1 00:00:01 --> 00:00:05 Thanksgivings. We're talking today about rational 2 00:00:05 --> 00:00:10 medicine, and really what we're talking about is an understanding of 3 00:00:10 --> 00:00:16 the molecular biology of disease has actually helped to revolutionize the 4 00:00:16 --> 00:00:21 new science of therapeutic medicine. And here, more often than not, the 5 00:00:21 --> 00:00:27 discussions are focused around cancer. And so, 6 00:00:27 --> 00:00:32 I will therefore talk about an interesting story, 7 00:00:32 --> 00:00:37 vis-à-vis the modern treatment of cancer, and how our understanding of 8 00:00:37 --> 00:00:43 the molecular biology of the disease really helps in developing radically 9 00:00:43 --> 00:00:48 new kinds of therapies. By way of background, 10 00:00:48 --> 00:00:53 let's just mention that most of the chemotherapeutics that we use today 11 00:00:53 --> 00:00:58 to treat cancer were developed over the last 40-50 years at a time when 12 00:00:58 --> 00:01:03 the molecular and biochemical defects inside cancer cells 13 00:01:03 --> 00:01:07 were totally obscure. And therefore, 14 00:01:07 --> 00:01:11 to the extent that one developed chemotherapeutics, 15 00:01:11 --> 00:01:14 they were developed simply empirically, trial and error. 16 00:01:14 --> 00:01:18 For example, some of the most effective chemotherapeutics against 17 00:01:18 --> 00:01:22 childhood leukemia are alkylating agents, 18 00:01:22 --> 00:01:30 which attach methyl and ethyl groups 19 00:01:30 --> 00:01:35 to target molecules inside cells. And their utility in cancer was 20 00:01:35 --> 00:01:41 first discerned because of an explosion in a container. 21 00:01:41 --> 00:01:46 I think it was in a ship off Naples in World War II where a bin of 22 00:01:46 --> 00:01:51 alkylating agents was dispersed. Many people were exposed to it, and 23 00:01:51 --> 00:01:57 these people, as a consequence of that, came down with what's 24 00:01:57 --> 00:02:02 called leucopoenia. Poenia generally means a depression, 25 00:02:02 --> 00:02:07 in this case, a depression of their white blood cells. 26 00:02:07 --> 00:02:12 Such alkylating agents had actually been used during the First World War 27 00:02:12 --> 00:02:17 in gas warfare because during the First World War, 28 00:02:17 --> 00:02:22 one used so-called mustard gases, which was a very effective way, even 29 00:02:22 --> 00:02:27 more effective than artillery in killing vast numbers 30 00:02:27 --> 00:02:32 of enemy soldiers. And, somebody noticed this 31 00:02:32 --> 00:02:36 leucopoenia in 1946-47 as a consequence of inadvertent exposure 32 00:02:36 --> 00:02:41 to these alkylating agents, which became dispersed as a gas. 33 00:02:41 --> 00:02:45 And about five years later, somebody made the logical leap that 34 00:02:45 --> 00:02:49 if these agents were able to suppress normal white blood 35 00:02:49 --> 00:02:54 concentrations that perhaps they might also be effective against what 36 00:02:54 --> 00:02:58 seemed to be ostensibly a related problem, which is the problem 37 00:02:58 --> 00:03:03 of leukemia. And keep in mind that when we talk 38 00:03:03 --> 00:03:08 about leukemia, the suffix -emia refers to blood 39 00:03:08 --> 00:03:13 generally, and leuk- once again refers to white blood, 40 00:03:13 --> 00:03:18 i.e. an excess of white blood cells in the blood. And so, 41 00:03:18 --> 00:03:22 through this accidental discovery, one began to develop alkylating 42 00:03:22 --> 00:03:27 agents that turned out to be extremely successful in treating, 43 00:03:27 --> 00:03:32 and often curing, childhood leukemias, most notably acute 44 00:03:32 --> 00:03:37 lymphocytic leukemia, which turns out to be very sensitive 45 00:03:37 --> 00:03:42 to this and other related agents. So, this is a very common form of 46 00:03:42 --> 00:03:46 childhood leukemia, which is now actually cured in 60 or 47 00:03:46 --> 00:03:50 70% of the children who were treated, which would have been unheard of 48 00:03:50 --> 00:03:55 half a century ago. But I return to what I said before, 49 00:03:55 --> 00:03:59 which is that this kind of treatment was developed in the face of total 50 00:03:59 --> 00:04:04 ignorance concerning the nature of the disease, the molecular defects 51 00:04:04 --> 00:04:08 that were present in the disease, and that were responsible for the 52 00:04:08 --> 00:04:12 runaway, can you still hear me OK, were responsible for the runaway 53 00:04:12 --> 00:04:17 proliferation of the cancer cells. So having said that, 54 00:04:17 --> 00:04:22 I want to go to a different kind of leukemia, and this is called chronic 55 00:04:22 --> 00:04:28 myelogenous leukemia, to give you an indication of the 56 00:04:28 --> 00:04:33 path of discovery that led from its original description to the 57 00:04:33 --> 00:04:39 development of rather successful treatments. 58 00:04:39 --> 00:04:45 So, chronic myelogenous leukemia, I mentioned the prefix myelo- last 59 00:04:45 --> 00:04:51 time or the time before referring to bone marrow, and this is a leukemia 60 00:04:51 --> 00:04:57 of cells coming from the bone marrow from the myeloid cells in the bone 61 00:04:57 --> 00:05:03 marrow, which are the precursors of things like macrophages 62 00:05:03 --> 00:05:08 and granulocytes. So, these are cells which are 63 00:05:08 --> 00:05:12 playing an important role in the immune response, 64 00:05:12 --> 00:05:16 and during this chronic myelogenous leukemia disease, 65 00:05:16 --> 00:05:21 which is called CML, there could be a period of three or 66 00:05:21 --> 00:05:25 four years where individuals develop large numbers of these cells in 67 00:05:25 --> 00:05:30 their blood stream. And after a period of about three or 68 00:05:30 --> 00:05:34 four years, all of a sudden there is an eruption into what's called blast 69 00:05:34 --> 00:05:38 crisis. And you may recall I mentioned the word blast also on one 70 00:05:38 --> 00:05:42 occasion earlier. This all fits together in a nice 71 00:05:42 --> 00:05:46 puzzle. Blast refers to primitive, embryonic-like cells, and all of a 72 00:05:46 --> 00:05:50 sudden there is an eruption of primitive, embryonic-like cells, 73 00:05:50 --> 00:05:54 less differentiated like these macrophages and granulocytes, 74 00:05:54 --> 00:05:58 which until this point had been present in vastly excessive numbers 75 00:05:58 --> 00:06:03 in the blood. There's blast crisis. 76 00:06:03 --> 00:06:08 This leads to acute myelogenous leukemia, and death ensues usually 77 00:06:08 --> 00:06:13 within a year or two, or that's been traditionally the 78 00:06:13 --> 00:06:17 case. No one really had any idea about the possible causative 79 00:06:17 --> 00:06:22 mechanisms of the disease, and that allows me to use another 80 00:06:22 --> 00:06:27 word which you might one day come across if you should stay in 81 00:06:27 --> 00:06:32 biomedical research. And that is the etiologic agents. 