WEBVTT

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Thanksgivings. We're
talking today about rational

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medicine, and really what we're
talking about is an understanding of

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the molecular biology of disease has
actually helped to revolutionize the

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new science of therapeutic medicine.
And here, more often than not, the

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discussions are focused
around cancer. And so,

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I will therefore talk
about an interesting story,

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vis-à-vis the modern treatment of
cancer, and how our understanding of

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the molecular biology of the disease
really helps in developing radically

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new kinds of therapies.
By way of background,

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let's just mention that most of the
chemotherapeutics that we use today

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to treat cancer were developed over
the last 40-50 years at a time when

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the molecular and biochemical
defects inside cancer cells

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were totally obscure.
And therefore,

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to the extent that one
developed chemotherapeutics,

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they were developed simply
empirically, trial and error.

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For example, some of the most
effective chemotherapeutics against

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childhood leukemia
are alkylating agents,

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which attach methyl
and ethyl groups

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to target molecules inside cells.
And their utility in cancer was

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first discerned because of
an explosion in a container.

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I think it was in a ship off Naples
in World War II where a bin of

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alkylating agents was dispersed.
Many people were exposed to it, and

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these people, as a consequence
of that, came down with what's

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called leucopoenia. Poenia
generally means a depression,

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in this case, a depression
of their white blood cells.

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Such alkylating agents had actually
been used during the First World War

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in gas warfare because
during the First World War,

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one used so-called mustard gases,
which was a very effective way, even

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more effective than artillery
in killing vast numbers

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of enemy soldiers. And,
somebody noticed this

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leucopoenia in 1946-47 as a
consequence of inadvertent exposure

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to these alkylating agents,
which became dispersed as a gas.

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And about five years later,
somebody made the logical leap that

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if these agents were able to
suppress normal white blood

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concentrations that perhaps they
might also be effective against what

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seemed to be ostensibly a related
problem, which is the problem

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of leukemia. And keep
in mind that when we talk

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about leukemia, the suffix
-emia refers to blood

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generally, and leuk- once
again refers to white blood,

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i.e. an excess of white blood
cells in the blood. And so,

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through this accidental discovery,
one began to develop alkylating

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agents that turned out to be
extremely successful in treating,

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and often curing, childhood
leukemias, most notably acute

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lymphocytic leukemia, which
turns out to be very sensitive

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to this and other related agents.
So, this is a very common form of

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childhood leukemia, which is
now actually cured in 60 or

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70% of the children who were treated,
which would have been unheard of

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half a century ago. But I
return to what I said before,

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which is that this kind of treatment
was developed in the face of total

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ignorance concerning the nature of
the disease, the molecular defects

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that were present in the disease,
and that were responsible for the

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runaway, can you still hear me OK,
were responsible for the runaway

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proliferation of the cancer
cells. So having said that,

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I want to go to a different kind of
leukemia, and this is called chronic

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myelogenous leukemia, to
give you an indication of the

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path of discovery that led from
its original description to the

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development of rather
successful treatments.

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So, chronic myelogenous leukemia,
I mentioned the prefix myelo- last

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time or the time before referring to
bone marrow, and this is a leukemia

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of cells coming from the bone marrow
from the myeloid cells in the bone

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marrow, which are the precursors
of things like macrophages

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and granulocytes. So,
these are cells which are

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playing an important role
in the immune response,

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and during this chronic
myelogenous leukemia disease,

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which is called CML, there
could be a period of three or

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four years where individuals develop
large numbers of these cells in

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their blood stream. And after
a period of about three or

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four years, all of a sudden there is
an eruption into what's called blast

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crisis. And you may recall I
mentioned the word blast also on one

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occasion earlier. This
all fits together in a nice

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puzzle. Blast refers to primitive,
embryonic-like cells, and all of a

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sudden there is an eruption of
primitive, embryonic-like cells,

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less differentiated like these
macrophages and granulocytes,

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which until this point had been
present in vastly excessive numbers

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in the blood.
There's blast crisis.

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This leads to acute myelogenous
leukemia, and death ensues usually

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within a year or two, or
that's been traditionally the

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case. No one really had any
idea about the possible causative

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mechanisms of the disease, and
that allows me to use another

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word which you might one day
come across if you should stay in

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biomedical research. And
that is the etiologic agents.

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When we talk about etiologic agents,
we talk about the agents which are

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causally responsible for inducing
a disease. These can be external

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agents, or they could
even be internal agents,

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molecules inside cells which are
responsible for the creation of the

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disease. And the key discovery was
made in 1960 when individuals were

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looking at the chromosomal
makeup of the CML cells.

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The chromosomal makeup, I'll
use another word just so we

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could expand our vocabulary this
morning, the chromosomal makeup is

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often called the karyotype, that
is to say the constellation of

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chromosomes that one can see
at mitosis under the microscope.

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Keep in mind, as we've said before,
that during the interphase of the

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cell cycle, chromosomes
are essentially invisible,

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but during the metaphase of
mitosis they become condensed,

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and on that occasion, individuals
noticed a 9-22 translocation.

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So here is chromosome nine normally.
Here's chromosome 22. And as you

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may know, the numbering system
with human chromosomes goes from the

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largest number one all the
way down to the smallest.

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So, this is the smallest,
with the exception of the Y

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chromosome. And what
they notice was instead of

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seeing this regular chromosomal
array, they noticed instead what

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looked very much like a
structure of this sort here, i.e.

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a translocation.

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And this translocation resulted in
a swapping of sequences between these

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two chromosomes.
Note, by the way,

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this is reciprocal, i.e. in
the sense that nine donates

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something to 22, and 22
donates something to nine.

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However, the segments that are
swapped are not necessarily of equal

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size. So, it turns out here that
in this case, chromosome nine has

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actually gained a lot more
than chromosome 22 gained as a

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consequence of this
exchange of genetic segments.

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And this 9-22 translocation made
the smallest chromosome even smaller.

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So, this was already the smallest
chromosome as I mentioned besides

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the smallest autosome, the
smallest non-sex chromosome.

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Now it got even smaller because
it lost some of its bulk as a

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consequence of this
chromosomal translocation.

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And because this discovery
was made in Philadelphia,

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it became known as the Philadelphia
chromosome. This is now about 40

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years ago, or as it's sometimes
called, PH-1 for reasons,

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I don't know why it's called
PH-1 except for Philadelphia.

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And, as investigators began to
look at other cases of chronic

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myelogenous leukemia,
they discovered that this

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translocation was present at
the Philadelphia chromosome most

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importantly was identifiable
in virtually all cases,

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more than 95% of the cases of
chronic myelogenous leukemia.

