furthermore, addition of CSF-1 appeared to activate the serine/threonine kinase, as judged by phosphorylation of the synthetic peptide QRTPPVAR.

Transforming mechanism of the feline sarcoma virus encoded v-fms oncogene product.

The v-fms oncogene product encoded by the McDonough strain of feline sarcoma virus (SM-FeSV) is a transmembrane glycoprotein which belongs to the tyrosine kinase receptor family. The cellular counterpart, the c-fms product, is the receptor for macrophage colony stimulating factor (M-CSF or CSF-1). The v-fms and the c-fms product differ structurally only in seven point mutations and in their C-terminal domains. We have corrected the published sequence of the v-fms product and found that the new C-terminal end contains a threonine phosphorylation site (Thr939). This site is phosphorylated in vivo leading to an enhancement of the v-fms-specific tyrosine kinase activity. The extracellular domain of the v-fms product contains 11 N-glycosylation sites. Glycosylation and transport of the v-fms molecules to the plasma membrane are prerequisites for the transforming potential of the virus. Phosphorylation of the v-fms molecules in tyrosine, serine and threonine residues takes place only at the plasma membrane. Coexpression showed that the overexpression of M-CSF and c-fms in fibroblasts leads to cell transformation by an autocrine loop mechanism. This interaction between M-CSF and the c-fms protein also takes place at the plasma membrane. To study the v-fms transforming mechanisms, we have expressed the v-fms oncogene in chicken fibroblasts which are free of the cross-reactive M-CSF. The expression of the v-fms oncogene alone did not cause transformation. However, upon addition of M-CSF, these cells became completely transformed.(ABSTRACT TRUNCATED AT 250 WORDS)

Preferential expression of c-kit protooncogene transcripts in small cell lung cancer.

As an initial step to understand rapid growth of small cell lung cancer (SCLC), a complementary DNA library prepared from a SCLC cell line was screened with viral oncogene probes encoding protein-tyrosine kinases, which are known to play an important role in regulation of cell growth. Fifteen clones hybridizing with v-fms probe were isolated, and, by partial sequence analysis, four of them were identified to be c-kit protooncogenes. Northern blot study demonstrated that most of the SCLC tumors and cell lines expressed c-kit transcripts, while non-SCLC tumors and cell lines did not. Neither amplification nor rearrangement of the c-kit gene was demonstrated in SCLC cell lines by Southern blot analysis, however. Our results suggested that c-kit expression in SCLC reflects the unique biological nature of the tumor cells different from non-SCLC and further suggested that the c-kit product may participate in autocrine or paracrine stimulation of SCLC growth.

Structure, biosynthesis and biological roles of monocyte-macrophage colony stimulating factor (CSF-1 or M-CSF).

CSF-1 or M-CSF is a homodimeric glycosylated protein purified to homogeneity through its hematopoietic growth factor properties. It has recently been cloned and sequenced as a unique gene encoding several m-RNAs and peptidic species. Its receptor has been identified as the c-fms protooncogene product expressed at the surface of all CSF-1 responsive cells as a transmembrane tyrosine kinase responsible for CSF-1 induced signal transduction. CSF-1 is permanently present in serum and several biological fluids at stable concentrations and can thus be considered as a hormone. In vivo concentrations are mainly regulated by binding, internalization and degradation of the CSF-1 molecule by hepatic and splenic macrophage receptors. Very little is known about the organs and cell types responsible for in vivo CSF-1 synthesis. In vitro, CSF-1 regulates the survival, proliferation and differentiation of the monocyte-macrophage lineage from progenitors to mature cells and activates several important functions of mature tissular macrophages. Recent results obtained with injections of recombinant CSF-1 show that interesting activities, e.g., as ADCC, NK or antiinfectious effects, that had been initially observed in vitro can also be induced in vivo. All these in vitro and in vivo data and the recent availability of large amounts of pure recombinant human CSF-1 point to the diagnostic value of CSF-1 concentration measurements in biological fluids and suggest a therapeutic role for CSF-1 infusion in cancer and infection therapy.

Transformation of chicken fibroblasts by the v-fms oncogene.

The v-fms oncogene of the McDonough strain of feline sarcoma virus (SM-FeSV) encodes a plasma-membrane-associated tyrosine kinase (gp140v-fms) which is closely related, both structurally and functionally, to the c-fms-specified receptor for the macrophage colony stimulating factor (CSF-1). In mammalian fibroblasts, the natural producers of CSF-1, expression of v-fms leads to cell transformation. To study the interaction between CSF-1 and gp140v-fms molecules in a cell system that does not produce endogenous cross-reactive CSF-1, we have expressed the entire v-fms gene as well as a nontransforming deletion mutant (SC2) in chicken embryo cells (CEC). For this purpose the avian retroviral vectors pDS3 and pREP, based on Rous sarcoma virus, were used to isolate recombinant virus particles. CEC infected with virus that carried the entire v-fms gene expressed high amounts of gp140v-fms, comparable to those in SM-FeSV transformed NRK cells. However, these CEC remained flat, retained their fibronectin network, and did not produce enhanced levels of plasminogen activator. The cells grew faster than control CEC for more than 8 weeks but failed to form colonies in soft agar. Within 2 days after addition of CSF-1 to the growth medium, a transformed cell phenotype was induced, as judged by loss of the fibronectin network, again with a growth rate fourfold faster than that of the parental cells and with colony formation in soft agar. Moreover, human CSF-1 caused a rapid tyrosine phosphorylation of v-fms molecules detectable within 5 min after addition of the growth factor. In contrast, CSF-1 had none of the above effects on cells that expressed the SC2 v-fms deletion mutant.

Growth factor modulation of metastatic lung colonization.

Altered production and response to growth factors is often involved in neoplasia but little is known about their effect on the dissemination of tumors. Therefore, we have examined the effect of growth factors on metastatic lung colonization. Autocrine induction of the metastatic phenotype was demonstrated in NIH-3T3 cells transformed by a signal peptide-bFGF gene but not bFGF in the absence of the signal peptide. In addition, exogenous growth factor regulation of metastasis was demonstrated. Treatment of ras transformed C3H-10T 1/2 cells and ras or src transformed NIH-3T3 cells with bFGF prior to intravenous injection resulted in potent inhibition of metastatic potential. Stimulation of v-fms transformed cells by the natural fms-ligand, CSF-1, resulted in potent stimulation of metastatic behavior in freshly plated or refed cells, whereas following autocrine conditioning of the medium, the metastatic properties of these cells were sensitive to inhibitory effects of CSF-1. These observations indicate that specific growth factors can regulate the metastatic phenotype and, depending on the oncogenes responsible for cell transformation and autocrine conditioning, these effects can be either stimulatory or inhibitory.