82 00:06:32 --> 00:06:37 When we talk about etiologic agents, we talk about the agents which are 83 00:06:37 --> 00:06:41 causally responsible for inducing a disease. These can be external 84 00:06:41 --> 00:06:46 agents, or they could even be internal agents, 85 00:06:46 --> 00:06:51 molecules inside cells which are responsible for the creation of the 86 00:06:51 --> 00:06:56 disease. And the key discovery was made in 1960 when individuals were 87 00:06:56 --> 00:07:01 looking at the chromosomal makeup of the CML cells. 88 00:07:01 --> 00:07:05 The chromosomal makeup, I'll use another word just so we 89 00:07:05 --> 00:07:09 could expand our vocabulary this morning, the chromosomal makeup is 90 00:07:09 --> 00:07:14 often called the karyotype, that is to say the constellation of 91 00:07:14 --> 00:07:18 chromosomes that one can see at mitosis under the microscope. 92 00:07:18 --> 00:07:22 Keep in mind, as we've said before, that during the interphase of the 93 00:07:22 --> 00:07:27 cell cycle, chromosomes are essentially invisible, 94 00:07:27 --> 00:07:31 but during the metaphase of mitosis they become condensed, 95 00:07:31 --> 00:07:36 and on that occasion, individuals noticed a 9-22 translocation. 96 00:07:36 --> 00:07:41 So here is chromosome nine normally. Here's chromosome 22. And as you 97 00:07:41 --> 00:07:46 may know, the numbering system with human chromosomes goes from the 98 00:07:46 --> 00:07:51 largest number one all the way down to the smallest. 99 00:07:51 --> 00:07:57 So, this is the smallest, with the exception of the Y 100 00:07:57 --> 00:08:01 chromosome. And what they notice was instead of 101 00:08:01 --> 00:08:04 seeing this regular chromosomal array, they noticed instead what 102 00:08:04 --> 00:08:07 looked very much like a structure of this sort here, i.e. 103 00:08:07 --> 00:08:35 a translocation. 104 00:08:35 --> 00:08:39 And this translocation resulted in a swapping of sequences between these 105 00:08:39 --> 00:08:43 two chromosomes. Note, by the way, 106 00:08:43 --> 00:08:47 this is reciprocal, i.e. in the sense that nine donates 107 00:08:47 --> 00:08:51 something to 22, and 22 donates something to nine. 108 00:08:51 --> 00:08:55 However, the segments that are swapped are not necessarily of equal 109 00:08:55 --> 00:08:59 size. So, it turns out here that in this case, chromosome nine has 110 00:08:59 --> 00:09:03 actually gained a lot more than chromosome 22 gained as a 111 00:09:03 --> 00:09:08 consequence of this exchange of genetic segments. 112 00:09:08 --> 00:09:12 And this 9-22 translocation made the smallest chromosome even smaller. 113 00:09:12 --> 00:09:16 So, this was already the smallest chromosome as I mentioned besides 114 00:09:16 --> 00:09:20 the smallest autosome, the smallest non-sex chromosome. 115 00:09:20 --> 00:09:24 Now it got even smaller because it lost some of its bulk as a 116 00:09:24 --> 00:09:29 consequence of this chromosomal translocation. 117 00:09:29 --> 00:09:33 And because this discovery was made in Philadelphia, 118 00:09:33 --> 00:09:37 it became known as the Philadelphia chromosome. This is now about 40 119 00:09:37 --> 00:09:42 years ago, or as it's sometimes called, PH-1 for reasons, 120 00:09:42 --> 00:09:46 I don't know why it's called PH-1 except for Philadelphia. 121 00:09:46 --> 00:09:51 And, as investigators began to look at other cases of chronic 122 00:09:51 --> 00:09:55 myelogenous leukemia, they discovered that this 123 00:09:55 --> 00:10:00 translocation was present at the Philadelphia chromosome most 124 00:10:00 --> 00:10:04 importantly was identifiable in virtually all cases, 125 00:10:04 --> 00:10:09 more than 95% of the cases of chronic myelogenous leukemia. 126 00:10:09 --> 00:10:13 And moreover, this chromosome was present as well in the more 127 00:10:13 --> 00:10:18 differentiated macrophages and granulocytes that were present and 128 00:10:18 --> 00:10:22 circulating in the blood of the CML patients. And that began to suggest 129 00:10:22 --> 00:10:27 the notion that there was a stem cell of some sort, 130 00:10:27 --> 00:10:31 oligopotential stem cell that created various kinds of more 131 00:10:31 --> 00:10:36 differentiated white blood cells that had sustained this chromosomal 132 00:10:36 --> 00:10:41 translocation because that's what it is, a translocalization, 133 00:10:41 --> 00:10:46 a translocation, that all the cells of these patients had sustained this 134 00:10:46 --> 00:10:51 chromosomal translocation. And that began to suggest the 135 00:10:51 --> 00:10:56 notion that somehow as a consequence of a random genetic accident 136 00:10:56 --> 00:11:01 happening in these people's blood, this particular chromosome was 137 00:11:01 --> 00:11:07 repeatedly identified. And it was with great likelihood 138 00:11:07 --> 00:11:13 causally or etiologically important in the genesis of the disease. 139 00:11:13 --> 00:11:20 But that in itself led nowhere. One could simply talk about its 140 00:11:20 --> 00:11:27 association until work from a totally unrelated area, 141 00:11:27 --> 00:11:33 which is to say the study of retroviruses discovered Abelson 142 00:11:33 --> 00:11:39 murine leukemia virus. And Abelson was named after the 143 00:11:39 --> 00:11:44 fellow, Herb Abelson, who first discovered it at NIH and 144 00:11:44 --> 00:11:48 undertook its molecular characterization here in our own 145 00:11:48 --> 00:11:53 cancer center, and Abelson discovered that this 146 00:11:53 --> 00:11:58 virus which he studied carried the ends of the murine leukemia virus, 147 00:11:58 --> 00:12:03 which was a parental virus. It was as hybrid virus. 148 00:12:03 --> 00:12:08 And into the middle of it, Abelson leukemia virus has acquired 149 00:12:08 --> 00:12:13 a cellular proto-oncogene, which it had activated into an 150 00:12:13 --> 00:12:18 oncogene. And therefore, here we have a situation where a 151 00:12:18 --> 00:12:23 cellular gene like sarc in the case of Rous Sarcoma Virus has been 152 00:12:23 --> 00:12:28 activated. This became called ABL for obvious reasons. 153 00:12:28 --> 00:12:33 And this gene, it turned out, was critically important in 154 00:12:33 --> 00:12:38 understanding hwo the chromosomal translocation led to cancer. 155 00:12:38 --> 00:12:43 In fact, if one infected mice with a retrovirus carrying this genome, 156 00:12:43 --> 00:12:48 this is just to indicate the fact that the repeat ends, 157 00:12:48 --> 00:12:53 the long terminal repeat ends of this provirus, 158 00:12:53 --> 00:12:58 they occur twice at the ends of this retrovirus. If one infected a mouse 159 00:12:58 --> 00:13:03 with the Abelson virus, out came a disease which was 160 00:13:03 --> 00:13:08 superficially similar at least to chronic myelogenous leukemia. 161 00:13:08 --> 00:13:13 And that began a search, then, for the chromosomal 162 00:13:13 --> 00:13:18 localization of the Abel proto-oncogene. 