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And moreover, this chromosome
was present as well in the more

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differentiated macrophages and
granulocytes that were present and

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circulating in the blood of the CML
patients. And that began to suggest

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the notion that there was
a stem cell of some sort,

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oligopotential stem cell that
created various kinds of more

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differentiated white blood cells
that had sustained this chromosomal

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translocation because that's
what it is, a translocalization,

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a translocation, that all the cells
of these patients had sustained this

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chromosomal translocation.
And that began to suggest the

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notion that somehow as a consequence
of a random genetic accident

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happening in these people's blood,
this particular chromosome was

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repeatedly identified. And
it was with great likelihood

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causally or etiologically important
in the genesis of the disease.

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But that in itself led nowhere.
One could simply talk about its

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association until work from
a totally unrelated area,

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which is to say the study of
retroviruses discovered Abelson

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murine leukemia virus. And
Abelson was named after the

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fellow, Herb Abelson, who
first discovered it at NIH and

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undertook its molecular
characterization here in our own

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cancer center, and Abelson
discovered that this

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virus which he studied carried the
ends of the murine leukemia virus,

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which was a parental virus.
It was as hybrid virus.

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And into the middle of it,
Abelson leukemia virus has acquired

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a cellular proto-oncogene,
which it had activated into an

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oncogene. And therefore, here
we have a situation where a

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cellular gene like sarc in the
case of Rous Sarcoma Virus has been

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activated. This became called
ABL for obvious reasons.

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And this gene, it turned out,
was critically important in

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understanding hwo the chromosomal
translocation led to cancer.

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In fact, if one infected mice with
a retrovirus carrying this genome,

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this is just to indicate the
fact that the repeat ends,

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the long terminal repeat
ends of this provirus,

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they occur twice at the ends of this
retrovirus. If one infected a mouse

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with the Abelson virus,
out came a disease which was

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superficially similar at least
to chronic myelogenous leukemia.

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And that began a search,
then, for the chromosomal

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localization of the
Abel proto-oncogene.

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And what was discovered
subsequently, fascinatingly enough,

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was that the Abel proto-oncogene was
right at the break point between two

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chromosomes, nine and 22. And
what happened as a consequence

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of this translocation, and
the resulting fusion of this

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chromosome with this chromosome
was the creation of a fuse gene,

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a hybrid gene that now carried the
reading frames of two previously

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unconnected gene, one
on chromosome nine,

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and one on chromosome 22.
Here's the normal, Abel protein.

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It's called C Abel, meaning the
cellular or the normal form of Abel.

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And you see it up here. It's
shown in a very schematic way.

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And here's a second protein which
is encoded on the other chromosome.

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So, Abel is encoded here, and the
other gene, which is called BCR is

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encoded here, and as a
consequence of the translocation,

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Abel is encoded here. BCR is
encoded here. As a consequence of

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the translocation, one now
has not only the fusion of

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chromosomal segments. But
one has the fusion of the

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reading frames of two
previously unlinked genes.

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And here, one creates as a
consequence of these fusions,

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any one of a series of three
quite distinct fusion proteins,

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which do not naturally
preexist in the normal cell.

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And there shown here is P-1
85, P-2 10, and P-2 30. These

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translocations allow different
parts of a second gene called BCR.

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BCR refers to breakpoint cluster
region. The area of the point of

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fusion is called the breakpoint
between the two genes.

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So, the point where each gene is
cut and fused with the other is

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called the breakpoint. And
it turns out that within the

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region of the chromosome where BCR
maps, there's actually three sites

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at which the fusion can occur.
If you look carefully at this

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diagram, you see that there's
differing extents of the BCR protein,

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which can be contributed
to the fusion protein.

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And, what this says, in
effect, is the following,

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that here, let's just refer
to this diagram right up here.

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Notice, by the way, in all three
of these, that the Abel protein is

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present at the C terminal end of the
protein. The BCR is present at the

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end terminal end. So,
here's the BCR gene.

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Here's the Abel gene down here.
And what investigators found is that

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there could be a break at
this part of the BCR gene,

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at this part of the BCR gene,
or at this part of the BCR gene,

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resulting always in the
fusion of Abel, to one, or two,

00:16:13.000 --> 00:16:17.000
or three different kinds
of BCR proteins. And,

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breakpoint cluster region signified
the fact that there was a whole

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cluster of sites in the previously
existing BCR gene to which this

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fusion could take place,
resulting, if the break occurred

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here, the breakpoint occurred
here, and BCR to get the longer one.

00:16:33.000 --> 00:16:37.000
Here you get the medium-sized one;
here you'd get the shortest one.

00:16:37.000 --> 00:16:41.000
And, interestingly enough,
as one explored virtually,

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other kinds of different leukemias,
one could see different of these

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fusion proteins that were produced.
Here's chronic, myelogenous

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leukemia, which I talked to
you about before. Here is acute

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lymphocytic leukemia, and
here's chronic and neutrophylic

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leukemia, three different kinds
of leukemia. We don't have to worry

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about the details of these diseases,
aside from the fact to say that the

00:17:05.000 --> 00:17:09.000
structure of this fusion protein
encourages the outgrowth of

00:17:09.000 --> 00:17:13.000
different kinds of stem cells in
the bone marrow, which in turn create

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three different kinds of diseases.
Most importantly for our discussion

00:17:17.000 --> 00:17:21.000
was an attempt to understand the
nature of the resulting fusion

00:17:21.000 --> 00:17:25.000
protein, which as a consequence
of this fusion caused by the

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chromosomal translocation now
clearly acquired biological powers

00:17:29.000 --> 00:17:33.000
that did not preexist in either
of the two parental proteins.

00:17:33.000 --> 00:17:37.000
These various notations here
indicate a whole series of different

00:17:37.000 --> 00:17:41.000
functions which are associated
with the Abel protein,

00:17:41.000 --> 00:17:45.000
and alternatively with the BCR
protein. And we don't need to get

00:17:45.000 --> 00:17:49.000
into them, except to say that each
one of these different names here

00:17:49.000 --> 00:17:53.000
allows the protein on its own to
associate with other proteins and do

00:17:53.000 --> 00:17:58.000
activated downstream
signaling cascade.

00:17:58.000 --> 00:18:03.000
What's most important about our
discussion is the realization that

00:18:03.000 --> 00:18:09.000
this SH-1 domain, indicated
here, SH-1 refers to the

00:18:09.000 --> 00:18:15.000
sarcomology domain, equals
sarcomology, equals a

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tyrosine kinase. And
therefore, what one has here is

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a protein, which is much
more elaborate than sarc,

00:18:27.000 --> 00:18:33.000
has vastly more
signaling capabilities,

00:18:33.000 --> 00:18:37.000
by virtue of the fact that these
different domains that are indicated

00:18:37.000 --> 00:18:42.000
here allow the resulting fusion
protein to grab hold of a whole

00:18:42.000 --> 00:18:47.000
bunch of different signally partners
so that it can send out a diverse

00:18:47.000 --> 00:18:52.000
array of downstream activating
signals. If one examined the

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structure of the SH-1 domain,
it had a tyrosine kinase activity

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very much like sarc,
and most importantly,

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if one introduced this fusion
protein into a retrovirus,

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now instead of Abel, one could
make a BCR Abel fusion protein.

