1
00:00:01,000 --> 00:00:05,000
Thanksgivings. We're
talking today about rational

2
00:00:05,000 --> 00:00:10,000
medicine, and really what we're
talking about is an understanding of

3
00:00:10,000 --> 00:00:16,000
the molecular biology of disease has
actually helped to revolutionize the

4
00:00:16,000 --> 00:00:21,000
new science of therapeutic medicine.
And here, more often than not, the

5
00:00:21,000 --> 00:00:27,000
discussions are focused
around cancer. And so,

6
00:00:27,000 --> 00:00:32,000
I will therefore talk
about an interesting story,

7
00:00:32,000 --> 00:00:37,000
vis-à-vis the modern treatment of
cancer, and how our understanding of

8
00:00:37,000 --> 00:00:43,000
the molecular biology of the disease
really helps in developing radically

9
00:00:43,000 --> 00:00:48,000
new kinds of therapies.
By way of background,

10
00:00:48,000 --> 00:00:53,000
let's just mention that most of the
chemotherapeutics that we use today

11
00:00:53,000 --> 00:00:58,000
to treat cancer were developed over
the last 40-50 years at a time when

12
00:00:58,000 --> 00:01:03,000
the molecular and biochemical
defects inside cancer cells

13
00:01:03,000 --> 00:01:07,000
were totally obscure.
And therefore,

14
00:01:07,000 --> 00:01:11,000
to the extent that one
developed chemotherapeutics,

15
00:01:11,000 --> 00:01:14,000
they were developed simply
empirically, trial and error.

16
00:01:14,000 --> 00:01:18,000
For example, some of the most
effective chemotherapeutics against

17
00:01:18,000 --> 00:01:22,000
childhood leukemia
are alkylating agents,

18
00:01:22,000 --> 00:01:30,000
which attach methyl
and ethyl groups

19
00:01:30,000 --> 00:01:35,000
to target molecules inside cells.
And their utility in cancer was

20
00:01:35,000 --> 00:01:41,000
first discerned because of
an explosion in a container.

21
00:01:41,000 --> 00:01:46,000
I think it was in a ship off Naples
in World War II where a bin of

22
00:01:46,000 --> 00:01:51,000
alkylating agents was dispersed.
Many people were exposed to it, and

23
00:01:51,000 --> 00:01:57,000
these people, as a consequence
of that, came down with what's

24
00:01:57,000 --> 00:02:02,000
called leucopoenia. Poenia
generally means a depression,

25
00:02:02,000 --> 00:02:07,000
in this case, a depression
of their white blood cells.

26
00:02:07,000 --> 00:02:12,000
Such alkylating agents had actually
been used during the First World War

27
00:02:12,000 --> 00:02:17,000
in gas warfare because
during the First World War,

28
00:02:17,000 --> 00:02:22,000
one used so-called mustard gases,
which was a very effective way, even

29
00:02:22,000 --> 00:02:27,000
more effective than artillery
in killing vast numbers

30
00:02:27,000 --> 00:02:32,000
of enemy soldiers. And,
somebody noticed this

31
00:02:32,000 --> 00:02:36,000
leucopoenia in 1946-47 as a
consequence of inadvertent exposure

32
00:02:36,000 --> 00:02:41,000
to these alkylating agents,
which became dispersed as a gas.

33
00:02:41,000 --> 00:02:45,000
And about five years later,
somebody made the logical leap that

34
00:02:45,000 --> 00:02:49,000
if these agents were able to
suppress normal white blood

35
00:02:49,000 --> 00:02:54,000
concentrations that perhaps they
might also be effective against what

36
00:02:54,000 --> 00:02:58,000
seemed to be ostensibly a related
problem, which is the problem

37
00:02:58,000 --> 00:03:03,000
of leukemia. And keep
in mind that when we talk

38
00:03:03,000 --> 00:03:08,000
about leukemia, the suffix
-emia refers to blood

39
00:03:08,000 --> 00:03:13,000
generally, and leuk- once
again refers to white blood,

40
00:03:13,000 --> 00:03:18,000
i.e. an excess of white blood
cells in the blood. And so,

41
00:03:18,000 --> 00:03:22,000
through this accidental discovery,
one began to develop alkylating

42
00:03:22,000 --> 00:03:27,000
agents that turned out to be
extremely successful in treating,

43
00:03:27,000 --> 00:03:32,000
and often curing, childhood
leukemias, most notably acute

44
00:03:32,000 --> 00:03:37,000
lymphocytic leukemia, which
turns out to be very sensitive

45
00:03:37,000 --> 00:03:42,000
to this and other related agents.
So, this is a very common form of

46
00:03:42,000 --> 00:03:46,000
childhood leukemia, which is
now actually cured in 60 or

47
00:03:46,000 --> 00:03:50,000
70% of the children who were treated,
which would have been unheard of

48
00:03:50,000 --> 00:03:55,000
half a century ago. But I
return to what I said before,

49
00:03:55,000 --> 00:03:59,000
which is that this kind of treatment
was developed in the face of total

50
00:03:59,000 --> 00:04:04,000
ignorance concerning the nature of
the disease, the molecular defects

51
00:04:04,000 --> 00:04:08,000
that were present in the disease,
and that were responsible for the

52
00:04:08,000 --> 00:04:12,000
runaway, can you still hear me OK,
were responsible for the runaway

53
00:04:12,000 --> 00:04:17,000
proliferation of the cancer
cells. So having said that,

54
00:04:17,000 --> 00:04:22,000
I want to go to a different kind of
leukemia, and this is called chronic

55
00:04:22,000 --> 00:04:28,000
myelogenous leukemia, to
give you an indication of the

56
00:04:28,000 --> 00:04:33,000
path of discovery that led from
its original description to the

57
00:04:33,000 --> 00:04:39,000
development of rather
successful treatments.

58
00:04:39,000 --> 00:04:45,000
So, chronic myelogenous leukemia,
I mentioned the prefix myelo- last

59
00:04:45,000 --> 00:04:51,000
time or the time before referring to
bone marrow, and this is a leukemia

60
00:04:51,000 --> 00:04:57,000
of cells coming from the bone marrow
from the myeloid cells in the bone

61
00:04:57,000 --> 00:05:03,000
marrow, which are the precursors
of things like macrophages

62
00:05:03,000 --> 00:05:08,000
and granulocytes. So,
these are cells which are

63
00:05:08,000 --> 00:05:12,000
playing an important role
in the immune response,

64
00:05:12,000 --> 00:05:16,000
and during this chronic
myelogenous leukemia disease,

65
00:05:16,000 --> 00:05:21,000
which is called CML, there
could be a period of three or

66
00:05:21,000 --> 00:05:25,000
four years where individuals develop
large numbers of these cells in

67
00:05:25,000 --> 00:05:30,000
their blood stream. And after
a period of about three or

68
00:05:30,000 --> 00:05:34,000
four years, all of a sudden there is
an eruption into what's called blast

69
00:05:34,000 --> 00:05:38,000
crisis. And you may recall I
mentioned the word blast also on one

70
00:05:38,000 --> 00:05:42,000
occasion earlier. This
all fits together in a nice

71
00:05:42,000 --> 00:05:46,000
puzzle. Blast refers to primitive,
embryonic-like cells, and all of a

72
00:05:46,000 --> 00:05:50,000
sudden there is an eruption of
primitive, embryonic-like cells,

73
00:05:50,000 --> 00:05:54,000
less differentiated like these
macrophages and granulocytes,

74
00:05:54,000 --> 00:05:58,000
which until this point had been
present in vastly excessive numbers

75
00:05:58,000 --> 00:06:03,000
in the blood.
There's blast crisis.

76
00:06:03,000 --> 00:06:08,000
This leads to acute myelogenous
leukemia, and death ensues usually

77
00:06:08,000 --> 00:06:13,000
within a year or two, or
that's been traditionally the

78
00:06:13,000 --> 00:06:17,000
case. No one really had any
idea about the possible causative

79
00:06:17,000 --> 00:06:22,000
mechanisms of the disease, and
that allows me to use another

80
00:06:22,000 --> 00:06:27,000
word which you might one day
come across if you should stay in

81
00:06:27,000 --> 00:06:32,000
biomedical research. And
that is the etiologic agents.

