"Clinical Research Opportunities in Brain Tumors" and "Molecular Basis of Gut Microbiome-associated Carcinogenesis"

May 05, 2021

Information

Yale Cancer Center Grand Rounds | May 4, 2021

Antonio Omuro, MD and Seth Herzon, PhD

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To CiteDCA Citation Guide

00:00We have two speakers today covering
00:02quite a diverse array of topics,
00:04which is terrific. Both internal
00:05speakers and I encourage people.
00:07If you have questions to
00:09type them into the chat.
00:11And then we'll get those questions
00:13answered when the talks are are finished.
00:15So our first speaker is Antonio Omuro.
00:17You may you may know he he is a professor
00:19of neurology and the chief of Neuro
00:22Oncology here and Clinical Leader Program
00:24leader of the shin of your family,
00:26can bring tumor center,
00:28which is a new program here.
00:30He received his initial
00:31medical training in Brazil,
00:32then worked at Memorial
00:33Sloan Kettering for a while,
00:35and began his faculty career
00:37at University of Miami.
00:38He joined us in 2012.
00:40He's an international leader in their
00:42clinical care and research on brain tumors.
00:45Leading leading pivotal research
00:46programs and treatment of these cancers,
00:48the Genevier family Brain Tumor Center is a
00:50new yellow initiative for the Comprehensive,
00:52multidisciplinary brain tumor.
00:53Karen, perhaps you might hear a
00:55little bit about that from Antonio,
00:57so Antonio, the floor is yours.
00:59Thank you for speaking today.
01:03Thank you very much,
01:04then super like to thank the
01:06organizers for inviting me to
01:07talk to you today and for today.
01:10Specifically, I was asked to
01:11share with you what's happening.
01:13Our division in terms of clinical
01:15trials and how we're tapping into
01:16Yale talent to build our portfolio,
01:18but I would also like to share
01:20with you the state of our fields
01:22and the spirit of almost like an
01:25invitation to even more investigators
01:26and labs to join us in this task.
01:33So today we're going to concentrate
01:35on gliomas and the reason for that is
01:38that they account for the vast majority
01:41of the brain tumors and as you can
01:44see here this is a fight chart showing
01:47all malignant intracranial tumors,
01:48and the vast majority of the patients
01:51have either glioblastoma or other
01:53forms of gliomas which for the
01:55most part our IDH mutants,
01:57which account for grades two and three
01:59others like Thomas and grades too.
02:02In three algorithms, this is 3 or
02:04by semester form as many Germans.
02:07In order rare tumors.
02:09But the bottom line here is that
02:11this even the most common tumor,
02:14which is unfortunately the great
02:16for glioma or glioblastoma,
02:18still is a relatively rare disease
02:20with only three point 1 patients
02:22for each 100,000 people.
02:24So it is again relatively rare disease
02:27Fortunately, but it is, as you know,
02:30a very devastating disease and.
02:32The reason why this is such a charming
02:35diseases that you know the anatomic
02:37location really doesn't help us.
02:40So these are places that presents
02:42with these large tumors with
02:44lots of surrounding edema,
02:46an infiltrative microscopic disease.
02:47These terms are highly vascularized,
02:49so we're at the same time dealing
02:52with an uncle logic disease,
02:54but truly we're dealing with
02:56a neurologic disease as well,
02:58and you can imagine how challenging
03:01it is to manage.
03:03All of these symptoms were still
03:04trying to make a difference in
03:06terms of uncle logic treatments.
03:11Ends reflecting this challenge is
03:13the fact that the only drug that
03:16has shown to improve survival so
03:18far is this alkylating alkylating
03:20agent that is more than 20 years old.
03:23So this is the most dolomite and in
03:25controllers here is saying that the
03:28Missouri might improves both work
03:30for survival and overall survival,
03:32but even the experimental arm
03:34in the pivotal trial,
03:35which was published in 2005.
03:38Survival remained only 15 months for again,
03:41newly diagnosed disease and further
03:43analysis of this data has shown that
03:46this survivor benefit is mostly driven
03:49by tumors that have this afternoon.
03:52Check silence of the Mt gene
03:54promoter by methylation so these
03:56patients with math Laden GMT tend
03:59to respond better to Tim's or mine,
04:02but they account for only
04:04about 30% of the patients.
04:06So for the remainder of the patients.
04:09The only real treatment that
04:11is available is radiation.
04:15And we did try a lot of agents,
04:18and here you're looking at a slide
04:21from 2005 where we were talking
04:23about all of these clinical trials in
04:25glioblastoma and in other diseases.
04:27Testing these novel target therapist.
04:29So we're very excited that for
04:31the first time in would be able
04:33to treat these patients with
04:35therapies that would carry minimal
04:37toxicities and tremendous efficacy,
04:39but as you know, the story was much more.
04:44You know, harder than than what we
04:47originally thought, and one by one.
04:49All of these stars went on to
04:53fail in recurrent disease.
04:55The sad thing is that or maybe the
04:57lucky thing for other diseases is that
05:00the majority of these drugs ended up
05:02being approved for other indications,
05:04but all of that rise in glucose
05:08Thomas have failed.
05:10And more challenging is the fact that
05:12we're not really sure what is it about
05:15the omens that all of these drugs
05:18actually fail one after the other,
05:20is that because we are targeting
05:23the wrong targets,
05:24maybe they're not sufficiently
05:25relevant for Uncle Genesis,
05:27or there are too many feedback
05:29loops and redundant pathways were
05:31now more and more learning about
05:34temporal spatial variations?
05:35Or is it be 'cause these
05:37are the wrong drugs and?
05:39We have problems you know,
05:42of achieving adequate concentrations,
05:43especially for drugs.
05:45They are not very potent.
05:47We do need to have better blood
05:51brain barrier penetration because a
05:53lot of these microscopic disease is
05:56behind an intact blood brain barrier.