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One could put this into a retrovirus
as before, just like up here.

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One could infect mice with it,
and now get out of a disease which

00:19:21.000 --> 00:19:25.000
was indistinguishable, in
essence, from chronic myelogenous

00:19:25.000 --> 00:19:30.000
leukemia in humans. If one
put a subtle point mutation

00:19:30.000 --> 00:19:34.000
in the tyrosine kinase domain, all
of the able protein, here is the

00:19:34.000 --> 00:19:38.000
tyrosine kinase domain, SH-1,
up here. Here, we see the

00:19:38.000 --> 00:19:43.000
tyrosine kinase domains represented
in the three different fusion

00:19:43.000 --> 00:19:47.000
proteins. Keep in mind SH-1 is
always the tyrosine kinase domain.

00:19:47.000 --> 00:19:51.000
If one put a subtle, inactivating
point mutation in the tyrosine

00:19:51.000 --> 00:19:56.000
kinase domain, that
immediately wiped out all

00:19:56.000 --> 00:20:00.000
biological powers of creating
leukemias on the part of this

00:20:00.000 --> 00:20:04.000
retrovirus here, or any
one of the other closely

00:20:04.000 --> 00:20:09.000
related kinds of fusion
proteins. And therefore,

00:20:09.000 --> 00:20:13.000
that indicated that the tyrosine
kinase domain indicated right here

00:20:13.000 --> 00:20:17.000
was really critical to creating the
tumor, and that any effects on its

00:20:17.000 --> 00:20:21.000
tyrosine kinase signaling
ability would, in the end,

00:20:21.000 --> 00:20:25.000
result in the collapse of the tumor,
or the inability of the resulting

00:20:25.000 --> 00:20:30.000
retrovirus to
actually create cancer.

00:20:30.000 --> 00:20:34.000
And so, now one had,
really for the first time,

00:20:34.000 --> 00:20:38.000
a clear demonstration of how a
commonly occurring human cancer,

00:20:38.000 --> 00:20:42.000
chronic myelogenous leukemia,
is unfortunately not so rare,

00:20:42.000 --> 00:20:46.000
could arise as a consequence of some
random, chromosomal translocation

00:20:46.000 --> 00:20:50.000
event. You might ask, why
does one always get this

00:20:50.000 --> 00:20:54.000
particular kind of translocation?
Well, the answer is, we don't

00:20:54.000 --> 00:20:58.000
really know. It would almost seem
as if there's a homing device which

00:20:58.000 --> 00:21:02.000
causes this fragment and this
fragment to target each other and to

00:21:02.000 --> 00:21:07.000
exchange one another.
It's probably not the case.

00:21:07.000 --> 00:21:11.000
What probably happens is that
chromosomal translocations take

00:21:11.000 --> 00:21:15.000
place rather randomly within the
bone marrow, and on rare occasion

00:21:15.000 --> 00:21:19.000
there is a chromosomal translocation
that creates exactly this kind of

00:21:19.000 --> 00:21:23.000
fusion. And this kind of fusion,
in turn, is what's responsible for

00:21:23.000 --> 00:21:27.000
creating this fusion protein,
and this fusion protein in turn

00:21:27.000 --> 00:21:31.000
creates the outgrowth of this CML,
the chronic myelogenous leukemia

00:21:31.000 --> 00:21:36.000
disease. So what that
means is really that a

00:21:36.000 --> 00:21:40.000
randomly occurring chromosomal
translocation on rare occasion hits

00:21:40.000 --> 00:21:44.000
a genetic jackpot, and the
cell which happens to have

00:21:44.000 --> 00:21:48.000
acquired this kind of chromosomal
translocation now begins to

00:21:48.000 --> 00:21:52.000
proliferate wildly, creating
first chronic myelogenous

00:21:52.000 --> 00:21:56.000
leukemia, and then subsequently
erupting into a subsequent acute

00:21:56.000 --> 00:22:00.000
phase where there are seemingly
additional genetic alterations

00:22:00.000 --> 00:22:04.000
beyond this chromosomal
translocation that conspire with the

00:22:04.000 --> 00:22:08.000
initially present chromosomal
translocation to create a very

00:22:08.000 --> 00:22:12.000
aggressive disease which
rapidly leads to the death of the

00:22:12.000 --> 00:22:16.000
leukemia patient. That
offered, in principle,

00:22:16.000 --> 00:22:21.000
an attractive way of beginning to
develop an anti-cancer therapeutic

00:22:21.000 --> 00:22:26.000
because what one might imagine was
that one could develop a tyrosine

00:22:26.000 --> 00:22:38.000
kinase inhibitor.

00:22:38.000 --> 00:22:42.000
Now keep in mind that tyrosine
kinases are a class of enzymes which

00:22:42.000 --> 00:22:46.000
attach phosphate groups onto
the tyrosine residues of various

00:22:46.000 --> 00:22:51.000
substrate proteins. And
keep in mind as well the fact

00:22:51.000 --> 00:22:55.000
that we drew a series of growth
factor receptors which have tyrosine

00:22:55.000 --> 00:23:00.000
kinase domains in them. And
I'm drawing the tyrosine kinase

00:23:00.000 --> 00:23:06.000
domains here like this,
that when these growth factor

00:23:06.000 --> 00:23:11.000
receptors become activated,
they attach phosphate groups onto

00:23:11.000 --> 00:23:17.000
the tails of one another.
And I'll draw those phosphate

00:23:17.000 --> 00:23:22.000
groups like this, i.e. the
binding of ligand or let's

00:23:22.000 --> 00:23:28.000
say epidermal growth factor ligand
or plate ligand causes the two

00:23:28.000 --> 00:23:33.000
receptors, which are normally
mobilized in the plasma membrane to

00:23:33.000 --> 00:23:39.000
come together to
transphosphorylate one another,

00:23:39.000 --> 00:23:44.000
and having done so, to acquire
actively signaling powers,

00:23:44.000 --> 00:23:49.000
because once these phosphates become
attached, they now represent sites

00:23:49.000 --> 00:23:54.000
where other molecules can anchor
themselves and send out downstream

00:23:54.000 --> 00:23:59.000
signals. In fact, there
are altogether 90 different

00:23:59.000 --> 00:24:05.000
tyrosine kinases encoded
in the human genome.