82
00:06:32,000 --> 00:06:37,000
When we talk about etiologic agents,
we talk about the agents which are

83
00:06:37,000 --> 00:06:41,000
causally responsible for inducing
a disease. These can be external

84
00:06:41,000 --> 00:06:46,000
agents, or they could
even be internal agents,

85
00:06:46,000 --> 00:06:51,000
molecules inside cells which are
responsible for the creation of the

86
00:06:51,000 --> 00:06:56,000
disease. And the key discovery was
made in 1960 when individuals were

87
00:06:56,000 --> 00:07:01,000
looking at the chromosomal
makeup of the CML cells.

88
00:07:01,000 --> 00:07:05,000
The chromosomal makeup, I'll
use another word just so we

89
00:07:05,000 --> 00:07:09,000
could expand our vocabulary this
morning, the chromosomal makeup is

90
00:07:09,000 --> 00:07:14,000
often called the karyotype, that
is to say the constellation of

91
00:07:14,000 --> 00:07:18,000
chromosomes that one can see
at mitosis under the microscope.

92
00:07:18,000 --> 00:07:22,000
Keep in mind, as we've said before,
that during the interphase of the

93
00:07:22,000 --> 00:07:27,000
cell cycle, chromosomes
are essentially invisible,

94
00:07:27,000 --> 00:07:31,000
but during the metaphase of
mitosis they become condensed,

95
00:07:31,000 --> 00:07:36,000
and on that occasion, individuals
noticed a 9-22 translocation.

96
00:07:36,000 --> 00:07:41,000
So here is chromosome nine normally.
Here's chromosome 22. And as you

97
00:07:41,000 --> 00:07:46,000
may know, the numbering system
with human chromosomes goes from the

98
00:07:46,000 --> 00:07:51,000
largest number one all the
way down to the smallest.

99
00:07:51,000 --> 00:07:57,000
So, this is the smallest,
with the exception of the Y

100
00:07:57,000 --> 00:08:01,000
chromosome. And what
they notice was instead of

101
00:08:01,000 --> 00:08:04,000
seeing this regular chromosomal
array, they noticed instead what

102
00:08:04,000 --> 00:08:07,000
looked very much like a
structure of this sort here, i.e.

103
00:08:07,000 --> 00:08:35,000
a translocation.

104
00:08:35,000 --> 00:08:39,000
And this translocation resulted in
a swapping of sequences between these

105
00:08:39,000 --> 00:08:43,000
two chromosomes.
Note, by the way,

106
00:08:43,000 --> 00:08:47,000
this is reciprocal, i.e. in
the sense that nine donates

107
00:08:47,000 --> 00:08:51,000
something to 22, and 22
donates something to nine.

108
00:08:51,000 --> 00:08:55,000
However, the segments that are
swapped are not necessarily of equal

109
00:08:55,000 --> 00:08:59,000
size. So, it turns out here that
in this case, chromosome nine has

110
00:08:59,000 --> 00:09:03,000
actually gained a lot more
than chromosome 22 gained as a

111
00:09:03,000 --> 00:09:08,000
consequence of this
exchange of genetic segments.

112
00:09:08,000 --> 00:09:12,000
And this 9-22 translocation made
the smallest chromosome even smaller.

113
00:09:12,000 --> 00:09:16,000
So, this was already the smallest
chromosome as I mentioned besides

114
00:09:16,000 --> 00:09:20,000
the smallest autosome, the
smallest non-sex chromosome.

115
00:09:20,000 --> 00:09:24,000
Now it got even smaller because
it lost some of its bulk as a

116
00:09:24,000 --> 00:09:29,000
consequence of this
chromosomal translocation.

117
00:09:29,000 --> 00:09:33,000
And because this discovery
was made in Philadelphia,

118
00:09:33,000 --> 00:09:37,000
it became known as the Philadelphia
chromosome. This is now about 40

119
00:09:37,000 --> 00:09:42,000
years ago, or as it's sometimes
called, PH-1 for reasons,

120
00:09:42,000 --> 00:09:46,000
I don't know why it's called
PH-1 except for Philadelphia.

121
00:09:46,000 --> 00:09:51,000
And, as investigators began to
look at other cases of chronic

122
00:09:51,000 --> 00:09:55,000
myelogenous leukemia,
they discovered that this

123
00:09:55,000 --> 00:10:00,000
translocation was present at
the Philadelphia chromosome most

124
00:10:00,000 --> 00:10:04,000
importantly was identifiable
in virtually all cases,

125
00:10:04,000 --> 00:10:09,000
more than 95% of the cases of
chronic myelogenous leukemia.

126
00:10:09,000 --> 00:10:13,000
And moreover, this chromosome
was present as well in the more

127
00:10:13,000 --> 00:10:18,000
differentiated macrophages and
granulocytes that were present and

128
00:10:18,000 --> 00:10:22,000
circulating in the blood of the CML
patients. And that began to suggest

129
00:10:22,000 --> 00:10:27,000
the notion that there was
a stem cell of some sort,

130
00:10:27,000 --> 00:10:31,000
oligopotential stem cell that
created various kinds of more

131
00:10:31,000 --> 00:10:36,000
differentiated white blood cells
that had sustained this chromosomal

132
00:10:36,000 --> 00:10:41,000
translocation because that's
what it is, a translocalization,

133
00:10:41,000 --> 00:10:46,000
a translocation, that all the cells
of these patients had sustained this

134
00:10:46,000 --> 00:10:51,000
chromosomal translocation.
And that began to suggest the

135
00:10:51,000 --> 00:10:56,000
notion that somehow as a consequence
of a random genetic accident

136
00:10:56,000 --> 00:11:01,000
happening in these people's blood,
this particular chromosome was

137
00:11:01,000 --> 00:11:07,000
repeatedly identified. And
it was with great likelihood

138
00:11:07,000 --> 00:11:13,000
causally or etiologically important
in the genesis of the disease.

139
00:11:13,000 --> 00:11:20,000
But that in itself led nowhere.
One could simply talk about its

140
00:11:20,000 --> 00:11:27,000
association until work from
a totally unrelated area,

141
00:11:27,000 --> 00:11:33,000
which is to say the study of
retroviruses discovered Abelson

142
00:11:33,000 --> 00:11:39,000
murine leukemia virus. And
Abelson was named after the

143
00:11:39,000 --> 00:11:44,000
fellow, Herb Abelson, who
first discovered it at NIH and

144
00:11:44,000 --> 00:11:48,000
undertook its molecular
characterization here in our own

145
00:11:48,000 --> 00:11:53,000
cancer center, and Abelson
discovered that this

146
00:11:53,000 --> 00:11:58,000
virus which he studied carried the
ends of the murine leukemia virus,

147
00:11:58,000 --> 00:12:03,000
which was a parental virus.
It was as hybrid virus.

148
00:12:03,000 --> 00:12:08,000
And into the middle of it,
Abelson leukemia virus has acquired

149
00:12:08,000 --> 00:12:13,000
a cellular proto-oncogene,
which it had activated into an

150
00:12:13,000 --> 00:12:18,000
oncogene. And therefore, here
we have a situation where a

151
00:12:18,000 --> 00:12:23,000
cellular gene like sarc in the
case of Rous Sarcoma Virus has been

152
00:12:23,000 --> 00:12:28,000
activated. This became called
ABL for obvious reasons.

153
00:12:28,000 --> 00:12:33,000
And this gene, it turned out,
was critically important in

154
00:12:33,000 --> 00:12:38,000
understanding hwo the chromosomal
translocation led to cancer.

155
00:12:38,000 --> 00:12:43,000
In fact, if one infected mice with
a retrovirus carrying this genome,

156
00:12:43,000 --> 00:12:48,000
this is just to indicate the
fact that the repeat ends,

157
00:12:48,000 --> 00:12:53,000
the long terminal repeat
ends of this provirus,

158
00:12:53,000 --> 00:12:58,000
they occur twice at the ends of this
retrovirus. If one infected a mouse

159
00:12:58,000 --> 00:13:03,000
with the Abelson virus,
out came a disease which was

160
00:13:03,000 --> 00:13:08,000
superficially similar at least
to chronic myelogenous leukemia.

161
00:13:08,000 --> 00:13:13,000
And that began a search,
then, for the chromosomal

162
00:13:13,000 --> 00:13:18,000
localization of the
Abel proto-oncogene.

