HEMATOPOIETIC GROWTH CONTROL AND ONCOGENESIS 
Charles J. Sherr, M.D., Ph.D., Investigator 
Colony-stimulating factor- 1 (CSF-1) supports the 
growth, differentiation, and survival of mononu- 
clear phagocytes by binding to a high-affinity cell 
surface receptor (CSF-IR) encoded by the c-fms 
proto-oncogene. CSF-IR is expressed on mono- 
cytes, macrophages, and their committed precur- 
sors, as well as on placental trophoblasts during 
fetal development. Ligand binding activates the re- 
ceptor tyrosine-specific protein kinase, leading to 
phosphorylation of cellular substrates that convey 
mitogenic signals from the plasma membrane to 
the cell nucleus. The major goals of this laboratory 
have been to understand the mechanism of recep- 
tor kinase activation, to identify substrates of the ki- 
nase that play a physiologic role in signal transduc- 
tion, and to pinpoint genetic alterations in the 
c-fms gene that constitutively activate the CSF-IR 
kinase and induce oncogenic transformation. 
I. Transforming Potential of Human Colony-stimu- 
lating Factor- 1 Receptor. 
Human CSF-IR is an integral transmembrane gly- 
coprotein consisting of a 5 12 -amino acid extracel- 
lular ligand-binding domain, a 2 5 -amino acid trans- 
membrane segment, and a 435-amino acid 
cytoplasmic tyrosine kinase domain. Recombination 
of cat c-fms sequences into feline sarcoma virus 
(FeSV) is responsible for its oncogenicity. Although 
analogous in structure to CSF-IR, the retroviral 
\-fms oncogene encodes a variant glycoprotein that 
exhibits ligand-independent tyrosine kinase activity. 
This year Dr. Sherr's laboratory identified critical 
genetic alterations in the human c-fms proto- 
oncogene that unmask its oncogenic potential. Ho- 
mologous recombination in lambda phage was 
used to generate chimeric receptor genes between 
feline v-fms and human c-fms cDNAs. When ex- 
pressed in mouse NIH 3T3 cells, glycoproteins con- 
taining residues 1-308 of human CSF-IR fused to 
the remainder of the y-fms-coAcd glycoprotein 
functioned as normal receptors, whereas reciprocal 
chimeras elicited ligand-independent cell transfor- 
mation. Thus "activating mutation(s)" were pre- 
dicted to reside within the first 308 v-fms codons. 
Based on differences in the nucleotide sequences 
of the feline y-fms and c-fms genes in this region, 
human c-fms mutants were generated and tested 
for transforming activity. Foci of transformed cells 
were induced by receptors bearing a serine-for-leu- 
cine substitution at codon 301. Although located in 
the CSF-IR extracellular domain, the activating mu- 
tation did not affect the high-affinity ligand-binding 
site, so that the growth of transformed cells was 
further stimulated by CSF-1. Truncations or muta- 
tions affecting the distal CSF-IR carboxyl-terminal 
tail, although themselves insufficient to activate on- 
cogenicity, enhanced the transforming efficiency of 
receptors containing the serine 301 mutation. 
Therefore multiple genetic events within c-fms can 
collaborate to generate a fully transformed pheno- 
type. 
Analogous mutations within the c-fms proto- 
oncogene in situ might contribute to leukemias in- 
volving cells of the mononuclear phagocyte series. 
Monoclonal antibodies to extracellular epitopes in 
human CSF-IR, developed in Dr. Sherr's laboratory, 
were used to detect receptor expression on leuke- 
mic blasts from —40% of pediatric and adult acute 
myelogenous leukemia (AML) patients. Surprisingly, 
this did not correlate with other characteristics of 
monocyte differentiation. The possibility that c-fms 
genes in receptor-positive cases have acquired acti- 
vating mutations is under investigation. Four 
monoclonal antibodies inhibited CSF-1 binding to 
its receptor, and one inhibited the ligand-indepen- 
dent growth of NIH 3T3 cells transformed by acti- 
vated c-fms genes without affecting CSF-IR inter- 
nalization or degradation. The latter result suggests 
that mutations that activate the CSF-IR kinase alter 
the aggregation state of the receptor at the cell sur- 
face. 
II. Genomic Organization of the Human c-fms 
Gene. 
The complete nucleotide sequence of the human 
c-fms gene revealed that CSF-IR is encoded by 21 
exons interspersed over 32 kilobases (kb) on the 
long arm of chromosome 5. Although transcription 
of c-fms mRNA in monocytes is initiated from a pro- 
moter located in close proximity to the CSF-IR initi- 
ator codon, transcripts in trophoblasts contain 
spliced 5' noncoding sequences originating from an 
exon located 26 kb upstream. This upstream exon 
was located <0.5 kb from the 3' end of the gene 
encoding the B-type platelet-derived growth factor 
receptor (PDGF-R). Based on the known intron- 
exon organization of c-fms and its sequence similar- 
ity to B-type PDGF-R, alignment of the two cDNAs 
Continued 
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