00:24:05.000 --> 00:24:09.000
And so, to the extent that
these tyrosine kinases become

00:24:09.000 --> 00:24:13.000
hyperactivated in various
kinds of human cancers,

00:24:13.000 --> 00:24:17.000
this represents in principle a
very attractive way of developing an

00:24:17.000 --> 00:24:21.000
anti-cancer therapeutic. But
let's think about the problems

00:24:21.000 --> 00:24:25.000
that are inherent in such
a compound. First of all,

00:24:25.000 --> 00:24:29.000
if one wants to develop
an anti-cancer therapeutic,

00:24:29.000 --> 00:24:34.000
it must be reasonably specific
for the Abelson tyrosine kinase,

00:24:34.000 --> 00:24:38.000
and not the 89 kinds of tyrosine
kinases that also coexist in the

00:24:38.000 --> 00:24:43.000
human genome, and are active,
and apparently responsible for

00:24:43.000 --> 00:24:48.000
normal cell metabolism in a whole
variety of normal cell types.

00:24:48.000 --> 00:24:53.000
So, one has to begin to think
about the issue of cell activity.

00:24:53.000 --> 00:24:58.000
How can one possibly make a
low molecular weight compound,

00:24:58.000 --> 00:25:03.000
which is selectively able to
inactivate the Abelson tyrosine

00:25:03.000 --> 00:25:08.000
kinase as indicated
here, the SH-1 group,

00:25:08.000 --> 00:25:11.000
but doesn't disturb a whole variety
of other tyrosine kinases that are

00:25:11.000 --> 00:25:15.000
responsible for other normal
physiological mechanisms.

00:25:15.000 --> 00:25:19.000
Well, you'll say that's pretty
easy. We have 90 different genes.

00:25:19.000 --> 00:25:23.000
Each of the 90 different
genes makes a distinct protein,

00:25:23.000 --> 00:25:27.000
and these proteins should be
very different. And therefore,

00:25:27.000 --> 00:25:31.000
if one can, in fact, if one does the
three-dimensional structure of these

00:25:31.000 --> 00:25:35.000
proteins, all the tyrosine
kinases look quite similar.

00:25:35.000 --> 00:25:40.000
They have a biload structure.
Here is the active site of the

00:25:40.000 --> 00:25:46.000
enzyme. That is to say, in
here is the catalytic cleft,

00:25:46.000 --> 00:25:51.000
the site where the actual catalysis
takes place, the site where the

00:25:51.000 --> 00:25:57.000
gamma phosphate of ATP is taken from
the ATP and attached to a substrate

00:25:57.000 --> 00:26:03.000
protein to the hydroxyl of a
tyrosine of a protein that's about

00:26:03.000 --> 00:26:07.000
to be phosphorylated. So,
you just make a low moleculoid

00:26:07.000 --> 00:26:11.000
chemical that's specific for the
tyrosine kinase domain of the Abel

00:26:11.000 --> 00:26:15.000
protein. And when I draw
this biload structure,

00:26:15.000 --> 00:26:18.000
this biload structure is carried
here within the SH-1 domain right

00:26:18.000 --> 00:26:22.000
here. So, this has a biload
structure. It's obviously not

00:26:22.000 --> 00:26:26.000
indicated here in this
very schematic drawing.

00:26:26.000 --> 00:26:30.000
The problem with
that is the following.

00:26:30.000 --> 00:26:33.000
All of the SH-1 domains, all
of the tyrosine kinase domains

00:26:33.000 --> 00:26:37.000
are evolutionarily closely
related to one another.

00:26:37.000 --> 00:26:41.000
They're all derived from the
precursors of the tyrosine kinase

00:26:41.000 --> 00:26:45.000
domain that probably existed
maybe 600 or 700 million years ago,

00:26:45.000 --> 00:26:49.000
and has as a consequence of gene
duplication been diversified to make

00:26:49.000 --> 00:26:53.000
90 different tyrosine kinases.
And if you look under x-ray

00:26:53.000 --> 00:26:57.000
crystallography at the three
dimensional structure of all these

00:26:57.000 --> 00:27:01.000
tyrosine kinases, they all
pretty much look like this,

00:27:01.000 --> 00:27:04.000
i.e. they all have rather similar
catalytic clefts because they

00:27:04.000 --> 00:27:08.000
diverge from a common ancestral
protein, and they retain this three

00:27:08.000 --> 00:27:12.000
dimensional configuration because
this three dimensional configuration

00:27:12.000 --> 00:27:15.000
seems to be important for the
retention of their function.

00:27:15.000 --> 00:27:19.000
You could imagine, conversely,
that if there were some descendants

00:27:19.000 --> 00:27:23.000
of the ancestral tyrosine kinase
domain that some of them became

00:27:23.000 --> 00:27:26.000
mutant and lost its three
dimensional structure.

00:27:26.000 --> 00:27:30.000
Those descendant kinases would
lose their ability to phosphorylate

00:27:30.000 --> 00:27:34.000
tyrosines on substrate proteins,
and therefore would be eliminated

00:27:34.000 --> 00:27:38.000
from the gene pool because
they would be defective.

00:27:38.000 --> 00:27:42.000
And that explains the strong
conservatism in the structure of

00:27:42.000 --> 00:27:46.000
these 90 different enzymes.
They all look very similar to one

00:27:46.000 --> 00:27:50.000
another, and that creates a great
difficulty for the drug developer

00:27:50.000 --> 00:27:54.000
because a low molecular weight drug,
which one would like to develop,

00:27:54.000 --> 00:27:58.000
that fits in here. So, here
I'll draw a low molecular

00:27:58.000 --> 00:28:02.000
weight drug that interacts in a
stereo-specific fashion with the

00:28:02.000 --> 00:28:06.000
amino acid residues that
are aligning this pocket,

00:28:06.000 --> 00:28:10.000
this catalytic cleft, might bind
and nicely inactivate the tyrosine

00:28:10.000 --> 00:28:15.000
kinase domain of Abel.
But at the same time,

00:28:15.000 --> 00:28:19.000
it might also bind and inactivate
a whole series of other tyrosine

00:28:19.000 --> 00:28:23.000
kinases, and that in turn could
lead to therapeutic disaster.

00:28:23.000 --> 00:28:27.000
For instance, if you had
a non-selective agent,

00:28:27.000 --> 00:28:31.000
you could treat a chronic
myelogenous leukemia patient with a

00:28:31.000 --> 00:28:35.000
low molecular weight inhibitor,
a low molecular weight compound,

00:28:35.000 --> 00:28:39.000
which would get into this
pocket of the BCR Abel protein.

00:28:39.000 --> 00:28:43.000
But it might similarly get into the
catalytic cleft of the EGF receptor.