163
00:13:18,000 --> 00:13:23,000
And what was discovered
subsequently, fascinatingly enough,

164
00:13:23,000 --> 00:13:29,000
was that the Abel proto-oncogene was
right at the break point between two

165
00:13:29,000 --> 00:13:34,000
chromosomes, nine and 22. And
what happened as a consequence

166
00:13:34,000 --> 00:13:38,000
of this translocation, and
the resulting fusion of this

167
00:13:38,000 --> 00:13:42,000
chromosome with this chromosome
was the creation of a fuse gene,

168
00:13:42,000 --> 00:13:46,000
a hybrid gene that now carried the
reading frames of two previously

169
00:13:46,000 --> 00:13:50,000
unconnected gene, one
on chromosome nine,

170
00:13:50,000 --> 00:13:54,000
and one on chromosome 22.
Here's the normal, Abel protein.

171
00:13:54,000 --> 00:13:58,000
It's called C Abel, meaning the
cellular or the normal form of Abel.

172
00:13:58,000 --> 00:14:02,000
And you see it up here. It's
shown in a very schematic way.

173
00:14:02,000 --> 00:14:06,000
And here's a second protein which
is encoded on the other chromosome.

174
00:14:06,000 --> 00:14:11,000
So, Abel is encoded here, and the
other gene, which is called BCR is

175
00:14:11,000 --> 00:14:15,000
encoded here, and as a
consequence of the translocation,

176
00:14:15,000 --> 00:14:19,000
Abel is encoded here. BCR is
encoded here. As a consequence of

177
00:14:19,000 --> 00:14:24,000
the translocation, one now
has not only the fusion of

178
00:14:24,000 --> 00:14:28,000
chromosomal segments. But
one has the fusion of the

179
00:14:28,000 --> 00:14:33,000
reading frames of two
previously unlinked genes.

180
00:14:33,000 --> 00:14:38,000
And here, one creates as a
consequence of these fusions,

181
00:14:38,000 --> 00:14:43,000
any one of a series of three
quite distinct fusion proteins,

182
00:14:43,000 --> 00:14:48,000
which do not naturally
preexist in the normal cell.

183
00:14:48,000 --> 00:14:53,000
And there shown here is P-1
85, P-2 10, and P-2 30. These

184
00:14:53,000 --> 00:14:58,000
translocations allow different
parts of a second gene called BCR.

185
00:14:58,000 --> 00:15:02,000
BCR refers to breakpoint cluster
region. The area of the point of

186
00:15:02,000 --> 00:15:06,000
fusion is called the breakpoint
between the two genes.

187
00:15:06,000 --> 00:15:10,000
So, the point where each gene is
cut and fused with the other is

188
00:15:10,000 --> 00:15:14,000
called the breakpoint. And
it turns out that within the

189
00:15:14,000 --> 00:15:18,000
region of the chromosome where BCR
maps, there's actually three sites

190
00:15:18,000 --> 00:15:22,000
at which the fusion can occur.
If you look carefully at this

191
00:15:22,000 --> 00:15:26,000
diagram, you see that there's
differing extents of the BCR protein,

192
00:15:26,000 --> 00:15:30,000
which can be contributed
to the fusion protein.

193
00:15:30,000 --> 00:15:35,000
And, what this says, in
effect, is the following,

194
00:15:35,000 --> 00:15:40,000
that here, let's just refer
to this diagram right up here.

195
00:15:40,000 --> 00:15:46,000
Notice, by the way, in all three
of these, that the Abel protein is

196
00:15:46,000 --> 00:15:51,000
present at the C terminal end of the
protein. The BCR is present at the

197
00:15:51,000 --> 00:15:57,000
end terminal end. So,
here's the BCR gene.

198
00:15:57,000 --> 00:16:01,000
Here's the Abel gene down here.
And what investigators found is that

199
00:16:01,000 --> 00:16:05,000
there could be a break at
this part of the BCR gene,

200
00:16:05,000 --> 00:16:09,000
at this part of the BCR gene,
or at this part of the BCR gene,

201
00:16:09,000 --> 00:16:13,000
resulting always in the
fusion of Abel, to one, or two,

202
00:16:13,000 --> 00:16:17,000
or three different kinds
of BCR proteins. And,

203
00:16:17,000 --> 00:16:21,000
breakpoint cluster region signified
the fact that there was a whole

204
00:16:21,000 --> 00:16:25,000
cluster of sites in the previously
existing BCR gene to which this

205
00:16:25,000 --> 00:16:29,000
fusion could take place,
resulting, if the break occurred

206
00:16:29,000 --> 00:16:33,000
here, the breakpoint occurred
here, and BCR to get the longer one.

207
00:16:33,000 --> 00:16:37,000
Here you get the medium-sized one;
here you'd get the shortest one.

208
00:16:37,000 --> 00:16:41,000
And, interestingly enough,
as one explored virtually,

209
00:16:41,000 --> 00:16:45,000
other kinds of different leukemias,
one could see different of these

210
00:16:45,000 --> 00:16:49,000
fusion proteins that were produced.
Here's chronic, myelogenous

211
00:16:49,000 --> 00:16:53,000
leukemia, which I talked to
you about before. Here is acute

212
00:16:53,000 --> 00:16:57,000
lymphocytic leukemia, and
here's chronic and neutrophylic

213
00:16:57,000 --> 00:17:01,000
leukemia, three different kinds
of leukemia. We don't have to worry

214
00:17:01,000 --> 00:17:05,000
about the details of these diseases,
aside from the fact to say that the

215
00:17:05,000 --> 00:17:09,000
structure of this fusion protein
encourages the outgrowth of

216
00:17:09,000 --> 00:17:13,000
different kinds of stem cells in
the bone marrow, which in turn create

217
00:17:13,000 --> 00:17:17,000
three different kinds of diseases.
Most importantly for our discussion

218
00:17:17,000 --> 00:17:21,000
was an attempt to understand the
nature of the resulting fusion

219
00:17:21,000 --> 00:17:25,000
protein, which as a consequence
of this fusion caused by the

220
00:17:25,000 --> 00:17:29,000
chromosomal translocation now
clearly acquired biological powers

221
00:17:29,000 --> 00:17:33,000
that did not preexist in either
of the two parental proteins.

222
00:17:33,000 --> 00:17:37,000
These various notations here
indicate a whole series of different

223
00:17:37,000 --> 00:17:41,000
functions which are associated
with the Abel protein,

224
00:17:41,000 --> 00:17:45,000
and alternatively with the BCR
protein. And we don't need to get

225
00:17:45,000 --> 00:17:49,000
into them, except to say that each
one of these different names here

226
00:17:49,000 --> 00:17:53,000
allows the protein on its own to
associate with other proteins and do

227
00:17:53,000 --> 00:17:58,000
activated downstream
signaling cascade.

228
00:17:58,000 --> 00:18:03,000
What's most important about our
discussion is the realization that

229
00:18:03,000 --> 00:18:09,000
this SH-1 domain, indicated
here, SH-1 refers to the

230
00:18:09,000 --> 00:18:15,000
sarcomology domain, equals
sarcomology, equals a

231
00:18:15,000 --> 00:18:21,000
tyrosine kinase. And
therefore, what one has here is

232
00:18:21,000 --> 00:18:27,000
a protein, which is much
more elaborate than sarc,

233
00:18:27,000 --> 00:18:33,000
has vastly more
signaling capabilities,

234
00:18:33,000 --> 00:18:37,000
by virtue of the fact that these
different domains that are indicated

235
00:18:37,000 --> 00:18:42,000
here allow the resulting fusion
protein to grab hold of a whole

236
00:18:42,000 --> 00:18:47,000
bunch of different signally partners
so that it can send out a diverse

237
00:18:47,000 --> 00:18:52,000
array of downstream activating
signals. If one examined the

238
00:18:52,000 --> 00:18:57,000
structure of the SH-1 domain,
it had a tyrosine kinase activity

239
00:18:57,000 --> 00:19:02,000
very much like sarc,
and most importantly,

240
00:19:02,000 --> 00:19:07,000
if one introduced this fusion
protein into a retrovirus,

241
00:19:07,000 --> 00:19:12,000
now instead of Abel, one could
make a BCR Abel fusion protein.

242
00:19:12,000 --> 00:19:16,000
One could put this into a retrovirus
as before, just like up here.

243
00:19:16,000 --> 00:19:21,000
One could infect mice with it,
and now get out of a disease which

244
00:19:21,000 --> 00:19:25,000
was indistinguishable, in
essence, from chronic myelogenous

245
00:19:25,000 --> 00:19:30,000
leukemia in humans. If one
put a subtle point mutation

246
00:19:30,000 --> 00:19:34,000
in the tyrosine kinase domain, all
of the able protein, here is the

247
00:19:34,000 --> 00:19:38,000
tyrosine kinase domain, SH-1,
up here. Here, we see the

248
00:19:38,000 --> 00:19:43,000
tyrosine kinase domains represented
in the three different fusion

249
00:19:43,000 --> 00:19:47,000
proteins. Keep in mind SH-1 is
always the tyrosine kinase domain.