163 00:13:18 --> 00:13:23 And what was discovered subsequently, fascinatingly enough, 164 00:13:23 --> 00:13:29 was that the Abel proto-oncogene was right at the break point between two 165 00:13:29 --> 00:13:34 chromosomes, nine and 22. And what happened as a consequence 166 00:13:34 --> 00:13:38 of this translocation, and the resulting fusion of this 167 00:13:38 --> 00:13:42 chromosome with this chromosome was the creation of a fuse gene, 168 00:13:42 --> 00:13:46 a hybrid gene that now carried the reading frames of two previously 169 00:13:46 --> 00:13:50 unconnected gene, one on chromosome nine, 170 00:13:50 --> 00:13:54 and one on chromosome 22. Here's the normal, Abel protein. 171 00:13:54 --> 00:13:58 It's called C Abel, meaning the cellular or the normal form of Abel. 172 00:13:58 --> 00:14:02 And you see it up here. It's shown in a very schematic way. 173 00:14:02 --> 00:14:06 And here's a second protein which is encoded on the other chromosome. 174 00:14:06 --> 00:14:11 So, Abel is encoded here, and the other gene, which is called BCR is 175 00:14:11 --> 00:14:15 encoded here, and as a consequence of the translocation, 176 00:14:15 --> 00:14:19 Abel is encoded here. BCR is encoded here. As a consequence of 177 00:14:19 --> 00:14:24 the translocation, one now has not only the fusion of 178 00:14:24 --> 00:14:28 chromosomal segments. But one has the fusion of the 179 00:14:28 --> 00:14:33 reading frames of two previously unlinked genes. 180 00:14:33 --> 00:14:38 And here, one creates as a consequence of these fusions, 181 00:14:38 --> 00:14:43 any one of a series of three quite distinct fusion proteins, 182 00:14:43 --> 00:14:48 which do not naturally preexist in the normal cell. 183 00:14:48 --> 00:14:53 And there shown here is P-1 85, P-2 10, and P-2 30. These 184 00:14:53 --> 00:14:58 translocations allow different parts of a second gene called BCR. 185 00:14:58 --> 00:15:02 BCR refers to breakpoint cluster region. The area of the point of 186 00:15:02 --> 00:15:06 fusion is called the breakpoint between the two genes. 187 00:15:06 --> 00:15:10 So, the point where each gene is cut and fused with the other is 188 00:15:10 --> 00:15:14 called the breakpoint. And it turns out that within the 189 00:15:14 --> 00:15:18 region of the chromosome where BCR maps, there's actually three sites 190 00:15:18 --> 00:15:22 at which the fusion can occur. If you look carefully at this 191 00:15:22 --> 00:15:26 diagram, you see that there's differing extents of the BCR protein, 192 00:15:26 --> 00:15:30 which can be contributed to the fusion protein. 193 00:15:30 --> 00:15:35 And, what this says, in effect, is the following, 194 00:15:35 --> 00:15:40 that here, let's just refer to this diagram right up here. 195 00:15:40 --> 00:15:46 Notice, by the way, in all three of these, that the Abel protein is 196 00:15:46 --> 00:15:51 present at the C terminal end of the protein. The BCR is present at the 197 00:15:51 --> 00:15:57 end terminal end. So, here's the BCR gene. 198 00:15:57 --> 00:16:01 Here's the Abel gene down here. And what investigators found is that 199 00:16:01 --> 00:16:05 there could be a break at this part of the BCR gene, 200 00:16:05 --> 00:16:09 at this part of the BCR gene, or at this part of the BCR gene, 201 00:16:09 --> 00:16:13 resulting always in the fusion of Abel, to one, or two, 202 00:16:13 --> 00:16:17 or three different kinds of BCR proteins. And, 203 00:16:17 --> 00:16:21 breakpoint cluster region signified the fact that there was a whole 204 00:16:21 --> 00:16:25 cluster of sites in the previously existing BCR gene to which this 205 00:16:25 --> 00:16:29 fusion could take place, resulting, if the break occurred 206 00:16:29 --> 00:16:33 here, the breakpoint occurred here, and BCR to get the longer one. 207 00:16:33 --> 00:16:37 Here you get the medium-sized one; here you'd get the shortest one. 208 00:16:37 --> 00:16:41 And, interestingly enough, as one explored virtually, 209 00:16:41 --> 00:16:45 other kinds of different leukemias, one could see different of these 210 00:16:45 --> 00:16:49 fusion proteins that were produced. Here's chronic, myelogenous 211 00:16:49 --> 00:16:53 leukemia, which I talked to you about before. Here is acute 212 00:16:53 --> 00:16:57 lymphocytic leukemia, and here's chronic and neutrophylic 213 00:16:57 --> 00:17:01 leukemia, three different kinds of leukemia. We don't have to worry 214 00:17:01 --> 00:17:05 about the details of these diseases, aside from the fact to say that the 215 00:17:05 --> 00:17:09 structure of this fusion protein encourages the outgrowth of 216 00:17:09 --> 00:17:13 different kinds of stem cells in the bone marrow, which in turn create 217 00:17:13 --> 00:17:17 three different kinds of diseases. Most importantly for our discussion 218 00:17:17 --> 00:17:21 was an attempt to understand the nature of the resulting fusion 219 00:17:21 --> 00:17:25 protein, which as a consequence of this fusion caused by the 220 00:17:25 --> 00:17:29 chromosomal translocation now clearly acquired biological powers 221 00:17:29 --> 00:17:33 that did not preexist in either of the two parental proteins. 222 00:17:33 --> 00:17:37 These various notations here indicate a whole series of different 223 00:17:37 --> 00:17:41 functions which are associated with the Abel protein, 224 00:17:41 --> 00:17:45 and alternatively with the BCR protein. And we don't need to get 225 00:17:45 --> 00:17:49 into them, except to say that each one of these different names here 226 00:17:49 --> 00:17:53 allows the protein on its own to associate with other proteins and do 227 00:17:53 --> 00:17:58 activated downstream signaling cascade. 228 00:17:58 --> 00:18:03 What's most important about our discussion is the realization that 229 00:18:03 --> 00:18:09 this SH-1 domain, indicated here, SH-1 refers to the 230 00:18:09 --> 00:18:15 sarcomology domain, equals sarcomology, equals a 231 00:18:15 --> 00:18:21 tyrosine kinase. And therefore, what one has here is 232 00:18:21 --> 00:18:27 a protein, which is much more elaborate than sarc, 233 00:18:27 --> 00:18:33 has vastly more signaling capabilities, 234 00:18:33 --> 00:18:37 by virtue of the fact that these different domains that are indicated 235 00:18:37 --> 00:18:42 here allow the resulting fusion protein to grab hold of a whole 236 00:18:42 --> 00:18:47 bunch of different signally partners so that it can send out a diverse 237 00:18:47 --> 00:18:52 array of downstream activating signals. If one examined the 238 00:18:52 --> 00:18:57 structure of the SH-1 domain, it had a tyrosine kinase activity 239 00:18:57 --> 00:19:02 very much like sarc, and most importantly, 240 00:19:02 --> 00:19:07 if one introduced this fusion protein into a retrovirus, 241 00:19:07 --> 00:19:12 now instead of Abel, one could make a BCR Abel fusion protein. 242 00:19:12 --> 00:19:16 One could put this into a retrovirus as before, just like up here. 243 00:19:16 --> 00:19:21 One could infect mice with it, and now get out of a disease which 244 00:19:21 --> 00:19:25 was indistinguishable, in essence, from chronic myelogenous 245 00:19:25 --> 00:19:30 leukemia in humans. If one put a subtle point mutation 246 00:19:30 --> 00:19:34 in the tyrosine kinase domain, all of the able protein, here is the 247 00:19:34 --> 00:19:38 tyrosine kinase domain, SH-1, up here. Here, we see the 248 00:19:38 --> 00:19:43 tyrosine kinase domains represented in the three different fusion 249 00:19:43 --> 00:19:47 proteins. Keep in mind SH-1 is always the tyrosine kinase domain. 