00:28:43.000 --> 00:28:47.000
And if it shot down the EGF
receptor, it might cause a fatal

00:28:47.000 --> 00:28:51.000
diarrhea because after all, the
EGF receptor, I will tell you,

00:28:51.000 --> 00:28:56.000
is needed to maintain the structure
of the epithelial lining of the

00:28:56.000 --> 00:29:00.000
colon. And so, you might
kill the patient simply

00:29:00.000 --> 00:29:04.000
because you had deprived the cells
in that person's colon of their

00:29:04.000 --> 00:29:08.000
ability to maintain themselves.
There are a whole series of growth

00:29:08.000 --> 00:29:12.000
factor receptors that are required
for hematapoeisis that we discussed

00:29:12.000 --> 00:29:16.000
last time. And there,
once again, if you had a

00:29:16.000 --> 00:29:20.000
nonselective compound, which
got into the domain of one of

00:29:20.000 --> 00:29:23.000
the growth factor receptors that
is responsible for hematapoeisis,

00:29:23.000 --> 00:29:27.000
you might shut down the entire
bone marrow, and once again kill the

00:29:27.000 --> 00:29:31.000
patient. I'm just giving you those
as overly dramatic examples of the

00:29:31.000 --> 00:29:35.000
fact that cell activity is an
extremely important consideration in

00:29:35.000 --> 00:29:40.000
developing such a drug. The
other thing is affinity for the

00:29:40.000 --> 00:29:48.000
target, for the catalytic
cleft that is being targeted.

00:29:48.000 --> 00:29:56.000
What do I mean by affinity?
If you look at those response

00:29:56.000 --> 00:30:04.000
curves of various compounds,
what you see is the following.

00:30:04.000 --> 00:30:12.000
You can draw out a line that looks
like this, a graph that looks like

00:30:12.000 --> 00:30:20.000
this, where here we have
log of drug concentration.

00:30:20.000 --> 00:30:28.000
And here is 10-4, here,
let's do the other one,

00:30:28.000 --> 00:30:36.000
10-8 molar, 10-7
molar, 10-6, 10-5, 10-4.

00:30:36.000 --> 00:30:42.000
And this is molar drug concentration.
And here is the percentage of

00:30:42.000 --> 00:30:49.000
inhibition. Let's say, for
example, we were able to take

00:30:49.000 --> 00:30:55.000
the BCR Abel protein and study it
in a test tube. And let's say we were

00:30:55.000 --> 00:31:02.000
interested in studying how well its
tyrosine kinase activity responded

00:31:02.000 --> 00:31:09.000
to an applied drug that
we developed against it.

00:31:09.000 --> 00:31:16.000
So, here's the percentage of
inhibition of tyrosine kinase

00:31:16.000 --> 00:31:24.000
activity of BCR Abel protein.
Now, I might be able to develop a

00:31:24.000 --> 00:31:32.000
drug whose dose response
curve would look like this.

00:31:32.000 --> 00:31:36.000
And you'll say,
well, that's terrific.

00:31:36.000 --> 00:31:40.000
That's a drug which shuts down BCR
Abel. We haven't even dealt with

00:31:40.000 --> 00:31:44.000
the issue of cell activity, but
let's look at where one begins

00:31:44.000 --> 00:31:48.000
to see a dose response right here,
10-5 molar. And if you calculate

00:31:48.000 --> 00:31:52.000
back as to how much of the drug
you need to deliver in order to shut

00:31:52.000 --> 00:31:56.000
down the BCR Abel protein in a
patient, the size of the pill they'd

00:31:56.000 --> 00:32:00.000
have to get would probably
be this big everyday.

00:32:00.000 --> 00:32:04.000
So, what you need to do is you need
to be an acceptable range of drug

00:32:04.000 --> 00:32:08.000
concentrations is down in
this area here. And therefore,

00:32:08.000 --> 00:32:12.000
only until you get a drug which
has a dose response curve that looks

00:32:12.000 --> 00:32:16.000
like this, which is two or three
orders of magnitude more potent

00:32:16.000 --> 00:32:20.000
where it's able to shut down the
kinase activity already at 10-7 in a,

00:32:20.000 --> 00:32:24.000
this is called a
submicromolar concentration.

00:32:24.000 --> 00:32:28.000
Micromolar is 10-6. Here
already at a tenth of a

00:32:28.000 --> 00:32:32.000
micromolar, 10-7 molar, we're
already getting a shutdown of

00:32:32.000 --> 00:32:36.000
the enzyme function.
And if one can do that,

00:32:36.000 --> 00:32:40.000
then one might in principle be able
to develop a pill that's this big

00:32:40.000 --> 00:32:44.000
and give that to the patient rather
than a pill that's a size of a

00:32:44.000 --> 00:32:47.000
football. And by the way, if
you have to make lots of a very

00:32:47.000 --> 00:32:51.000
complex, organic molecule through
organic synthesis that has an

00:32:51.000 --> 00:32:55.000
affinity of this, it's
also very expensive.

00:32:55.000 --> 00:32:58.000
Obviously, if you can make a
compound that's a hundredfold more

00:32:58.000 --> 00:33:02.000
potent and requires a hundredfold
less material to deliver to the

00:33:02.000 --> 00:33:06.000
patient body, then you might
have some success in treating

00:33:06.000 --> 00:33:09.000
the patient. Here's
another issue.

00:33:09.000 --> 00:33:13.000
So, we've talked about cell
activity. We've talked about

00:33:13.000 --> 00:33:17.000
potency or affinity,
affinity for the substrate or

00:33:17.000 --> 00:33:20.000
potency. So, this would
be an acceptable drug.

00:33:20.000 --> 00:33:24.000
It works already at molar
concentration where the inflection

00:33:24.000 --> 00:33:28.000
point of this curve is. This
is an unacceptable drug at

00:33:28.000 --> 00:33:32.000
10-5. We can
also talk about

00:33:32.000 --> 00:33:36.000
pharmacokinetics. I want
to give you a feeling for

00:33:36.000 --> 00:33:40.000
how complex drug development is,
and why it so rarely succeeds. By

00:33:40.000 --> 00:33:44.000
the way, you know how much it costs
to develop a drug that's useful in

00:33:44.000 --> 00:33:49.000
the clinic these days and test it
on people? Anybody have any idea?

00:33:49.000 --> 00:33:53.000
How much? Yeah. It's
pretty close to $1 billion,

00:33:53.000 --> 00:33:57.000
between $900 million and $1 billion.
That's a lot of money. That's more

00:33:57.000 --> 00:34:01.000
money than you and I are going
to earn together, all of us

00:34:01.000 --> 00:34:06.000
maybe, in a lifetime. OK,
anyhow, pharmacokinetics,

00:34:06.000 --> 00:34:10.000
well what's pharmacokinetics?
Glad I asked that question.

00:34:10.000 --> 00:34:14.000
How long does the drug stay inside
of you after you take it if you're a

00:34:14.000 --> 00:34:18.000
cancer patient? What
happens if the drug is

00:34:18.000 --> 00:34:22.000
excreted by the kidneys within
minutes of its being taken,

00:34:22.000 --> 00:34:26.000
let's say, either by
injection or orally?

00:34:26.000 --> 00:34:32.000
So here we can imagine, let's
talk about drug concentration.

00:34:32.000 --> 00:34:38.000
I'll use the word drug
concentration in blood.

00:34:38.000 --> 00:34:44.000
And here's time. And here's what
some drugs look like when you give

00:34:44.000 --> 00:34:50.000
them, let's say, orally.
Here's what they look like.