250
00:19:47,000 --> 00:19:51,000
If one put a subtle, inactivating
point mutation in the tyrosine

251
00:19:51,000 --> 00:19:56,000
kinase domain, that
immediately wiped out all

252
00:19:56,000 --> 00:20:00,000
biological powers of creating
leukemias on the part of this

253
00:20:00,000 --> 00:20:04,000
retrovirus here, or any
one of the other closely

254
00:20:04,000 --> 00:20:09,000
related kinds of fusion
proteins. And therefore,

255
00:20:09,000 --> 00:20:13,000
that indicated that the tyrosine
kinase domain indicated right here

256
00:20:13,000 --> 00:20:17,000
was really critical to creating the
tumor, and that any effects on its

257
00:20:17,000 --> 00:20:21,000
tyrosine kinase signaling
ability would, in the end,

258
00:20:21,000 --> 00:20:25,000
result in the collapse of the tumor,
or the inability of the resulting

259
00:20:25,000 --> 00:20:30,000
retrovirus to
actually create cancer.

260
00:20:30,000 --> 00:20:34,000
And so, now one had,
really for the first time,

261
00:20:34,000 --> 00:20:38,000
a clear demonstration of how a
commonly occurring human cancer,

262
00:20:38,000 --> 00:20:42,000
chronic myelogenous leukemia,
is unfortunately not so rare,

263
00:20:42,000 --> 00:20:46,000
could arise as a consequence of some
random, chromosomal translocation

264
00:20:46,000 --> 00:20:50,000
event. You might ask, why
does one always get this

265
00:20:50,000 --> 00:20:54,000
particular kind of translocation?
Well, the answer is, we don't

266
00:20:54,000 --> 00:20:58,000
really know. It would almost seem
as if there's a homing device which

267
00:20:58,000 --> 00:21:02,000
causes this fragment and this
fragment to target each other and to

268
00:21:02,000 --> 00:21:07,000
exchange one another.
It's probably not the case.

269
00:21:07,000 --> 00:21:11,000
What probably happens is that
chromosomal translocations take

270
00:21:11,000 --> 00:21:15,000
place rather randomly within the
bone marrow, and on rare occasion

271
00:21:15,000 --> 00:21:19,000
there is a chromosomal translocation
that creates exactly this kind of

272
00:21:19,000 --> 00:21:23,000
fusion. And this kind of fusion,
in turn, is what's responsible for

273
00:21:23,000 --> 00:21:27,000
creating this fusion protein,
and this fusion protein in turn

274
00:21:27,000 --> 00:21:31,000
creates the outgrowth of this CML,
the chronic myelogenous leukemia

275
00:21:31,000 --> 00:21:36,000
disease. So what that
means is really that a

276
00:21:36,000 --> 00:21:40,000
randomly occurring chromosomal
translocation on rare occasion hits

277
00:21:40,000 --> 00:21:44,000
a genetic jackpot, and the
cell which happens to have

278
00:21:44,000 --> 00:21:48,000
acquired this kind of chromosomal
translocation now begins to

279
00:21:48,000 --> 00:21:52,000
proliferate wildly, creating
first chronic myelogenous

280
00:21:52,000 --> 00:21:56,000
leukemia, and then subsequently
erupting into a subsequent acute

281
00:21:56,000 --> 00:22:00,000
phase where there are seemingly
additional genetic alterations

282
00:22:00,000 --> 00:22:04,000
beyond this chromosomal
translocation that conspire with the

283
00:22:04,000 --> 00:22:08,000
initially present chromosomal
translocation to create a very

284
00:22:08,000 --> 00:22:12,000
aggressive disease which
rapidly leads to the death of the

285
00:22:12,000 --> 00:22:16,000
leukemia patient. That
offered, in principle,

286
00:22:16,000 --> 00:22:21,000
an attractive way of beginning to
develop an anti-cancer therapeutic

287
00:22:21,000 --> 00:22:26,000
because what one might imagine was
that one could develop a tyrosine

288
00:22:26,000 --> 00:22:38,000
kinase inhibitor.

289
00:22:38,000 --> 00:22:42,000
Now keep in mind that tyrosine
kinases are a class of enzymes which

290
00:22:42,000 --> 00:22:46,000
attach phosphate groups onto
the tyrosine residues of various

291
00:22:46,000 --> 00:22:51,000
substrate proteins. And
keep in mind as well the fact

292
00:22:51,000 --> 00:22:55,000
that we drew a series of growth
factor receptors which have tyrosine

293
00:22:55,000 --> 00:23:00,000
kinase domains in them. And
I'm drawing the tyrosine kinase

294
00:23:00,000 --> 00:23:06,000
domains here like this,
that when these growth factor

295
00:23:06,000 --> 00:23:11,000
receptors become activated,
they attach phosphate groups onto

296
00:23:11,000 --> 00:23:17,000
the tails of one another.
And I'll draw those phosphate

297
00:23:17,000 --> 00:23:22,000
groups like this, i.e. the
binding of ligand or let's

298
00:23:22,000 --> 00:23:28,000
say epidermal growth factor ligand
or plate ligand causes the two

299
00:23:28,000 --> 00:23:33,000
receptors, which are normally
mobilized in the plasma membrane to

300
00:23:33,000 --> 00:23:39,000
come together to
transphosphorylate one another,

301
00:23:39,000 --> 00:23:44,000
and having done so, to acquire
actively signaling powers,

302
00:23:44,000 --> 00:23:49,000
because once these phosphates become
attached, they now represent sites

303
00:23:49,000 --> 00:23:54,000
where other molecules can anchor
themselves and send out downstream

304
00:23:54,000 --> 00:23:59,000
signals. In fact, there
are altogether 90 different

305
00:23:59,000 --> 00:24:05,000
tyrosine kinases encoded
in the human genome.

306
00:24:05,000 --> 00:24:09,000
And so, to the extent that
these tyrosine kinases become

307
00:24:09,000 --> 00:24:13,000
hyperactivated in various
kinds of human cancers,

308
00:24:13,000 --> 00:24:17,000
this represents in principle a
very attractive way of developing an

309
00:24:17,000 --> 00:24:21,000
anti-cancer therapeutic. But
let's think about the problems

310
00:24:21,000 --> 00:24:25,000
that are inherent in such
a compound. First of all,

311
00:24:25,000 --> 00:24:29,000
if one wants to develop
an anti-cancer therapeutic,

312
00:24:29,000 --> 00:24:34,000
it must be reasonably specific
for the Abelson tyrosine kinase,

313
00:24:34,000 --> 00:24:38,000
and not the 89 kinds of tyrosine
kinases that also coexist in the

314
00:24:38,000 --> 00:24:43,000
human genome, and are active,
and apparently responsible for

315
00:24:43,000 --> 00:24:48,000
normal cell metabolism in a whole
variety of normal cell types.

316
00:24:48,000 --> 00:24:53,000
So, one has to begin to think
about the issue of cell activity.

317
00:24:53,000 --> 00:24:58,000
How can one possibly make a
low molecular weight compound,

318
00:24:58,000 --> 00:25:03,000
which is selectively able to
inactivate the Abelson tyrosine

319
00:25:03,000 --> 00:25:08,000
kinase as indicated
here, the SH-1 group,

320
00:25:08,000 --> 00:25:11,000
but doesn't disturb a whole variety
of other tyrosine kinases that are

321
00:25:11,000 --> 00:25:15,000
responsible for other normal
physiological mechanisms.

322
00:25:15,000 --> 00:25:19,000
Well, you'll say that's pretty
easy. We have 90 different genes.

323
00:25:19,000 --> 00:25:23,000
Each of the 90 different
genes makes a distinct protein,

324
00:25:23,000 --> 00:25:27,000
and these proteins should be
very different. And therefore,

325
00:25:27,000 --> 00:25:31,000
if one can, in fact, if one does the
three-dimensional structure of these

326
00:25:31,000 --> 00:25:35,000
proteins, all the tyrosine
kinases look quite similar.