250 00:19:47 --> 00:19:51 If one put a subtle, inactivating point mutation in the tyrosine 251 00:19:51 --> 00:19:56 kinase domain, that immediately wiped out all 252 00:19:56 --> 00:20:00 biological powers of creating leukemias on the part of this 253 00:20:00 --> 00:20:04 retrovirus here, or any one of the other closely 254 00:20:04 --> 00:20:09 related kinds of fusion proteins. And therefore, 255 00:20:09 --> 00:20:13 that indicated that the tyrosine kinase domain indicated right here 256 00:20:13 --> 00:20:17 was really critical to creating the tumor, and that any effects on its 257 00:20:17 --> 00:20:21 tyrosine kinase signaling ability would, in the end, 258 00:20:21 --> 00:20:25 result in the collapse of the tumor, or the inability of the resulting 259 00:20:25 --> 00:20:30 retrovirus to actually create cancer. 260 00:20:30 --> 00:20:34 And so, now one had, really for the first time, 261 00:20:34 --> 00:20:38 a clear demonstration of how a commonly occurring human cancer, 262 00:20:38 --> 00:20:42 chronic myelogenous leukemia, is unfortunately not so rare, 263 00:20:42 --> 00:20:46 could arise as a consequence of some random, chromosomal translocation 264 00:20:46 --> 00:20:50 event. You might ask, why does one always get this 265 00:20:50 --> 00:20:54 particular kind of translocation? Well, the answer is, we don't 266 00:20:54 --> 00:20:58 really know. It would almost seem as if there's a homing device which 267 00:20:58 --> 00:21:02 causes this fragment and this fragment to target each other and to 268 00:21:02 --> 00:21:07 exchange one another. It's probably not the case. 269 00:21:07 --> 00:21:11 What probably happens is that chromosomal translocations take 270 00:21:11 --> 00:21:15 place rather randomly within the bone marrow, and on rare occasion 271 00:21:15 --> 00:21:19 there is a chromosomal translocation that creates exactly this kind of 272 00:21:19 --> 00:21:23 fusion. And this kind of fusion, in turn, is what's responsible for 273 00:21:23 --> 00:21:27 creating this fusion protein, and this fusion protein in turn 274 00:21:27 --> 00:21:31 creates the outgrowth of this CML, the chronic myelogenous leukemia 275 00:21:31 --> 00:21:36 disease. So what that means is really that a 276 00:21:36 --> 00:21:40 randomly occurring chromosomal translocation on rare occasion hits 277 00:21:40 --> 00:21:44 a genetic jackpot, and the cell which happens to have 278 00:21:44 --> 00:21:48 acquired this kind of chromosomal translocation now begins to 279 00:21:48 --> 00:21:52 proliferate wildly, creating first chronic myelogenous 280 00:21:52 --> 00:21:56 leukemia, and then subsequently erupting into a subsequent acute 281 00:21:56 --> 00:22:00 phase where there are seemingly additional genetic alterations 282 00:22:00 --> 00:22:04 beyond this chromosomal translocation that conspire with the 283 00:22:04 --> 00:22:08 initially present chromosomal translocation to create a very 284 00:22:08 --> 00:22:12 aggressive disease which rapidly leads to the death of the 285 00:22:12 --> 00:22:16 leukemia patient. That offered, in principle, 286 00:22:16 --> 00:22:21 an attractive way of beginning to develop an anti-cancer therapeutic 287 00:22:21 --> 00:22:26 because what one might imagine was that one could develop a tyrosine 288 00:22:26 --> 00:22:38 kinase inhibitor. 289 00:22:38 --> 00:22:42 Now keep in mind that tyrosine kinases are a class of enzymes which 290 00:22:42 --> 00:22:46 attach phosphate groups onto the tyrosine residues of various 291 00:22:46 --> 00:22:51 substrate proteins. And keep in mind as well the fact 292 00:22:51 --> 00:22:55 that we drew a series of growth factor receptors which have tyrosine 293 00:22:55 --> 00:23:00 kinase domains in them. And I'm drawing the tyrosine kinase 294 00:23:00 --> 00:23:06 domains here like this, that when these growth factor 295 00:23:06 --> 00:23:11 receptors become activated, they attach phosphate groups onto 296 00:23:11 --> 00:23:17 the tails of one another. And I'll draw those phosphate 297 00:23:17 --> 00:23:22 groups like this, i.e. the binding of ligand or let's 298 00:23:22 --> 00:23:28 say epidermal growth factor ligand or plate ligand causes the two 299 00:23:28 --> 00:23:33 receptors, which are normally mobilized in the plasma membrane to 300 00:23:33 --> 00:23:39 come together to transphosphorylate one another, 301 00:23:39 --> 00:23:44 and having done so, to acquire actively signaling powers, 302 00:23:44 --> 00:23:49 because once these phosphates become attached, they now represent sites 303 00:23:49 --> 00:23:54 where other molecules can anchor themselves and send out downstream 304 00:23:54 --> 00:23:59 signals. In fact, there are altogether 90 different 305 00:23:59 --> 00:24:05 tyrosine kinases encoded in the human genome. 306 00:24:05 --> 00:24:09 And so, to the extent that these tyrosine kinases become 307 00:24:09 --> 00:24:13 hyperactivated in various kinds of human cancers, 308 00:24:13 --> 00:24:17 this represents in principle a very attractive way of developing an 309 00:24:17 --> 00:24:21 anti-cancer therapeutic. But let's think about the problems 310 00:24:21 --> 00:24:25 that are inherent in such a compound. First of all, 311 00:24:25 --> 00:24:29 if one wants to develop an anti-cancer therapeutic, 312 00:24:29 --> 00:24:34 it must be reasonably specific for the Abelson tyrosine kinase, 313 00:24:34 --> 00:24:38 and not the 89 kinds of tyrosine kinases that also coexist in the 314 00:24:38 --> 00:24:43 human genome, and are active, and apparently responsible for 315 00:24:43 --> 00:24:48 normal cell metabolism in a whole variety of normal cell types. 316 00:24:48 --> 00:24:53 So, one has to begin to think about the issue of cell activity. 317 00:24:53 --> 00:24:58 How can one possibly make a low molecular weight compound, 318 00:24:58 --> 00:25:03 which is selectively able to inactivate the Abelson tyrosine 319 00:25:03 --> 00:25:08 kinase as indicated here, the SH-1 group, 320 00:25:08 --> 00:25:11 but doesn't disturb a whole variety of other tyrosine kinases that are 321 00:25:11 --> 00:25:15 responsible for other normal physiological mechanisms. 322 00:25:15 --> 00:25:19 Well, you'll say that's pretty easy. We have 90 different genes. 323 00:25:19 --> 00:25:23 Each of the 90 different genes makes a distinct protein, 324 00:25:23 --> 00:25:27 and these proteins should be very different. And therefore, 325 00:25:27 --> 00:25:31 if one can, in fact, if one does the three-dimensional structure of these 326 00:25:31 --> 00:25:35 proteins, all the tyrosine kinases look quite similar. 