00:34:50.000 --> 00:34:56.000
So, let's say here's the effective
drug concentration: effective

00:34:56.000 --> 00:35:02.000
concentration. And
we know the effective

00:35:02.000 --> 00:35:06.000
concentration from doing
measurements like this.

00:35:06.000 --> 00:35:10.000
We just measure it, work that out.
So, let's say we develop a drug

00:35:10.000 --> 00:35:14.000
which is able to hit the BCR Abel
protein. What are the kinetics with

00:35:14.000 --> 00:35:18.000
which the drug becomes
soluble in the blood stream?

00:35:18.000 --> 00:35:22.000
And it might look like this,
where I'm drawing here now, this is

00:35:22.000 --> 00:35:26.000
one hour. This is two hours.
This is three hours, four hours.

00:35:26.000 --> 00:35:30.000
Is that
long enough?

00:35:30.000 --> 00:35:34.000
Well, the fact of the matter is,
if you're going to try to kill a

00:35:34.000 --> 00:35:38.000
cancer cell, and that's what
the name of this game is,

00:35:38.000 --> 00:35:41.000
you want to have it around for
a while because it turns out,

00:35:41.000 --> 00:35:45.000
as one learned, the continued
viability of the CML cancer cells of

00:35:45.000 --> 00:35:49.000
the leukemia cells was dependent on
the continued firing by the BCR Abel

00:35:49.000 --> 00:35:52.000
kinase protein. In
fact, as one learned,

00:35:52.000 --> 00:35:56.000
if one shut off firing by the
tyrosine kinase molecule in a

00:35:56.000 --> 00:36:00.000
chronic myelogenous leukemia
cell, the cells would implode.

00:36:00.000 --> 00:36:04.000
They would undergo apitosis.
So, this began to reveal that in

00:36:04.000 --> 00:36:08.000
fact the BCR Abel protein is not
only responsible for forcing these

00:36:08.000 --> 00:36:12.000
cells to proliferate, but it
also independently provides

00:36:12.000 --> 00:36:16.000
them with anti-apoptotic signal.
It keeps them from falling over the

00:36:16.000 --> 00:36:20.000
cliff into apitosis. It
keeps them from killing

00:36:20.000 --> 00:36:24.000
themselves, and that's obviously
critical for the ability of this

00:36:24.000 --> 00:36:28.000
tumor to proliferate, for the
number of cells to expand in

00:36:28.000 --> 00:36:32.000
the body of a patient. It
turns out that if you provide

00:36:32.000 --> 00:36:37.000
these cancer cells with an effective
way of shutting down their BCR Abel

00:36:37.000 --> 00:36:42.000
protein for 30 or 40 or 50 minutes,
not much happens to them. You need

00:36:42.000 --> 00:36:47.000
to deprive them of the drug
for a very long period of time,

00:36:47.000 --> 00:36:52.000
well, 15-20 hours, and therefore you
need pharmacokinetics that look like

00:36:52.000 --> 00:36:57.000
this. It needs to be present
for an extended period of time,

00:36:57.000 --> 00:37:03.000
or even better, let me re-draw
that, even better look like this.

00:37:03.000 --> 00:37:06.000
It stays in the blood for
an extended period of time.

00:37:06.000 --> 00:37:10.000
Some drugs stay in the
circulation for a long time.

00:37:10.000 --> 00:37:13.000
Other drugs stay in the circulation
for a very short period of time.

00:37:13.000 --> 00:37:17.000
There's another problem which we
haven't even begun to talk about,

00:37:17.000 --> 00:37:21.000
and that is the metabolism of the
drug. It turns out that many drugs

00:37:21.000 --> 00:37:24.000
that you give a patient are rapidly
converted by the enzymes and the

00:37:24.000 --> 00:37:28.000
liver which are normally responsible
for detoxifying chemicals that come

00:37:28.000 --> 00:37:32.000
into our body.
And therefore,

00:37:32.000 --> 00:37:36.000
many of the drugs that come into
our bodies are with greater or lesser

00:37:36.000 --> 00:37:40.000
speed altered into something else,
detoxified, and therefore rendered

00:37:40.000 --> 00:37:44.000
innocuous. Now you'll say, well,
you can figure that out too,

00:37:44.000 --> 00:37:48.000
but here's an additional fly in
the ointment. Because we are a

00:37:48.000 --> 00:37:52.000
polymorphic population, because
we humans are genetically

00:37:52.000 --> 00:37:56.000
heterogeneous,
one from the other,

00:37:56.000 --> 00:38:00.000
some of us metabolize a given drug
much more rapidly than others do.

00:38:00.000 --> 00:38:04.000
And here, we have a situation
where potentially, most of us might

00:38:04.000 --> 00:38:08.000
metabolize a drug very quickly,
in which case the physicians would

00:38:08.000 --> 00:38:13.000
want to give us a very high dose of
the drug so that we have enough of

00:38:13.000 --> 00:38:17.000
the drug around for a long enough
period of time to do some effect.

00:38:17.000 --> 00:38:22.000
So let's say that 97% of us are
able to metabolize the drug very

00:38:22.000 --> 00:38:26.000
quickly, and as a consequence,
we're given a very high dosage in

00:38:26.000 --> 00:38:31.000
order to have some effective dosage
reaching the tumor to compensate for

00:38:31.000 --> 00:38:35.000
the fact that much of this drug
is rapidly eliminated by metabolism

00:38:35.000 --> 00:38:40.000
in the liver. It's
inter-converted into another

00:38:40.000 --> 00:38:44.000
chemically innocuous compound.
Well, you'll say, that's good.

00:38:44.000 --> 00:38:48.000
We'll just take a large
dose of that compound,

00:38:48.000 --> 00:38:52.000
but let's think about the other
3% in the population who metabolize

00:38:52.000 --> 00:38:56.000
this compound very slowly.
Like the other 97%, these

00:38:56.000 --> 00:39:00.000
individuals will be given a high
dose of the drug because experience

00:39:00.000 --> 00:39:04.000
shows that in general, most
human beings metabolize a drug

00:39:04.000 --> 00:39:09.000
very quickly. These
individuals metabolize the

00:39:09.000 --> 00:39:13.000
drug very slowly, and what's
going to happen to them?

00:39:13.000 --> 00:39:18.000
Well, they might croak. Why?
Because that drug is going to be

00:39:18.000 --> 00:39:23.000
around in potent biologically active
form for an extended period of time

00:39:23.000 --> 00:39:27.000
in their bodies, and
might have in them a lethal

00:39:27.000 --> 00:39:32.000
outcome. So therefore, we have
to deal with the effects of

00:39:32.000 --> 00:39:37.000
variability in drug metabolism,
variability in metabolism because it

00:39:37.000 --> 00:39:43.000
turns out that different people
metabolize the drug differently and

00:39:43.000 --> 00:39:49.000
that variability in drug metabolism
is vastly greater if you compare the

00:39:49.000 --> 00:39:54.000
way we metabolize drugs to the
way that laboratory mice metabolize

00:39:54.000 --> 00:40:00.000
drugs. Well, you'll say,
why should we care about how

00:40:00.000 --> 00:40:06.000
laboratory mice metabolize
this or that drug?