327
00:25:35,000 --> 00:25:40,000
They have a biload structure.
Here is the active site of the

328
00:25:40,000 --> 00:25:46,000
enzyme. That is to say, in
here is the catalytic cleft,

329
00:25:46,000 --> 00:25:51,000
the site where the actual catalysis
takes place, the site where the

330
00:25:51,000 --> 00:25:57,000
gamma phosphate of ATP is taken from
the ATP and attached to a substrate

331
00:25:57,000 --> 00:26:03,000
protein to the hydroxyl of a
tyrosine of a protein that's about

332
00:26:03,000 --> 00:26:07,000
to be phosphorylated. So,
you just make a low moleculoid

333
00:26:07,000 --> 00:26:11,000
chemical that's specific for the
tyrosine kinase domain of the Abel

334
00:26:11,000 --> 00:26:15,000
protein. And when I draw
this biload structure,

335
00:26:15,000 --> 00:26:18,000
this biload structure is carried
here within the SH-1 domain right

336
00:26:18,000 --> 00:26:22,000
here. So, this has a biload
structure. It's obviously not

337
00:26:22,000 --> 00:26:26,000
indicated here in this
very schematic drawing.

338
00:26:26,000 --> 00:26:30,000
The problem with
that is the following.

339
00:26:30,000 --> 00:26:33,000
All of the SH-1 domains, all
of the tyrosine kinase domains

340
00:26:33,000 --> 00:26:37,000
are evolutionarily closely
related to one another.

341
00:26:37,000 --> 00:26:41,000
They're all derived from the
precursors of the tyrosine kinase

342
00:26:41,000 --> 00:26:45,000
domain that probably existed
maybe 600 or 700 million years ago,

343
00:26:45,000 --> 00:26:49,000
and has as a consequence of gene
duplication been diversified to make

344
00:26:49,000 --> 00:26:53,000
90 different tyrosine kinases.
And if you look under x-ray

345
00:26:53,000 --> 00:26:57,000
crystallography at the three
dimensional structure of all these

346
00:26:57,000 --> 00:27:01,000
tyrosine kinases, they all
pretty much look like this,

347
00:27:01,000 --> 00:27:04,000
i.e. they all have rather similar
catalytic clefts because they

348
00:27:04,000 --> 00:27:08,000
diverge from a common ancestral
protein, and they retain this three

349
00:27:08,000 --> 00:27:12,000
dimensional configuration because
this three dimensional configuration

350
00:27:12,000 --> 00:27:15,000
seems to be important for the
retention of their function.

351
00:27:15,000 --> 00:27:19,000
You could imagine, conversely,
that if there were some descendants

352
00:27:19,000 --> 00:27:23,000
of the ancestral tyrosine kinase
domain that some of them became

353
00:27:23,000 --> 00:27:26,000
mutant and lost its three
dimensional structure.

354
00:27:26,000 --> 00:27:30,000
Those descendant kinases would
lose their ability to phosphorylate

355
00:27:30,000 --> 00:27:34,000
tyrosines on substrate proteins,
and therefore would be eliminated

356
00:27:34,000 --> 00:27:38,000
from the gene pool because
they would be defective.

357
00:27:38,000 --> 00:27:42,000
And that explains the strong
conservatism in the structure of

358
00:27:42,000 --> 00:27:46,000
these 90 different enzymes.
They all look very similar to one

359
00:27:46,000 --> 00:27:50,000
another, and that creates a great
difficulty for the drug developer

360
00:27:50,000 --> 00:27:54,000
because a low molecular weight drug,
which one would like to develop,

361
00:27:54,000 --> 00:27:58,000
that fits in here. So, here
I'll draw a low molecular

362
00:27:58,000 --> 00:28:02,000
weight drug that interacts in a
stereo-specific fashion with the

363
00:28:02,000 --> 00:28:06,000
amino acid residues that
are aligning this pocket,

364
00:28:06,000 --> 00:28:10,000
this catalytic cleft, might bind
and nicely inactivate the tyrosine

365
00:28:10,000 --> 00:28:15,000
kinase domain of Abel.
But at the same time,

366
00:28:15,000 --> 00:28:19,000
it might also bind and inactivate
a whole series of other tyrosine

367
00:28:19,000 --> 00:28:23,000
kinases, and that in turn could
lead to therapeutic disaster.

368
00:28:23,000 --> 00:28:27,000
For instance, if you had
a non-selective agent,

369
00:28:27,000 --> 00:28:31,000
you could treat a chronic
myelogenous leukemia patient with a

370
00:28:31,000 --> 00:28:35,000
low molecular weight inhibitor,
a low molecular weight compound,

371
00:28:35,000 --> 00:28:39,000
which would get into this
pocket of the BCR Abel protein.

372
00:28:39,000 --> 00:28:43,000
But it might similarly get into the
catalytic cleft of the EGF receptor.

373
00:28:43,000 --> 00:28:47,000
And if it shot down the EGF
receptor, it might cause a fatal

374
00:28:47,000 --> 00:28:51,000
diarrhea because after all, the
EGF receptor, I will tell you,

375
00:28:51,000 --> 00:28:56,000
is needed to maintain the structure
of the epithelial lining of the

376
00:28:56,000 --> 00:29:00,000
colon. And so, you might
kill the patient simply

377
00:29:00,000 --> 00:29:04,000
because you had deprived the cells
in that person's colon of their

378
00:29:04,000 --> 00:29:08,000
ability to maintain themselves.
There are a whole series of growth

379
00:29:08,000 --> 00:29:12,000
factor receptors that are required
for hematapoeisis that we discussed

380
00:29:12,000 --> 00:29:16,000
last time. And there,
once again, if you had a

381
00:29:16,000 --> 00:29:20,000
nonselective compound, which
got into the domain of one of

382
00:29:20,000 --> 00:29:23,000
the growth factor receptors that
is responsible for hematapoeisis,

383
00:29:23,000 --> 00:29:27,000
you might shut down the entire
bone marrow, and once again kill the

384
00:29:27,000 --> 00:29:31,000
patient. I'm just giving you those
as overly dramatic examples of the

385
00:29:31,000 --> 00:29:35,000
fact that cell activity is an
extremely important consideration in

386
00:29:35,000 --> 00:29:40,000
developing such a drug. The
other thing is affinity for the

387
00:29:40,000 --> 00:29:48,000
target, for the catalytic
cleft that is being targeted.

388
00:29:48,000 --> 00:29:56,000
What do I mean by affinity?
If you look at those response

389
00:29:56,000 --> 00:30:04,000
curves of various compounds,
what you see is the following.

390
00:30:04,000 --> 00:30:12,000
You can draw out a line that looks
like this, a graph that looks like

391
00:30:12,000 --> 00:30:20,000
this, where here we have
log of drug concentration.

392
00:30:20,000 --> 00:30:28,000
And here is 10-4, here,
let's do the other one,

393
00:30:28,000 --> 00:30:36,000
10-8 molar, 10-7
molar, 10-6, 10-5, 10-4.

394
00:30:36,000 --> 00:30:42,000
And this is molar drug concentration.
And here is the percentage of

395
00:30:42,000 --> 00:30:49,000
inhibition. Let's say, for
example, we were able to take

396
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

397
00:30:55,000 --> 00:31:02,000
interested in studying how well its
tyrosine kinase activity responded

398
00:31:02,000 --> 00:31:09,000
to an applied drug that
we developed against it.

399
00:31:09,000 --> 00:31:16,000
So, here's the percentage of
inhibition of tyrosine kinase

400
00:31:16,000 --> 00:31:24,000
activity of BCR Abel protein.
Now, I might be able to develop a

401
00:31:24,000 --> 00:31:32,000
drug whose dose response
curve would look like this.

402
00:31:32,000 --> 00:31:36,000
And you'll say,
well, that's terrific.

403
00:31:36,000 --> 00:31:40,000
That's a drug which shuts down BCR
Abel. We haven't even dealt with

404
00:31:40,000 --> 00:31:44,000
the issue of cell activity, but
let's look at where one begins

405
00:31:44,000 --> 00:31:48,000
to see a dose response right here,
10-5 molar. And if you calculate

406
00:31:48,000 --> 00:31:52,000
back as to how much of the drug
you need to deliver in order to shut

407
00:31:52,000 --> 00:31:56,000
down the BCR Abel protein in a
patient, the size of the pill they'd

408
00:31:56,000 --> 00:32:00,000
have to get would probably
be this big everyday.

