May 09, 2003
Gene Screen Nabs New Colon-Cancer Genes
Howard Hughes Medical Institute researchers have combed through a
catalog of all known tyrosine kinase enzymes to identify new gene
mutations that occur in a significant fraction of colon cancers.
Researchers have known for years that abnormal activity of tyrosine
kinases can hasten the development of certain forms of cancer. In this
first-of-a-kind survey, researchers used a forward-looking approach to
screen the genes that produce tyrosine kinases — reasoning that
somewhere in this genomic landscape they might encounter novel
mutations that spur colon cancer and other types of cancer.
“The availability of the human genome sequence allows scientists to scan sequences to identify kinases, and the increased speed with which DNA can be sequenced enables us to rapidly search for mutations in those kinases in human cancers.”
Sanford Markowitz
The researchers — including Howard Hughes Medical Institute
investigators Bert
Vogelstein and Sanford
Markowitz — published their findings in the May 9, 2003, issue of
the journal Science. Vogelstein is at the Sidney Kimmel
Comprehensive Cancer Center at The Johns Hopkins University, and
Markowitz is at Case Western Reserve University and University
Hospitals of Cleveland.
The starting point for the studies was a detailed catalog of all
kinases in the cell, similar to one published in December 2002 in the
journal Science.
Kinases are important enzymes that activate other proteins by adding
a phosphate group to them. According to Vogelstein, this catalog, which
its developers named the “kinome,” constitutes an ideal
collection of targets for cancer researchers seeking to stop the
uncontrolled proliferation of cancer cells.
“The good news is that many of the genes responsible for
common cancers, like colon cancer, have been identified,” said
Vogelstein. “The bad news is that most of these genes are tumor
suppressor genes, the metabolic brakes on cells. These suppressor genes
are inactivated in tumors, and since cancer drugs work by reducing the
activity of enzymes, they won't work on such suppressors because a drug
can't inhibit a gene that is already inactivated.”
According to Vogelstein, genetic mutations can produce a tyrosine
kinase that is essentially “turned on” in the absence of a
normal activation signal, which is called constitutive activation. This
represents an ideal drug target, said Vogelstein, and is exemplified by
the success of the drug Gleevec in treating chronic myeloid
leukemia.
“In the past, the search for drug targets has been guided
mechanistically - by asking what karyotypic abnormalities are found in
a cancer cell or what's responsible for a hereditary cancer
predisposition,” said Vogelstein. “But with the completion
of the human genome sequence and the availability of the kinome, one
can begin to think about how to do this in a much wider and unbiased
sense.”
The approach taken in this work actually represents a marriage of
two technologies. “The availability of the human genome sequence
allows scientists to scan sequences to identify kinases, and the
increased speed with which DNA can be sequenced enables us to rapidly
search for mutations in those kinases in human cancers,” said
Markowitz.
In beginning their survey of the kinome for kinases activated in
colon cancers, the group first explored tyrosine kinases and related
enzymes. To reduce the amount of gene sequencing required, they focused
their search on mutations in the “kinase domain” of these
enzymes, which is the region that is principally responsible for
enzymatic activity.
“The kinase motifs are fairly stereotypical,” said
Markowitz. “They always include an adenosine triphosphate binding
site and a set of conserved amino acid residues. That motif makes it
possible to identify kinases using computer-based information and to
screen genome sequences for all of the possibilities.”
“We figured that if there were going to be mutations that
constitutively activated these enzymes, and that would thus be
targetable by drugs, the kinase domains would be the ones to go
for,” said Vogelstein.
The researchers first identified the kinase domains of 138 tyrosine
kinases and similar enzymes from the kinome database. They next
extracted these same domains from 35 colorectal cancer cell lines, most
of which had been generated by Markowitz and his colleagues. They then
sequenced those domains for comparison.
Their studies revealed mutations within the kinase domains in 14
genes. The researchers then analyzed another 147 colorectal cancer cell
lines in the same way for kinase-domain mutations in these genes, and
then sequenced the entire coding region of all kinases that were
mutated, in all discovering 46 new mutations.
The major difficulty, said Vogelstein, was distinguishing the
“signal from the noise,” in this case, the mutations that
would trigger cancer from the huge number of harmless variants in
kinase genes. “We saw hundreds of changes in genes in cancer
cells that had not been identified before, and for each of these, HHMI
research associate Alberto Bardelli had to perform comparisons with the
normal tissues of the same patient, to see if the mutation was specific
to the cancer,” he said.
Analyses of mutations unique to the cancer cells strongly indicated
that the mutations affect the function of the tyrosine kinases in the
cells, constitutively activating them, said Vogelstein. “Most
importantly, they were in positions of the kinase domains predicted to
alter function, based on analogous mutations that have been seen before
in other kinases,” he said.
According to Vogelstein, these studies of the mutations indicate
that about 30 percent of colorectal cancers have mutations in at least
one kinase, meaning that these cancers will theoretically be vulnerable
to drugs that can block the action of the kinases.
The survey method demonstrated by the researchers could have a
profound impact on colon cancer treatment strategies, said Vogelstein.
“We envision that in the future, there could be tailored cancer
therapies. For example, each patient with colon cancer could have a
diagnostic analysis to determine which kinases are activated by
mutation — an easy task once you know which ones to look for. Then,
that patient could be treated with a drug that specifically targets
that kinase.”
More broadly, he said, the approach could impact treatment of other
types of cancer. “I think it's a perfectly reasonable speculation
to suggest that ultimately a great majority of cancers will be found to
have at least one drug-targetable mutation, and this could lead to new
avenues for individualized therapy,” he said.
“Two of the major questions to be addressed by our
laboratories are the functional consequences of the mutations we are
finding, and whether these kinases are targets for drugs,” said
Markowitz. “Discovering a new oncogene in a tumor now allows one
to ask the next big questions, what happens when you turn it off? How
big an impact will it have on the behavior of the tumor? Gleevec works
so well in chronic myeloid leukemia because it turns off a tyrosine
kinase that dramatically reverses the cancer process.”
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