00:40:06.000 --> 00:40:10.000
Why is it important? The fact
is, the first tryouts of a

00:40:10.000 --> 00:40:15.000
candidate drug are tried out in
laboratory mice where laboratory

00:40:15.000 --> 00:40:20.000
mice are given a tumor, and
they're injected with the drug

00:40:20.000 --> 00:40:25.000
to see whether the tumor begins
to shrink. But if it's the case,

00:40:25.000 --> 00:40:29.000
if the laboratory mice metabolize a
drug in a vastly different way than

00:40:29.000 --> 00:40:34.000
do humans, then the outcome of
working with laboratory mice might

00:40:34.000 --> 00:40:39.000
be enormously misleading.
And these are just some of the

00:40:39.000 --> 00:40:44.000
problems that bedevil
the development of a drug.

00:40:44.000 --> 00:40:50.000
In any case, around 1994, at
a company which was a precursor

00:40:50.000 --> 00:40:55.000
of Novartis, it was
called Ciba-Geigy in Basel,

00:40:55.000 --> 00:41:00.000
Switzerland. They developed a
highly specific and potent anti-Abel,

00:41:00.000 --> 00:41:06.000
low molecular weight compound,
which came to be called Leveck.

00:41:06.000 --> 00:41:10.000
Or in Europe it's called Gleveck.
It's also pronounced Leveck, but

00:41:10.000 --> 00:41:15.000
it's spelled differently. In
fact, it was one of the other

00:41:15.000 --> 00:41:19.000
difficulties of developing
this drug was the following.

00:41:19.000 --> 00:41:24.000
The higher ups in the drug company
who were paying for this research

00:41:24.000 --> 00:41:28.000
wanted on repeated occasion to
scrub this entire drug development

00:41:28.000 --> 00:41:33.000
program. Why? Because
the number of cases of

00:41:33.000 --> 00:41:37.000
chronic myelogenous leukemia overall
worldwide is relatively small.

00:41:37.000 --> 00:41:42.000
How many are in this country
every year? I don't know,

00:41:42.000 --> 00:41:47.000
10 or 15,000. So, the question was,
economically speaking, would the

00:41:47.000 --> 00:41:51.000
relatively small number of cases of
this disease justify their investing

00:41:51.000 --> 00:41:56.000
$1 billion in the development of
the drug. Maybe it would take them a

00:41:56.000 --> 00:42:01.000
generation to get any payback
from their initial investment.

00:42:01.000 --> 00:42:05.000
And so, they tried time after time,
time and again, to shut down this

00:42:05.000 --> 00:42:09.000
development program because it
didn't seem to have any clear,

00:42:09.000 --> 00:42:14.000
long-term economic benefit. Of
course, now we're not talking about

00:42:14.000 --> 00:42:18.000
biology. We're talking about
economics, and rational economics.

00:42:18.000 --> 00:42:23.000
This is not avarice on their part.
A drug company like that cannot go

00:42:23.000 --> 00:42:27.000
on spending $1 billion here and $1
billion there without at one point

00:42:27.000 --> 00:42:32.000
or another leading to a
major financial hemorrhage.

00:42:32.000 --> 00:42:38.000
So, Gleveck turned out to be
highly specific for the Abel kinase,

00:42:38.000 --> 00:42:44.000
and as it turned out, for two
other kinds of kinases as well.

00:42:44.000 --> 00:42:50.000
Another kind of kinase is against a
tyrosine kinase receptor called KIT,

00:42:50.000 --> 00:42:56.000
this is a receptor tyrosine kinase,
and another receptor tyrosine kinase

00:42:56.000 --> 00:43:02.000
called the PDGF receptor,
which we've also encountered in

00:43:02.000 --> 00:43:07.000
passing earlier. These
two other growth factor

00:43:07.000 --> 00:43:12.000
receptors, KIT and the PDGF receptor
also have tyrosine kinase domains.

00:43:12.000 --> 00:43:16.000
They therefore follow this
overall structural plan here,

00:43:16.000 --> 00:43:21.000
and it turns out by evolutionary
quirk that the structures of their

00:43:21.000 --> 00:43:26.000
tyrosine kinase domains are actually
similar in certain ways to the

00:43:26.000 --> 00:43:31.000
tyrosine kinase domain of
Abel, and therefore of BCR Abel.

00:43:31.000 --> 00:43:35.000
So, in fact, they didn't actually
have a totally specific drug which

00:43:35.000 --> 00:43:39.000
would attack only one out of the
90-tyrosine kinases encoded in their

00:43:39.000 --> 00:43:44.000
genome. It attacked three
of the 90-tyrosine kinases,

00:43:44.000 --> 00:43:48.000
the Abel, the KITT, and the
PDGF receptor. And this might,

00:43:48.000 --> 00:43:53.000
on its own, have already proven to
be the death nail for the protein,

00:43:53.000 --> 00:43:57.000
except they began to try it out
for patients, and they saw some

00:43:57.000 --> 00:44:02.000
remarkable responses. It
turned out that the great

00:44:02.000 --> 00:44:08.000
majority of CML patients who were
treated with Gleveck at therapeutic

00:44:08.000 --> 00:44:13.000
concentrations ended up having a
rapid remission of their chronic

00:44:13.000 --> 00:44:18.000
myelogenous leukemia disease,
which ultimately resulted in their

00:44:18.000 --> 00:44:24.000
being outwardly free of the disease.
This is your question of the day.

00:44:45.000 --> 00:44:49.000
So, Gleveck goes into the catalytic
cleft of the Abel tyrosine kinase.

00:44:49.000 --> 00:44:54.000
It blocks the ATP binding site
because keep in mind that these

00:44:54.000 --> 00:44:59.000
enzymes need to grab the gamma
phosphate off of ATP and transfer it

00:44:59.000 --> 00:45:04.000
to a protein substrate, and
it does so because it hydrogen

00:45:04.000 --> 00:45:09.000
bonds to the amino acids which
are lining this catalytic cleft.