409
00:32:00,000 --> 00:32:04,000
So, what you need to do is you need
to be an acceptable range of drug

410
00:32:04,000 --> 00:32:08,000
concentrations is down in
this area here. And therefore,

411
00:32:08,000 --> 00:32:12,000
only until you get a drug which
has a dose response curve that looks

412
00:32:12,000 --> 00:32:16,000
like this, which is two or three
orders of magnitude more potent

413
00:32:16,000 --> 00:32:20,000
where it's able to shut down the
kinase activity already at 10-7 in a,

414
00:32:20,000 --> 00:32:24,000
this is called a
submicromolar concentration.

415
00:32:24,000 --> 00:32:28,000
Micromolar is 10-6. Here
already at a tenth of a

416
00:32:28,000 --> 00:32:32,000
micromolar, 10-7 molar, we're
already getting a shutdown of

417
00:32:32,000 --> 00:32:36,000
the enzyme function.
And if one can do that,

418
00:32:36,000 --> 00:32:40,000
then one might in principle be able
to develop a pill that's this big

419
00:32:40,000 --> 00:32:44,000
and give that to the patient rather
than a pill that's a size of a

420
00:32:44,000 --> 00:32:47,000
football. And by the way, if
you have to make lots of a very

421
00:32:47,000 --> 00:32:51,000
complex, organic molecule through
organic synthesis that has an

422
00:32:51,000 --> 00:32:55,000
affinity of this, it's
also very expensive.

423
00:32:55,000 --> 00:32:58,000
Obviously, if you can make a
compound that's a hundredfold more

424
00:32:58,000 --> 00:33:02,000
potent and requires a hundredfold
less material to deliver to the

425
00:33:02,000 --> 00:33:06,000
patient body, then you might
have some success in treating

426
00:33:06,000 --> 00:33:09,000
the patient. Here's
another issue.

427
00:33:09,000 --> 00:33:13,000
So, we've talked about cell
activity. We've talked about

428
00:33:13,000 --> 00:33:17,000
potency or affinity,
affinity for the substrate or

429
00:33:17,000 --> 00:33:20,000
potency. So, this would
be an acceptable drug.

430
00:33:20,000 --> 00:33:24,000
It works already at molar
concentration where the inflection

431
00:33:24,000 --> 00:33:28,000
point of this curve is. This
is an unacceptable drug at

432
00:33:28,000 --> 00:33:32,000
10-5. We can
also talk about

433
00:33:32,000 --> 00:33:36,000
pharmacokinetics. I want
to give you a feeling for

434
00:33:36,000 --> 00:33:40,000
how complex drug development is,
and why it so rarely succeeds. By

435
00:33:40,000 --> 00:33:44,000
the way, you know how much it costs
to develop a drug that's useful in

436
00:33:44,000 --> 00:33:49,000
the clinic these days and test it
on people? Anybody have any idea?

437
00:33:49,000 --> 00:33:53,000
How much? Yeah. It's
pretty close to $1 billion,

438
00:33:53,000 --> 00:33:57,000
between $900 million and $1 billion.
That's a lot of money. That's more

439
00:33:57,000 --> 00:34:01,000
money than you and I are going
to earn together, all of us

440
00:34:01,000 --> 00:34:06,000
maybe, in a lifetime. OK,
anyhow, pharmacokinetics,

441
00:34:06,000 --> 00:34:10,000
well what's pharmacokinetics?
Glad I asked that question.

442
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

443
00:34:14,000 --> 00:34:18,000
cancer patient? What
happens if the drug is

444
00:34:18,000 --> 00:34:22,000
excreted by the kidneys within
minutes of its being taken,

445
00:34:22,000 --> 00:34:26,000
let's say, either by
injection or orally?

446
00:34:26,000 --> 00:34:32,000
So here we can imagine, let's
talk about drug concentration.

447
00:34:32,000 --> 00:34:38,000
I'll use the word drug
concentration in blood.

448
00:34:38,000 --> 00:34:44,000
And here's time. And here's what
some drugs look like when you give

449
00:34:44,000 --> 00:34:50,000
them, let's say, orally.
Here's what they look like.

450
00:34:50,000 --> 00:34:56,000
So, let's say here's the effective
drug concentration: effective

451
00:34:56,000 --> 00:35:02,000
concentration. And
we know the effective

452
00:35:02,000 --> 00:35:06,000
concentration from doing
measurements like this.

453
00:35:06,000 --> 00:35:10,000
We just measure it, work that out.
So, let's say we develop a drug

454
00:35:10,000 --> 00:35:14,000
which is able to hit the BCR Abel
protein. What are the kinetics with

455
00:35:14,000 --> 00:35:18,000
which the drug becomes
soluble in the blood stream?

456
00:35:18,000 --> 00:35:22,000
And it might look like this,
where I'm drawing here now, this is

457
00:35:22,000 --> 00:35:26,000
one hour. This is two hours.
This is three hours, four hours.

458
00:35:26,000 --> 00:35:30,000
Is that
long enough?

459
00:35:30,000 --> 00:35:34,000
Well, the fact of the matter is,
if you're going to try to kill a

460
00:35:34,000 --> 00:35:38,000
cancer cell, and that's what
the name of this game is,

461
00:35:38,000 --> 00:35:41,000
you want to have it around for
a while because it turns out,

462
00:35:41,000 --> 00:35:45,000
as one learned, the continued
viability of the CML cancer cells of

463
00:35:45,000 --> 00:35:49,000
the leukemia cells was dependent on
the continued firing by the BCR Abel

464
00:35:49,000 --> 00:35:52,000
kinase protein. In
fact, as one learned,

465
00:35:52,000 --> 00:35:56,000
if one shut off firing by the
tyrosine kinase molecule in a

466
00:35:56,000 --> 00:36:00,000
chronic myelogenous leukemia
cell, the cells would implode.

467
00:36:00,000 --> 00:36:04,000
They would undergo apitosis.
So, this began to reveal that in

468
00:36:04,000 --> 00:36:08,000
fact the BCR Abel protein is not
only responsible for forcing these

469
00:36:08,000 --> 00:36:12,000
cells to proliferate, but it
also independently provides

470
00:36:12,000 --> 00:36:16,000
them with anti-apoptotic signal.
It keeps them from falling over the

471
00:36:16,000 --> 00:36:20,000
cliff into apitosis. It
keeps them from killing

472
00:36:20,000 --> 00:36:24,000
themselves, and that's obviously
critical for the ability of this

473
00:36:24,000 --> 00:36:28,000
tumor to proliferate, for the
number of cells to expand in

474
00:36:28,000 --> 00:36:32,000
the body of a patient. It
turns out that if you provide

475
00:36:32,000 --> 00:36:37,000
these cancer cells with an effective
way of shutting down their BCR Abel

476
00:36:37,000 --> 00:36:42,000
protein for 30 or 40 or 50 minutes,
not much happens to them. You need

477
00:36:42,000 --> 00:36:47,000
to deprive them of the drug
for a very long period of time,

478
00:36:47,000 --> 00:36:52,000
well, 15-20 hours, and therefore you
need pharmacokinetics that look like

479
00:36:52,000 --> 00:36:57,000
this. It needs to be present
for an extended period of time,

480
00:36:57,000 --> 00:37:03,000
or even better, let me re-draw
that, even better look like this.

481
00:37:03,000 --> 00:37:06,000
It stays in the blood for
an extended period of time.

482
00:37:06,000 --> 00:37:10,000
Some drugs stay in the
circulation for a long time.

483
00:37:10,000 --> 00:37:13,000
Other drugs stay in the circulation
for a very short period of time.

484
00:37:13,000 --> 00:37:17,000
There's another problem which we
haven't even begun to talk about,

485
00:37:17,000 --> 00:37:21,000
and that is the metabolism of the
drug. It turns out that many drugs

486
00:37:21,000 --> 00:37:24,000
that you give a patient are rapidly
converted by the enzymes and the

487
00:37:24,000 --> 00:37:28,000
liver which are normally responsible
for detoxifying chemicals that come

488
00:37:28,000 --> 00:37:32,000
into our body.
And therefore,

489
00:37:32,000 --> 00:37:36,000
many of the drugs that come into
our bodies are with greater or lesser

490
00:37:36,000 --> 00:37:40,000
speed altered into something else,
detoxified, and therefore rendered

491
00:37:40,000 --> 00:37:44,000
innocuous. Now you'll say, well,
you can figure that out too,

492
00:37:44,000 --> 00:37:48,000
but here's an additional fly in
the ointment. Because we are a

493
00:37:48,000 --> 00:37:52,000
polymorphic population, because
we humans are genetically

494
00:37:52,000 --> 00:37:56,000
heterogeneous,
one from the other,

495
00:37:56,000 --> 00:38:00,000
some of us metabolize a given drug
much more rapidly than others do.