327 00:25:35 --> 00:25:40 They have a biload structure. Here is the active site of the 328 00:25:40 --> 00:25:46 enzyme. That is to say, in here is the catalytic cleft, 329 00:25:46 --> 00:25:51 the site where the actual catalysis takes place, the site where the 330 00:25:51 --> 00:25:57 gamma phosphate of ATP is taken from the ATP and attached to a substrate 331 00:25:57 --> 00:26:03 protein to the hydroxyl of a tyrosine of a protein that's about 332 00:26:03 --> 00:26:07 to be phosphorylated. So, you just make a low moleculoid 333 00:26:07 --> 00:26:11 chemical that's specific for the tyrosine kinase domain of the Abel 334 00:26:11 --> 00:26:15 protein. And when I draw this biload structure, 335 00:26:15 --> 00:26:18 this biload structure is carried here within the SH-1 domain right 336 00:26:18 --> 00:26:22 here. So, this has a biload structure. It's obviously not 337 00:26:22 --> 00:26:26 indicated here in this very schematic drawing. 338 00:26:26 --> 00:26:30 The problem with that is the following. 339 00:26:30 --> 00:26:33 All of the SH-1 domains, all of the tyrosine kinase domains 340 00:26:33 --> 00:26:37 are evolutionarily closely related to one another. 341 00:26:37 --> 00:26:41 They're all derived from the precursors of the tyrosine kinase 342 00:26:41 --> 00:26:45 domain that probably existed maybe 600 or 700 million years ago, 343 00:26:45 --> 00:26:49 and has as a consequence of gene duplication been diversified to make 344 00:26:49 --> 00:26:53 90 different tyrosine kinases. And if you look under x-ray 345 00:26:53 --> 00:26:57 crystallography at the three dimensional structure of all these 346 00:26:57 --> 00:27:01 tyrosine kinases, they all pretty much look like this, 347 00:27:01 --> 00:27:04 i.e. they all have rather similar catalytic clefts because they 348 00:27:04 --> 00:27:08 diverge from a common ancestral protein, and they retain this three 349 00:27:08 --> 00:27:12 dimensional configuration because this three dimensional configuration 350 00:27:12 --> 00:27:15 seems to be important for the retention of their function. 351 00:27:15 --> 00:27:19 You could imagine, conversely, that if there were some descendants 352 00:27:19 --> 00:27:23 of the ancestral tyrosine kinase domain that some of them became 353 00:27:23 --> 00:27:26 mutant and lost its three dimensional structure. 354 00:27:26 --> 00:27:30 Those descendant kinases would lose their ability to phosphorylate 355 00:27:30 --> 00:27:34 tyrosines on substrate proteins, and therefore would be eliminated 356 00:27:34 --> 00:27:38 from the gene pool because they would be defective. 357 00:27:38 --> 00:27:42 And that explains the strong conservatism in the structure of 358 00:27:42 --> 00:27:46 these 90 different enzymes. They all look very similar to one 359 00:27:46 --> 00:27:50 another, and that creates a great difficulty for the drug developer 360 00:27:50 --> 00:27:54 because a low molecular weight drug, which one would like to develop, 361 00:27:54 --> 00:27:58 that fits in here. So, here I'll draw a low molecular 362 00:27:58 --> 00:28:02 weight drug that interacts in a stereo-specific fashion with the 363 00:28:02 --> 00:28:06 amino acid residues that are aligning this pocket, 364 00:28:06 --> 00:28:10 this catalytic cleft, might bind and nicely inactivate the tyrosine 365 00:28:10 --> 00:28:15 kinase domain of Abel. But at the same time, 366 00:28:15 --> 00:28:19 it might also bind and inactivate a whole series of other tyrosine 367 00:28:19 --> 00:28:23 kinases, and that in turn could lead to therapeutic disaster. 368 00:28:23 --> 00:28:27 For instance, if you had a non-selective agent, 369 00:28:27 --> 00:28:31 you could treat a chronic myelogenous leukemia patient with a 370 00:28:31 --> 00:28:35 low molecular weight inhibitor, a low molecular weight compound, 371 00:28:35 --> 00:28:39 which would get into this pocket of the BCR Abel protein. 372 00:28:39 --> 00:28:43 But it might similarly get into the catalytic cleft of the EGF receptor. 373 00:28:43 --> 00:28:47 And if it shot down the EGF receptor, it might cause a fatal 374 00:28:47 --> 00:28:51 diarrhea because after all, the EGF receptor, I will tell you, 375 00:28:51 --> 00:28:56 is needed to maintain the structure of the epithelial lining of the 376 00:28:56 --> 00:29:00 colon. And so, you might kill the patient simply 377 00:29:00 --> 00:29:04 because you had deprived the cells in that person's colon of their 378 00:29:04 --> 00:29:08 ability to maintain themselves. There are a whole series of growth 379 00:29:08 --> 00:29:12 factor receptors that are required for hematapoeisis that we discussed 380 00:29:12 --> 00:29:16 last time. And there, once again, if you had a 381 00:29:16 --> 00:29:20 nonselective compound, which got into the domain of one of 382 00:29:20 --> 00:29:23 the growth factor receptors that is responsible for hematapoeisis, 383 00:29:23 --> 00:29:27 you might shut down the entire bone marrow, and once again kill the 384 00:29:27 --> 00:29:31 patient. I'm just giving you those as overly dramatic examples of the 385 00:29:31 --> 00:29:35 fact that cell activity is an extremely important consideration in 386 00:29:35 --> 00:29:40 developing such a drug. The other thing is affinity for the 387 00:29:40 --> 00:29:48 target, for the catalytic cleft that is being targeted. 388 00:29:48 --> 00:29:56 What do I mean by affinity? If you look at those response 389 00:29:56 --> 00:30:04 curves of various compounds, what you see is the following. 390 00:30:04 --> 00:30:12 You can draw out a line that looks like this, a graph that looks like 391 00:30:12 --> 00:30:20 this, where here we have log of drug concentration. 392 00:30:20 --> 00:30:28 And here is 10-4, here, let's do the other one, 393 00:30:28 --> 00:30:36 10-8 molar, 10-7 molar, 10-6, 10-5, 10-4. 394 00:30:36 --> 00:30:42 And this is molar drug concentration. And here is the percentage of 395 00:30:42 --> 00:30:49 inhibition. Let's say, for example, we were able to take 396 00:30:49 --> 00:30:55 the BCR Abel protein and study it in a test tube. And let's say we were 397 00:30:55 --> 00:31:02 interested in studying how well its tyrosine kinase activity responded 398 00:31:02 --> 00:31:09 to an applied drug that we developed against it. 399 00:31:09 --> 00:31:16 So, here's the percentage of inhibition of tyrosine kinase 400 00:31:16 --> 00:31:24 activity of BCR Abel protein. Now, I might be able to develop a 401 00:31:24 --> 00:31:32 drug whose dose response curve would look like this. 402 00:31:32 --> 00:31:36 And you'll say, well, that's terrific. 403 00:31:36 --> 00:31:40 That's a drug which shuts down BCR Abel. We haven't even dealt with 404 00:31:40 --> 00:31:44 the issue of cell activity, but let's look at where one begins 405 00:31:44 --> 00:31:48 to see a dose response right here, 10-5 molar. And if you calculate 406 00:31:48 --> 00:31:52 back as to how much of the drug you need to deliver in order to shut 407 00:31:52 --> 00:31:56 down the BCR Abel protein in a patient, the size of the pill they'd 408 00:31:56 --> 00:32:00 have to get would probably be this big everyday. 409 00:32:00 --> 00:32:04 So, what you need to do is you need to be an acceptable range of drug 410 00:32:04 --> 00:32:08 concentrations is down in this area here. And therefore, 411 00:32:08 --> 00:32:12 only until you get a drug which has a dose response curve that looks 412 00:32:12 --> 00:32:16 like this, which is two or three orders of magnitude more potent 413 00:32:16 --> 00:32:20 where it's able to shut down the kinase activity already at 10-7 in a, 414 00:32:20 --> 00:32:24 this is called a submicromolar concentration. 