00:45:09.000 --> 00:45:12.000
In other words, this
catalytic cleft up here is

00:45:12.000 --> 00:45:16.000
obviously made of amino acids,
and there are hydrogen bonds which

00:45:16.000 --> 00:45:20.000
Gleveck can form with the amino acid
resides that you're lining on both

00:45:20.000 --> 00:45:23.000
sides of the cleft. I should
have brought you a picture

00:45:23.000 --> 00:45:27.000
of that. And, a similar
kind of hydrogen bonding

00:45:27.000 --> 00:45:31.000
can occur with the amino acids that
are aligning the catalytic clefts of

00:45:31.000 --> 00:45:35.000
the PDGF receptor and KIT, and
that hydrogen bonding can occur

00:45:35.000 --> 00:45:39.000
already at concentrations
that are submicromolar,

00:45:39.000 --> 00:45:43.000
less than 10-6 molar, 10-7,
even sometimes 10-8 molar

00:45:43.000 --> 00:45:48.000
under certain conditions. So,
it's a high affinity binding,

00:45:48.000 --> 00:45:52.000
and it's relatively specific.
Only three out of the 90 different

00:45:52.000 --> 00:45:56.000
kinases are bound. We can
do the following kind of

00:45:56.000 --> 00:46:02.000
experiment. If I were
to add Gleveck to cells

00:46:02.000 --> 00:46:10.000
with BCR Abel function, this
is the response that BCR Abel

00:46:10.000 --> 00:46:17.000
would show. Here is the response
that the EGF receptor would show.

00:46:17.000 --> 00:46:25.000
So, if I dose the patient at
this concentration of drug,

00:46:25.000 --> 00:46:33.000
Gleveck will shut down
the BCR Abel protein.

00:46:33.000 --> 00:46:37.000
But it won't shut down the EGF
receptor, which requires vastly

00:46:37.000 --> 00:46:41.000
higher concentrations of drug in
order to shut down its tyrosine

00:46:41.000 --> 00:46:45.000
kinase domain.
And right here,

00:46:45.000 --> 00:46:49.000
we can see what we call selectivity.
The fact that this enzyme responds

00:46:49.000 --> 00:46:53.000
at very log drug concentration,
this enzyme EGF receptor and its

00:46:53.000 --> 00:46:57.000
tyrosine kinase, it's a
growth factor receptor once

00:46:57.000 --> 00:47:01.000
again, requires a vastly higher
concentration drug in order to

00:47:01.000 --> 00:47:04.000
elicit an outcome. So,
what happened to the chronic

00:47:04.000 --> 00:47:08.000
myelogenous leukemia patients.
The great majority of them between

00:47:08.000 --> 00:47:12.000
70-80% had a miraculous
collapse of their disease.

00:47:12.000 --> 00:47:16.000
In most cases, this
disease could be monitored

00:47:16.000 --> 00:47:20.000
microscopically. One
could look for the immature

00:47:20.000 --> 00:47:24.000
myeloid cells in their blood and see
where they were previously present

00:47:24.000 --> 00:47:28.000
in vast numbers. They
were microscopically now

00:47:28.000 --> 00:47:32.000
indetectable (sic). However,
in those patients where the

00:47:32.000 --> 00:47:38.000
disease seemed to collapse, one
could still use the PCR test to

00:47:38.000 --> 00:47:44.000
demonstrate there were residual
cancer cells in their blood.

00:47:44.000 --> 00:47:49.000
How could one do that? Well,
let's imagine that here is the PCR

00:47:49.000 --> 00:47:55.000
Abel fusion protein. So,
here's PCR, and here's Abel

00:47:55.000 --> 00:48:00.000
over here. You can
make PCR primers,

00:48:00.000 --> 00:48:05.000
one of which is specific for a PCR
sequence, and the other of which is

00:48:05.000 --> 00:48:09.000
specific for an Abel sequence,
and the only time that you'll get a

00:48:09.000 --> 00:48:14.000
PCR product is if these two
sequences exist on the same

00:48:14.000 --> 00:48:19.000
messenger RNA molecule that's
reverse transcribed into a CDNA.

00:48:19.000 --> 00:48:23.000
You could even do this genomic DNA
as well, and so one can specifically

00:48:23.000 --> 00:48:28.000
detect using this PCR test the
presence of cells which have this

00:48:28.000 --> 00:48:33.000
chromosomal translocation. If
one of the PCR primers is against

00:48:33.000 --> 00:48:37.000
BCR and the other is Abel, and
the distances between these two

00:48:37.000 --> 00:48:42.000
primers is not too far away, not
more than, let's say, kilobase,

00:48:42.000 --> 00:48:46.000
so you get rather
efficient PCR amplification.

00:48:46.000 --> 00:48:51.000
So, it turned out that the great
majority of patients who were

00:48:51.000 --> 00:48:55.000
cytologically cured,
cytology means a cytological

00:48:55.000 --> 00:49:00.000
analysis represents what you
see through in microscopes.

00:49:00.000 --> 00:49:04.000
So these patients, if you
looked at a smear of their

00:49:04.000 --> 00:49:08.000
blood, cytologically they were
cured. But if you used PCR analysis,

00:49:08.000 --> 00:49:12.000
which is far more sensitive, one
could detect residual cancer cells

00:49:12.000 --> 00:49:17.000
that might be present in one out of
105 or one out of 106 cells moving

00:49:17.000 --> 00:49:21.000
around in circulation, which
are almost invisible if you're

00:49:21.000 --> 00:49:25.000
looking through a very complex
mixture of cells through the light

00:49:25.000 --> 00:49:29.000
microscope. And so, what
happened was that patients

00:49:29.000 --> 00:49:34.000
began to relapse, and after
a period of several years,

00:49:34.000 --> 00:49:38.000
a number of patients began to show
a restoration of their CML condition.

00:49:38.000 --> 00:49:43.000
In fact, in one recent European
study, indicates that between 10-12%

00:49:43.000 --> 00:49:48.000
of the CML patients who were treated
with Gleveck relapsed every year.

00:49:48.000 --> 00:49:52.000
What do I mean by relapse? I mean
they show a resurgence of their

00:49:52.000 --> 00:49:57.000
disease. The disease comes back
to life, and they once again

00:49:57.000 --> 00:50:02.000
have the disease. And
interestingly enough,

00:50:02.000 --> 00:50:08.000
if one now looks at their
cancer cells, what do you see?

00:50:08.000 --> 00:50:14.000
In virtually every case you see
some alteration in the BCR Abel

00:50:14.000 --> 00:50:19.000
protein. In the great majority of
instances, you see point mutations

00:50:19.000 --> 00:50:25.000
that affect amino acid
residues lining the cavity here,

00:50:25.000 --> 00:50:31.000
lining the cavity of
the Abel kinase protein.

00:50:31.000 --> 00:50:35.000
Those amino acid substitutions do
not compromise the tyrosine kinase

00:50:35.000 --> 00:50:40.000
activity of this enzyme. But
they do prevent Gleveck from

00:50:40.000 --> 00:50:44.000
binding, and as a consequence, now
you begin to have patients whose

00:50:44.000 --> 00:50:49.000
tumors are no longer responsive to
Gleveck. And what's happened now is

00:50:49.000 --> 00:50:53.000
one has developed a new generation
of compounds which binds into this

00:50:53.000 --> 00:50:58.000
pocket even in the presence of these
amino acid substitutions to retreat

00:50:58.000 --> 00:51:03.000
these patients. See
you on Wednesday.