496
00:38:00,000 --> 00:38:04,000
And here, we have a situation
where potentially, most of us might

497
00:38:04,000 --> 00:38:08,000
metabolize a drug very quickly,
in which case the physicians would

498
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

499
00:38:13,000 --> 00:38:17,000
the drug around for a long enough
period of time to do some effect.

500
00:38:17,000 --> 00:38:22,000
So let's say that 97% of us are
able to metabolize the drug very

501
00:38:22,000 --> 00:38:26,000
quickly, and as a consequence,
we're given a very high dosage in

502
00:38:26,000 --> 00:38:31,000
order to have some effective dosage
reaching the tumor to compensate for

503
00:38:31,000 --> 00:38:35,000
the fact that much of this drug
is rapidly eliminated by metabolism

504
00:38:35,000 --> 00:38:40,000
in the liver. It's
inter-converted into another

505
00:38:40,000 --> 00:38:44,000
chemically innocuous compound.
Well, you'll say, that's good.

506
00:38:44,000 --> 00:38:48,000
We'll just take a large
dose of that compound,

507
00:38:48,000 --> 00:38:52,000
but let's think about the other
3% in the population who metabolize

508
00:38:52,000 --> 00:38:56,000
this compound very slowly.
Like the other 97%, these

509
00:38:56,000 --> 00:39:00,000
individuals will be given a high
dose of the drug because experience

510
00:39:00,000 --> 00:39:04,000
shows that in general, most
human beings metabolize a drug

511
00:39:04,000 --> 00:39:09,000
very quickly. These
individuals metabolize the

512
00:39:09,000 --> 00:39:13,000
drug very slowly, and what's
going to happen to them?

513
00:39:13,000 --> 00:39:18,000
Well, they might croak. Why?
Because that drug is going to be

514
00:39:18,000 --> 00:39:23,000
around in potent biologically active
form for an extended period of time

515
00:39:23,000 --> 00:39:27,000
in their bodies, and
might have in them a lethal

516
00:39:27,000 --> 00:39:32,000
outcome. So therefore, we have
to deal with the effects of

517
00:39:32,000 --> 00:39:37,000
variability in drug metabolism,
variability in metabolism because it

518
00:39:37,000 --> 00:39:43,000
turns out that different people
metabolize the drug differently and

519
00:39:43,000 --> 00:39:49,000
that variability in drug metabolism
is vastly greater if you compare the

520
00:39:49,000 --> 00:39:54,000
way we metabolize drugs to the
way that laboratory mice metabolize

521
00:39:54,000 --> 00:40:00,000
drugs. Well, you'll say,
why should we care about how

522
00:40:00,000 --> 00:40:06,000
laboratory mice metabolize
this or that drug?

523
00:40:06,000 --> 00:40:10,000
Why is it important? The fact
is, the first tryouts of a

524
00:40:10,000 --> 00:40:15,000
candidate drug are tried out in
laboratory mice where laboratory

525
00:40:15,000 --> 00:40:20,000
mice are given a tumor, and
they're injected with the drug

526
00:40:20,000 --> 00:40:25,000
to see whether the tumor begins
to shrink. But if it's the case,

527
00:40:25,000 --> 00:40:29,000
if the laboratory mice metabolize a
drug in a vastly different way than

528
00:40:29,000 --> 00:40:34,000
do humans, then the outcome of
working with laboratory mice might

529
00:40:34,000 --> 00:40:39,000
be enormously misleading.
And these are just some of the

530
00:40:39,000 --> 00:40:44,000
problems that bedevil
the development of a drug.

531
00:40:44,000 --> 00:40:50,000
In any case, around 1994, at
a company which was a precursor

532
00:40:50,000 --> 00:40:55,000
of Novartis, it was
called Ciba-Geigy in Basel,

533
00:40:55,000 --> 00:41:00,000
Switzerland. They developed a
highly specific and potent anti-Abel,

534
00:41:00,000 --> 00:41:06,000
low molecular weight compound,
which came to be called Leveck.

535
00:41:06,000 --> 00:41:10,000
Or in Europe it's called Gleveck.
It's also pronounced Leveck, but

536
00:41:10,000 --> 00:41:15,000
it's spelled differently. In
fact, it was one of the other

537
00:41:15,000 --> 00:41:19,000
difficulties of developing
this drug was the following.

538
00:41:19,000 --> 00:41:24,000
The higher ups in the drug company
who were paying for this research

539
00:41:24,000 --> 00:41:28,000
wanted on repeated occasion to
scrub this entire drug development

540
00:41:28,000 --> 00:41:33,000
program. Why? Because
the number of cases of

541
00:41:33,000 --> 00:41:37,000
chronic myelogenous leukemia overall
worldwide is relatively small.

542
00:41:37,000 --> 00:41:42,000
How many are in this country
every year? I don't know,

543
00:41:42,000 --> 00:41:47,000
10 or 15,000. So, the question was,
economically speaking, would the

544
00:41:47,000 --> 00:41:51,000
relatively small number of cases of
this disease justify their investing

545
00:41:51,000 --> 00:41:56,000
$1 billion in the development of
the drug. Maybe it would take them a

546
00:41:56,000 --> 00:42:01,000
generation to get any payback
from their initial investment.

547
00:42:01,000 --> 00:42:05,000
And so, they tried time after time,
time and again, to shut down this

548
00:42:05,000 --> 00:42:09,000
development program because it
didn't seem to have any clear,

549
00:42:09,000 --> 00:42:14,000
long-term economic benefit. Of
course, now we're not talking about

550
00:42:14,000 --> 00:42:18,000
biology. We're talking about
economics, and rational economics.

551
00:42:18,000 --> 00:42:23,000
This is not avarice on their part.
A drug company like that cannot go

552
00:42:23,000 --> 00:42:27,000
on spending $1 billion here and $1
billion there without at one point

553
00:42:27,000 --> 00:42:32,000
or another leading to a
major financial hemorrhage.

554
00:42:32,000 --> 00:42:38,000
So, Gleveck turned out to be
highly specific for the Abel kinase,

555
00:42:38,000 --> 00:42:44,000
and as it turned out, for two
other kinds of kinases as well.

556
00:42:44,000 --> 00:42:50,000
Another kind of kinase is against a
tyrosine kinase receptor called KIT,

557
00:42:50,000 --> 00:42:56,000
this is a receptor tyrosine kinase,
and another receptor tyrosine kinase

558
00:42:56,000 --> 00:43:02,000
called the PDGF receptor,
which we've also encountered in

559
00:43:02,000 --> 00:43:07,000
passing earlier. These
two other growth factor

560
00:43:07,000 --> 00:43:12,000
receptors, KIT and the PDGF receptor
also have tyrosine kinase domains.

561
00:43:12,000 --> 00:43:16,000
They therefore follow this
overall structural plan here,

562
00:43:16,000 --> 00:43:21,000
and it turns out by evolutionary
quirk that the structures of their

563
00:43:21,000 --> 00:43:26,000
tyrosine kinase domains are actually
similar in certain ways to the

564
00:43:26,000 --> 00:43:31,000
tyrosine kinase domain of
Abel, and therefore of BCR Abel.

565
00:43:31,000 --> 00:43:35,000
So, in fact, they didn't actually
have a totally specific drug which

566
00:43:35,000 --> 00:43:39,000
would attack only one out of the
90-tyrosine kinases encoded in their

567
00:43:39,000 --> 00:43:44,000
genome. It attacked three
of the 90-tyrosine kinases,

568
00:43:44,000 --> 00:43:48,000
the Abel, the KITT, and the
PDGF receptor. And this might,

569
00:43:48,000 --> 00:43:53,000
on its own, have already proven to
be the death nail for the protein,

570
00:43:53,000 --> 00:43:57,000
except they began to try it out
for patients, and they saw some

571
00:43:57,000 --> 00:44:02,000
remarkable responses. It
turned out that the great

572
00:44:02,000 --> 00:44:08,000
majority of CML patients who were
treated with Gleveck at therapeutic

573
00:44:08,000 --> 00:44:13,000
concentrations ended up having a
rapid remission of their chronic

574
00:44:13,000 --> 00:44:18,000
myelogenous leukemia disease,
which ultimately resulted in their

575
00:44:18,000 --> 00:44:24,000
being outwardly free of the disease.
This is your question of the day.