415 00:32:24 --> 00:32:28 Micromolar is 10-6. Here already at a tenth of a 416 00:32:28 --> 00:32:32 micromolar, 10-7 molar, we're already getting a shutdown of 417 00:32:32 --> 00:32:36 the enzyme function. And if one can do that, 418 00:32:36 --> 00:32:40 then one might in principle be able to develop a pill that's this big 419 00:32:40 --> 00:32:44 and give that to the patient rather than a pill that's a size of a 420 00:32:44 --> 00:32:47 football. And by the way, if you have to make lots of a very 421 00:32:47 --> 00:32:51 complex, organic molecule through organic synthesis that has an 422 00:32:51 --> 00:32:55 affinity of this, it's also very expensive. 423 00:32:55 --> 00:32:58 Obviously, if you can make a compound that's a hundredfold more 424 00:32:58 --> 00:33:02 potent and requires a hundredfold less material to deliver to the 425 00:33:02 --> 00:33:06 patient body, then you might have some success in treating 426 00:33:06 --> 00:33:09 the patient. Here's another issue. 427 00:33:09 --> 00:33:13 So, we've talked about cell activity. We've talked about 428 00:33:13 --> 00:33:17 potency or affinity, affinity for the substrate or 429 00:33:17 --> 00:33:20 potency. So, this would be an acceptable drug. 430 00:33:20 --> 00:33:24 It works already at molar concentration where the inflection 431 00:33:24 --> 00:33:28 point of this curve is. This is an unacceptable drug at 432 00:33:28 --> 00:33:32 10-5. We can also talk about 433 00:33:32 --> 00:33:36 pharmacokinetics. I want to give you a feeling for 434 00:33:36 --> 00:33:40 how complex drug development is, and why it so rarely succeeds. By 435 00:33:40 --> 00:33:44 the way, you know how much it costs to develop a drug that's useful in 436 00:33:44 --> 00:33:49 the clinic these days and test it on people? Anybody have any idea? 437 00:33:49 --> 00:33:53 How much? Yeah. It's pretty close to $1 billion, 438 00:33:53 --> 00:33:57 between $900 million and $1 billion. That's a lot of money. That's more 439 00:33:57 --> 00:34:01 money than you and I are going to earn together, all of us 440 00:34:01 --> 00:34:06 maybe, in a lifetime. OK, anyhow, pharmacokinetics, 441 00:34:06 --> 00:34:10 well what's pharmacokinetics? Glad I asked that question. 442 00:34:10 --> 00:34:14 How long does the drug stay inside of you after you take it if you're a 443 00:34:14 --> 00:34:18 cancer patient? What happens if the drug is 444 00:34:18 --> 00:34:22 excreted by the kidneys within minutes of its being taken, 445 00:34:22 --> 00:34:26 let's say, either by injection or orally? 446 00:34:26 --> 00:34:32 So here we can imagine, let's talk about drug concentration. 447 00:34:32 --> 00:34:38 I'll use the word drug concentration in blood. 448 00:34:38 --> 00:34:44 And here's time. And here's what some drugs look like when you give 449 00:34:44 --> 00:34:50 them, let's say, orally. Here's what they look like. 450 00:34:50 --> 00:34:56 So, let's say here's the effective drug concentration: effective 451 00:34:56 --> 00:35:02 concentration. And we know the effective 452 00:35:02 --> 00:35:06 concentration from doing measurements like this. 453 00:35:06 --> 00:35:10 We just measure it, work that out. So, let's say we develop a drug 454 00:35:10 --> 00:35:14 which is able to hit the BCR Abel protein. What are the kinetics with 455 00:35:14 --> 00:35:18 which the drug becomes soluble in the blood stream? 456 00:35:18 --> 00:35:22 And it might look like this, where I'm drawing here now, this is 457 00:35:22 --> 00:35:26 one hour. This is two hours. This is three hours, four hours. 458 00:35:26 --> 00:35:30 Is that long enough? 459 00:35:30 --> 00:35:34 Well, the fact of the matter is, if you're going to try to kill a 460 00:35:34 --> 00:35:38 cancer cell, and that's what the name of this game is, 461 00:35:38 --> 00:35:41 you want to have it around for a while because it turns out, 462 00:35:41 --> 00:35:45 as one learned, the continued viability of the CML cancer cells of 463 00:35:45 --> 00:35:49 the leukemia cells was dependent on the continued firing by the BCR Abel 464 00:35:49 --> 00:35:52 kinase protein. In fact, as one learned, 465 00:35:52 --> 00:35:56 if one shut off firing by the tyrosine kinase molecule in a 466 00:35:56 --> 00:36:00 chronic myelogenous leukemia cell, the cells would implode. 467 00:36:00 --> 00:36:04 They would undergo apitosis. So, this began to reveal that in 468 00:36:04 --> 00:36:08 fact the BCR Abel protein is not only responsible for forcing these 469 00:36:08 --> 00:36:12 cells to proliferate, but it also independently provides 470 00:36:12 --> 00:36:16 them with anti-apoptotic signal. It keeps them from falling over the 471 00:36:16 --> 00:36:20 cliff into apitosis. It keeps them from killing 472 00:36:20 --> 00:36:24 themselves, and that's obviously critical for the ability of this 473 00:36:24 --> 00:36:28 tumor to proliferate, for the number of cells to expand in 474 00:36:28 --> 00:36:32 the body of a patient. It turns out that if you provide 475 00:36:32 --> 00:36:37 these cancer cells with an effective way of shutting down their BCR Abel 476 00:36:37 --> 00:36:42 protein for 30 or 40 or 50 minutes, not much happens to them. You need 477 00:36:42 --> 00:36:47 to deprive them of the drug for a very long period of time, 478 00:36:47 --> 00:36:52 well, 15-20 hours, and therefore you need pharmacokinetics that look like 479 00:36:52 --> 00:36:57 this. It needs to be present for an extended period of time, 480 00:36:57 --> 00:37:03 or even better, let me re-draw that, even better look like this. 481 00:37:03 --> 00:37:06 It stays in the blood for an extended period of time. 482 00:37:06 --> 00:37:10 Some drugs stay in the circulation for a long time. 483 00:37:10 --> 00:37:13 Other drugs stay in the circulation for a very short period of time. 484 00:37:13 --> 00:37:17 There's another problem which we haven't even begun to talk about, 485 00:37:17 --> 00:37:21 and that is the metabolism of the drug. It turns out that many drugs 486 00:37:21 --> 00:37:24 that you give a patient are rapidly converted by the enzymes and the 487 00:37:24 --> 00:37:28 liver which are normally responsible for detoxifying chemicals that come 488 00:37:28 --> 00:37:32 into our body. And therefore, 489 00:37:32 --> 00:37:36 many of the drugs that come into our bodies are with greater or lesser 490 00:37:36 --> 00:37:40 speed altered into something else, detoxified, and therefore rendered 491 00:37:40 --> 00:37:44 innocuous. Now you'll say, well, you can figure that out too, 492 00:37:44 --> 00:37:48 but here's an additional fly in the ointment. Because we are a 493 00:37:48 --> 00:37:52 polymorphic population, because we humans are genetically 494 00:37:52 --> 00:37:56 heterogeneous, one from the other, 495 00:37:56 --> 00:38:00 some of us metabolize a given drug much more rapidly than others do. 496 00:38:00 --> 00:38:04 And here, we have a situation where potentially, most of us might 497 00:38:04 --> 00:38:08 metabolize a drug very quickly, in which case the physicians would 498 00:38:08 --> 00:38:13 want to give us a very high dose of the drug so that we have enough of 499 00:38:13 --> 00:38:17 the drug around for a long enough period of time to do some effect. 