576
00:44:45,000 --> 00:44:49,000
So, Gleveck goes into the catalytic
cleft of the Abel tyrosine kinase.

577
00:44:49,000 --> 00:44:54,000
It blocks the ATP binding site
because keep in mind that these

578
00:44:54,000 --> 00:44:59,000
enzymes need to grab the gamma
phosphate off of ATP and transfer it

579
00:44:59,000 --> 00:45:04,000
to a protein substrate, and
it does so because it hydrogen

580
00:45:04,000 --> 00:45:09,000
bonds to the amino acids which
are lining this catalytic cleft.

581
00:45:09,000 --> 00:45:12,000
In other words, this
catalytic cleft up here is

582
00:45:12,000 --> 00:45:16,000
obviously made of amino acids,
and there are hydrogen bonds which

583
00:45:16,000 --> 00:45:20,000
Gleveck can form with the amino acid
resides that you're lining on both

584
00:45:20,000 --> 00:45:23,000
sides of the cleft. I should
have brought you a picture

585
00:45:23,000 --> 00:45:27,000
of that. And, a similar
kind of hydrogen bonding

586
00:45:27,000 --> 00:45:31,000
can occur with the amino acids that
are aligning the catalytic clefts of

587
00:45:31,000 --> 00:45:35,000
the PDGF receptor and KIT, and
that hydrogen bonding can occur

588
00:45:35,000 --> 00:45:39,000
already at concentrations
that are submicromolar,

589
00:45:39,000 --> 00:45:43,000
less than 10-6 molar, 10-7,
even sometimes 10-8 molar

590
00:45:43,000 --> 00:45:48,000
under certain conditions. So,
it's a high affinity binding,

591
00:45:48,000 --> 00:45:52,000
and it's relatively specific.
Only three out of the 90 different

592
00:45:52,000 --> 00:45:56,000
kinases are bound. We can
do the following kind of

593
00:45:56,000 --> 00:46:02,000
experiment. If I were
to add Gleveck to cells

594
00:46:02,000 --> 00:46:10,000
with BCR Abel function, this
is the response that BCR Abel

595
00:46:10,000 --> 00:46:17,000
would show. Here is the response
that the EGF receptor would show.

596
00:46:17,000 --> 00:46:25,000
So, if I dose the patient at
this concentration of drug,

597
00:46:25,000 --> 00:46:33,000
Gleveck will shut down
the BCR Abel protein.

598
00:46:33,000 --> 00:46:37,000
But it won't shut down the EGF
receptor, which requires vastly

599
00:46:37,000 --> 00:46:41,000
higher concentrations of drug in
order to shut down its tyrosine

600
00:46:41,000 --> 00:46:45,000
kinase domain.
And right here,

601
00:46:45,000 --> 00:46:49,000
we can see what we call selectivity.
The fact that this enzyme responds

602
00:46:49,000 --> 00:46:53,000
at very log drug concentration,
this enzyme EGF receptor and its

603
00:46:53,000 --> 00:46:57,000
tyrosine kinase, it's a
growth factor receptor once

604
00:46:57,000 --> 00:47:01,000
again, requires a vastly higher
concentration drug in order to

605
00:47:01,000 --> 00:47:04,000
elicit an outcome. So,
what happened to the chronic

606
00:47:04,000 --> 00:47:08,000
myelogenous leukemia patients.
The great majority of them between

607
00:47:08,000 --> 00:47:12,000
70-80% had a miraculous
collapse of their disease.

608
00:47:12,000 --> 00:47:16,000
In most cases, this
disease could be monitored

609
00:47:16,000 --> 00:47:20,000
microscopically. One
could look for the immature

610
00:47:20,000 --> 00:47:24,000
myeloid cells in their blood and see
where they were previously present

611
00:47:24,000 --> 00:47:28,000
in vast numbers. They
were microscopically now

612
00:47:28,000 --> 00:47:32,000
indetectable (sic). However,
in those patients where the

613
00:47:32,000 --> 00:47:38,000
disease seemed to collapse, one
could still use the PCR test to

614
00:47:38,000 --> 00:47:44,000
demonstrate there were residual
cancer cells in their blood.

615
00:47:44,000 --> 00:47:49,000
How could one do that? Well,
let's imagine that here is the PCR

616
00:47:49,000 --> 00:47:55,000
Abel fusion protein. So,
here's PCR, and here's Abel

617
00:47:55,000 --> 00:48:00,000
over here. You can
make PCR primers,

618
00:48:00,000 --> 00:48:05,000
one of which is specific for a PCR
sequence, and the other of which is

619
00:48:05,000 --> 00:48:09,000
specific for an Abel sequence,
and the only time that you'll get a

620
00:48:09,000 --> 00:48:14,000
PCR product is if these two
sequences exist on the same

621
00:48:14,000 --> 00:48:19,000
messenger RNA molecule that's
reverse transcribed into a CDNA.

622
00:48:19,000 --> 00:48:23,000
You could even do this genomic DNA
as well, and so one can specifically

623
00:48:23,000 --> 00:48:28,000
detect using this PCR test the
presence of cells which have this

624
00:48:28,000 --> 00:48:33,000
chromosomal translocation. If
one of the PCR primers is against

625
00:48:33,000 --> 00:48:37,000
BCR and the other is Abel, and
the distances between these two

626
00:48:37,000 --> 00:48:42,000
primers is not too far away, not
more than, let's say, kilobase,

627
00:48:42,000 --> 00:48:46,000
so you get rather
efficient PCR amplification.

628
00:48:46,000 --> 00:48:51,000
So, it turned out that the great
majority of patients who were

629
00:48:51,000 --> 00:48:55,000
cytologically cured,
cytology means a cytological

630
00:48:55,000 --> 00:49:00,000
analysis represents what you
see through in microscopes.

631
00:49:00,000 --> 00:49:04,000
So these patients, if you
looked at a smear of their

632
00:49:04,000 --> 00:49:08,000
blood, cytologically they were
cured. But if you used PCR analysis,

633
00:49:08,000 --> 00:49:12,000
which is far more sensitive, one
could detect residual cancer cells

634
00:49:12,000 --> 00:49:17,000
that might be present in one out of
105 or one out of 106 cells moving

635
00:49:17,000 --> 00:49:21,000
around in circulation, which
are almost invisible if you're

636
00:49:21,000 --> 00:49:25,000
looking through a very complex
mixture of cells through the light

637
00:49:25,000 --> 00:49:29,000
microscope. And so, what
happened was that patients

638
00:49:29,000 --> 00:49:34,000
began to relapse, and after
a period of several years,

639
00:49:34,000 --> 00:49:38,000
a number of patients began to show
a restoration of their CML condition.

640
00:49:38,000 --> 00:49:43,000
In fact, in one recent European
study, indicates that between 10-12%

641
00:49:43,000 --> 00:49:48,000
of the CML patients who were treated
with Gleveck relapsed every year.

642
00:49:48,000 --> 00:49:52,000
What do I mean by relapse? I mean
they show a resurgence of their

643
00:49:52,000 --> 00:49:57,000
disease. The disease comes back
to life, and they once again

644
00:49:57,000 --> 00:50:02,000
have the disease. And
interestingly enough,

645
00:50:02,000 --> 00:50:08,000
if one now looks at their
cancer cells, what do you see?

646
00:50:08,000 --> 00:50:14,000
In virtually every case you see
some alteration in the BCR Abel

647
00:50:14,000 --> 00:50:19,000
protein. In the great majority of
instances, you see point mutations

648
00:50:19,000 --> 00:50:25,000
that affect amino acid
residues lining the cavity here,

649
00:50:25,000 --> 00:50:31,000
lining the cavity of
the Abel kinase protein.

650
00:50:31,000 --> 00:50:35,000
Those amino acid substitutions do
not compromise the tyrosine kinase

651
00:50:35,000 --> 00:50:40,000
activity of this enzyme. But
they do prevent Gleveck from

652
00:50:40,000 --> 00:50:44,000
binding, and as a consequence, now
you begin to have patients whose

653
00:50:44,000 --> 00:50:49,000
tumors are no longer responsive to
Gleveck. And what's happened now is

654
00:50:49,000 --> 00:50:53,000
one has developed a new generation
of compounds which binds into this

655
00:50:53,000 --> 00:50:58,000
pocket even in the presence of these
amino acid substitutions to retreat

656
00:50:58,000 --> 00:51:03,000
these patients. See
you on Wednesday.