500 00:38:17 --> 00:38:22 So let's say that 97% of us are able to metabolize the drug very 501 00:38:22 --> 00:38:26 quickly, and as a consequence, we're given a very high dosage in 502 00:38:26 --> 00:38:31 order to have some effective dosage reaching the tumor to compensate for 503 00:38:31 --> 00:38:35 the fact that much of this drug is rapidly eliminated by metabolism 504 00:38:35 --> 00:38:40 in the liver. It's inter-converted into another 505 00:38:40 --> 00:38:44 chemically innocuous compound. Well, you'll say, that's good. 506 00:38:44 --> 00:38:48 We'll just take a large dose of that compound, 507 00:38:48 --> 00:38:52 but let's think about the other 3% in the population who metabolize 508 00:38:52 --> 00:38:56 this compound very slowly. Like the other 97%, these 509 00:38:56 --> 00:39:00 individuals will be given a high dose of the drug because experience 510 00:39:00 --> 00:39:04 shows that in general, most human beings metabolize a drug 511 00:39:04 --> 00:39:09 very quickly. These individuals metabolize the 512 00:39:09 --> 00:39:13 drug very slowly, and what's going to happen to them? 513 00:39:13 --> 00:39:18 Well, they might croak. Why? Because that drug is going to be 514 00:39:18 --> 00:39:23 around in potent biologically active form for an extended period of time 515 00:39:23 --> 00:39:27 in their bodies, and might have in them a lethal 516 00:39:27 --> 00:39:32 outcome. So therefore, we have to deal with the effects of 517 00:39:32 --> 00:39:37 variability in drug metabolism, variability in metabolism because it 518 00:39:37 --> 00:39:43 turns out that different people metabolize the drug differently and 519 00:39:43 --> 00:39:49 that variability in drug metabolism is vastly greater if you compare the 520 00:39:49 --> 00:39:54 way we metabolize drugs to the way that laboratory mice metabolize 521 00:39:54 --> 00:40:00 drugs. Well, you'll say, why should we care about how 522 00:40:00 --> 00:40:06 laboratory mice metabolize this or that drug? 523 00:40:06 --> 00:40:10 Why is it important? The fact is, the first tryouts of a 524 00:40:10 --> 00:40:15 candidate drug are tried out in laboratory mice where laboratory 525 00:40:15 --> 00:40:20 mice are given a tumor, and they're injected with the drug 526 00:40:20 --> 00:40:25 to see whether the tumor begins to shrink. But if it's the case, 527 00:40:25 --> 00:40:29 if the laboratory mice metabolize a drug in a vastly different way than 528 00:40:29 --> 00:40:34 do humans, then the outcome of working with laboratory mice might 529 00:40:34 --> 00:40:39 be enormously misleading. And these are just some of the 530 00:40:39 --> 00:40:44 problems that bedevil the development of a drug. 531 00:40:44 --> 00:40:50 In any case, around 1994, at a company which was a precursor 532 00:40:50 --> 00:40:55 of Novartis, it was called Ciba-Geigy in Basel, 533 00:40:55 --> 00:41:00 Switzerland. They developed a highly specific and potent anti-Abel, 534 00:41:00 --> 00:41:06 low molecular weight compound, which came to be called Leveck. 535 00:41:06 --> 00:41:10 Or in Europe it's called Gleveck. It's also pronounced Leveck, but 536 00:41:10 --> 00:41:15 it's spelled differently. In fact, it was one of the other 537 00:41:15 --> 00:41:19 difficulties of developing this drug was the following. 538 00:41:19 --> 00:41:24 The higher ups in the drug company who were paying for this research 539 00:41:24 --> 00:41:28 wanted on repeated occasion to scrub this entire drug development 540 00:41:28 --> 00:41:33 program. Why? Because the number of cases of 541 00:41:33 --> 00:41:37 chronic myelogenous leukemia overall worldwide is relatively small. 542 00:41:37 --> 00:41:42 How many are in this country every year? I don't know, 543 00:41:42 --> 00:41:47 10 or 15,000. So, the question was, economically speaking, would the 544 00:41:47 --> 00:41:51 relatively small number of cases of this disease justify their investing 545 00:41:51 --> 00:41:56 $1 billion in the development of the drug. Maybe it would take them a 546 00:41:56 --> 00:42:01 generation to get any payback from their initial investment. 547 00:42:01 --> 00:42:05 And so, they tried time after time, time and again, to shut down this 548 00:42:05 --> 00:42:09 development program because it didn't seem to have any clear, 549 00:42:09 --> 00:42:14 long-term economic benefit. Of course, now we're not talking about 550 00:42:14 --> 00:42:18 biology. We're talking about economics, and rational economics. 551 00:42:18 --> 00:42:23 This is not avarice on their part. A drug company like that cannot go 552 00:42:23 --> 00:42:27 on spending $1 billion here and $1 billion there without at one point 553 00:42:27 --> 00:42:32 or another leading to a major financial hemorrhage. 554 00:42:32 --> 00:42:38 So, Gleveck turned out to be highly specific for the Abel kinase, 555 00:42:38 --> 00:42:44 and as it turned out, for two other kinds of kinases as well. 556 00:42:44 --> 00:42:50 Another kind of kinase is against a tyrosine kinase receptor called KIT, 557 00:42:50 --> 00:42:56 this is a receptor tyrosine kinase, and another receptor tyrosine kinase 558 00:42:56 --> 00:43:02 called the PDGF receptor, which we've also encountered in 559 00:43:02 --> 00:43:07 passing earlier. These two other growth factor 560 00:43:07 --> 00:43:12 receptors, KIT and the PDGF receptor also have tyrosine kinase domains. 561 00:43:12 --> 00:43:16 They therefore follow this overall structural plan here, 562 00:43:16 --> 00:43:21 and it turns out by evolutionary quirk that the structures of their 563 00:43:21 --> 00:43:26 tyrosine kinase domains are actually similar in certain ways to the 564 00:43:26 --> 00:43:31 tyrosine kinase domain of Abel, and therefore of BCR Abel. 565 00:43:31 --> 00:43:35 So, in fact, they didn't actually have a totally specific drug which 566 00:43:35 --> 00:43:39 would attack only one out of the 90-tyrosine kinases encoded in their 567 00:43:39 --> 00:43:44 genome. It attacked three of the 90-tyrosine kinases, 568 00:43:44 --> 00:43:48 the Abel, the KITT, and the PDGF receptor. And this might, 569 00:43:48 --> 00:43:53 on its own, have already proven to be the death nail for the protein, 570 00:43:53 --> 00:43:57 except they began to try it out for patients, and they saw some 571 00:43:57 --> 00:44:02 remarkable responses. It turned out that the great 572 00:44:02 --> 00:44:08 majority of CML patients who were treated with Gleveck at therapeutic 573 00:44:08 --> 00:44:13 concentrations ended up having a rapid remission of their chronic 574 00:44:13 --> 00:44:18 myelogenous leukemia disease, which ultimately resulted in their 575 00:44:18 --> 00:44:24 being outwardly free of the disease. This is your question of the day. 576 00:44:24 --> 00:44:45 577 00:44:45 --> 00:44:49 So, Gleveck goes into the catalytic cleft of the Abel tyrosine kinase. 578 00:44:49 --> 00:44:54 It blocks the ATP binding site because keep in mind that these 579 00:44:54 --> 00:44:59 enzymes need to grab the gamma phosphate off of ATP and transfer it 580 00:44:59 --> 00:45:04 to a protein substrate, and it does so because it hydrogen 581 00:45:04 --> 00:45:09 bonds to the amino acids which are lining this catalytic cleft. 582 00:45:09 --> 00:45:12 In other words, this catalytic cleft up here is 583 00:45:12 --> 00:45:16 obviously made of amino acids, and there are hydrogen bonds which 584 00:45:16 --> 00:45:20 Gleveck can form with the amino acid resides that you're lining on both 585 00:45:20 --> 00:45:23 sides of the cleft. I should have brought you a picture 586 00:45:23 --> 00:45:27 of that. And, a similar kind of hydrogen bonding 587 00:45:27 --> 00:45:31 can occur with the amino acids that are aligning the catalytic clefts of 588 00:45:31 --> 00:45:35 the PDGF receptor and KIT, and that hydrogen bonding can occur 589 00:45:35 --> 00:45:39 already at concentrations that are submicromolar, 590 00:45:39 --> 00:45:43 less than 10-6 molar, 10-7, even sometimes 10-8 molar 591 00:45:43 --> 00:45:48 under certain conditions. So, it's a high affinity binding, 592 00:45:48 --> 00:45:52 and it's relatively specific. Only three out of the 90 different 593 00:45:52 --> 00:45:56 kinases are bound. We can do the following kind of 594 00:45:56 --> 00:46:02 experiment. If I were to add Gleveck to cells 595 00:46:02 --> 00:46:10 with BCR Abel function, this is the response that BCR Abel 596 00:46:10 --> 00:46:17 would show. Here is the response that the EGF receptor would show. 597 00:46:17 --> 00:46:25 So, if I dose the patient at this concentration of drug, 598 00:46:25 --> 00:46:33 Gleveck will shut down the BCR Abel protein. 599 00:46:33 --> 00:46:37 But it won't shut down the EGF receptor, which requires vastly 600 00:46:37 --> 00:46:41 higher concentrations of drug in order to shut down its tyrosine 601 00:46:41 --> 00:46:45 kinase domain. And right here, 602 00:46:45 --> 00:46:49 we can see what we call selectivity. The fact that this enzyme responds 603 00:46:49 --> 00:46:53 at very log drug concentration, this enzyme EGF receptor and its 604 00:46:53 --> 00:46:57 tyrosine kinase, it's a growth factor receptor once 605 00:46:57 --> 00:47:01 again, requires a vastly higher concentration drug in order to 606 00:47:01 --> 00:47:04 elicit an outcome. So, what happened to the chronic 607 00:47:04 --> 00:47:08 myelogenous leukemia patients. The great majority of them between 608 00:47:08 --> 00:47:12 70-80% had a miraculous collapse of their disease. 609 00:47:12 --> 00:47:16 In most cases, this disease could be monitored 610 00:47:16 --> 00:47:20 microscopically. One could look for the immature 611 00:47:20 --> 00:47:24 myeloid cells in their blood and see where they were previously present 612 00:47:24 --> 00:47:28 in vast numbers. They were microscopically now 613 00:47:28 --> 00:47:32 indetectable (sic). However, in those patients where the 614 00:47:32 --> 00:47:38 disease seemed to collapse, one could still use the PCR test to 615 00:47:38 --> 00:47:44 demonstrate there were residual cancer cells in their blood. 616 00:47:44 --> 00:47:49 How could one do that? Well, let's imagine that here is the PCR 617 00:47:49 --> 00:47:55 Abel fusion protein. So, here's PCR, and here's Abel 618 00:47:55 --> 00:48:00 over here. You can make PCR primers, 619 00:48:00 --> 00:48:05 one of which is specific for a PCR sequence, and the other of which is 620 00:48:05 --> 00:48:09 specific for an Abel sequence, and the only time that you'll get a 621 00:48:09 --> 00:48:14 PCR product is if these two sequences exist on the same 622 00:48:14 --> 00:48:19 messenger RNA molecule that's reverse transcribed into a CDNA. 623 00:48:19 --> 00:48:23 You could even do this genomic DNA as well, and so one can specifically 624 00:48:23 --> 00:48:28 detect using this PCR test the presence of cells which have this 625 00:48:28 --> 00:48:33 chromosomal translocation. If one of the PCR primers is against 626 00:48:33 --> 00:48:37 BCR and the other is Abel, and the distances between these two 627 00:48:37 --> 00:48:42 primers is not too far away, not more than, let's say, kilobase, 628 00:48:42 --> 00:48:46 so you get rather efficient PCR amplification. 629 00:48:46 --> 00:48:51 So, it turned out that the great majority of patients who were 630 00:48:51 --> 00:48:55 cytologically cured, cytology means a cytological 631 00:48:55 --> 00:49:00 analysis represents what you see through in microscopes. 632 00:49:00 --> 00:49:04 So these patients, if you looked at a smear of their 633 00:49:04 --> 00:49:08 blood, cytologically they were cured. But if you used PCR analysis, 634 00:49:08 --> 00:49:12 which is far more sensitive, one could detect residual cancer cells 635 00:49:12 --> 00:49:17 that might be present in one out of 105 or one out of 106 cells moving 636 00:49:17 --> 00:49:21 around in circulation, which are almost invisible if you're 637 00:49:21 --> 00:49:25 looking through a very complex mixture of cells through the light 638 00:49:25 --> 00:49:29 microscope. And so, what happened was that patients 639 00:49:29 --> 00:49:34 began to relapse, and after a period of several years, 640 00:49:34 --> 00:49:38 a number of patients began to show a restoration of their CML condition. 641 00:49:38 --> 00:49:43 In fact, in one recent European study, indicates that between 10-12% 642 00:49:43 --> 00:49:48 of the CML patients who were treated with Gleveck relapsed every year. 643 00:49:48 --> 00:49:52 What do I mean by relapse? I mean they show a resurgence of their 644 00:49:52 --> 00:49:57 disease. The disease comes back to life, and they once again 645 00:49:57 --> 00:50:02 have the disease. And interestingly enough, 646 00:50:02 --> 00:50:08 if one now looks at their cancer cells, what do you see? 647 00:50:08 --> 00:50:14 In virtually every case you see some alteration in the BCR Abel 648 00:50:14 --> 00:50:19 protein. In the great majority of instances, you see point mutations 649 00:50:19 --> 00:50:25 that affect amino acid residues lining the cavity here, 650 00:50:25 --> 00:50:31 lining the cavity of the Abel kinase protein. 651 00:50:31 --> 00:50:35 Those amino acid substitutions do not compromise the tyrosine kinase 652 00:50:35 --> 00:50:40 activity of this enzyme. But they do prevent Gleveck from 653 00:50:40 --> 00:50:44 binding, and as a consequence, now you begin to have patients whose 654 00:50:44 --> 00:50:49 tumors are no longer responsive to Gleveck. And what's happened now is 655 00:50:49 --> 00:50:53 one has developed a new generation of compounds which binds into this 656 00:50:53 --> 00:50:58 pocket even in the presence of these amino acid substitutions to retreat 657 00:50:58 --> 51:03 these patients. See you on Wednesday.