05:59Also,
05:59we still don't know how to
06:01select basis for these drugs.
06:03We're still not sure if it should select
06:06based on specific mutations or should
06:08we go through transcription subgroups or not?
06:10Do any selection whatsoever and treat
06:12a large number of patients then then
06:15identify the responders and then go after
06:17the phenotypes that predict response.
06:19So in other words,
06:20regardless of what we do,
06:22we certainly need to improve translation
06:24components within our trials,
06:25improve the science before, during,
06:27and after the trial and this is.
06:30Actually,
06:30paradigm that we have
06:31been following artificial.
06:36So the low hanging fruit is to try
06:39to use the genomic information
06:41that is now widely available.
06:43Only streamers to see if we
06:46can improve our outcomes.
06:47So as you know, global someone was the very
06:51first tumor sequenced by the TSJ effort,
06:54and since then gene sequencing
06:55has become the norm when managing
06:58these patients and here looking
06:59at all types of biome's and these
07:02different colors here represents
07:04the different subtypes of gliomas.
07:06And you have no difficulty to see that.
07:10The genomic signatures are very
07:13distinct across the different
07:15histologies you can see here.
07:17The quintessential signature of the
07:19algal blooms, which is more penalty?
07:22Q coalition ideate mutation Sir
07:25promoter mutation and see I see
07:27and if you put P1 and here is the
07:31quintessential signature of Astros
07:33with guided meditation AT Rex,
07:35Magician or lost interpretive fermentation.
07:37And here's this essential signatures
07:40of global stoma.
07:41Now we start to see Jeff Farm
07:44to fication or mutation Pete.
07:46Then loss or mutation and lots
07:49of formalities in CD case.
07:51So putting those patients now,
07:54arranging them into what kind of pathways
07:57ended up being abnormally disturbers,
08:00we can see the vast majority of
08:04patients follow this cake recipe.
08:06So basically 1000 kinase pathway with
08:10PKU KTM Tor pathway activation and F1
08:13you see also a lot of these patients
08:17with arousing in the T3 pathway leading to.
08:22Abnormalities in senescence and a pop
08:24ptosis and a lot of these patients
08:27having a normality's in cell cycle control.
08:31But then when we put all of these
08:34patients a match to actually which
08:36mutations have a track record of
08:39being drug and what you can see is
08:42unfortunately each of these mutations
08:44is actually very where we're not
08:47being very good at identifying
08:49drugs for those specific phenotypes
08:51we heavily rely on basket trials.
08:53But unfortunately basket trials
08:55typically exclude patients with
08:57brain tumors were left with no
09:00trials or very trials that address.
09:02These questions we do have
09:03some low hanging fruits.
09:05Of course ideas mutation will
09:06talk a little bit more about that,
09:09but again,
09:09the message here is that it is
09:12very difficult to run start
09:14therapy trials of these days.
09:16Because you really need to have
09:18strategies to tackle the rarity
09:20of each of these phenotypes.
09:24And adding to our challenges are how
09:27these tumors evolve overtime and how
09:30they are heterogeneous to begin with.
09:33So this is a patient, for example,
09:36that at diagnosis she was enrolled in
09:39one of my trials of a notch inhibitor
09:42and she had a very typical signature
09:46of astrocytomas with identification
09:48interaxon to 53 mutations and several
09:51potential target targetable abnormalities
09:52with other abnormalities, but.
09:54When this patient again,
09:57she received the nearly diagnosed.
10:01Trial and then when she recovered,
10:03she was operated on again,
10:05even though she had a very small tumor.
10:08And what we found is that all of those
10:11potential target mutations are actually gone.
10:13We're seeing some passengers here.
10:15But the reality that's what's driving
10:17this tumor now is actually probably about
10:20Melanie's at the OBGYN attic level,
10:22and you can imagine that if at this
10:25point in time of her disease we work
10:28to enroll her in a clinical trial.
10:31Most patients do not have another brain
10:33surgery to have another sequence,
10:35so you go to archive tissue and we
10:38would have selected her for trials
10:40that probably were irrelevant for
10:42her at this point in time.
10:44Again,
10:45those are males that we thought
10:47were present were actually gone.
10:50This is another example of potentially
10:54targetable mutations that actually were
10:57very different at the time of recurrence.
11:00And.
11:00Another difficult challenge are
11:03these patients here.
11:05So these are patients that we serve created.
11:08These is a result of the use of the
11:12Mozilla might that can cause mutations in
11:15mismatch repair genes at typically MSH.
11:186 and what happens is that these patients
11:22with mismatch repair defects start
11:24accumulating all of these mutations
11:26and you can imagine that developing
11:29target therapies for these folks.
11:32Is much harder.
11:33And one of the surprising findings
11:36of our studies have been that
11:39these are actually much more common
11:41than we previously thought.
11:47So in moving forward what we're trying to
11:49do is to again improve the science linked
11:52through the early development trials,
11:55so we more and more relying Phase
11:57Zero tries to show us if our drugs
12:00are actually getting into the brain,
12:02especially in areas with intact
12:04blood brain barrier.
12:05We also want to see if the.
12:09The drugs are hitting their targets and
12:12we like to look at the pharmacodynamic
12:14effects in these resected specimens.
12:17Be more and more have we have to
12:19work with their companies to have
12:21basket trials that actually include
12:24patients with our rare phenotypes.
12:26There's a shift towards more
12:28of a newly diagnosed disease.
12:30Be 'cause these are easier patients,
12:32and the genomics information is
12:34actually fresh, and where we're
12:36dealing with recurrent disease,
12:37we typically like to re
12:39sample specials for target.
12:41Therapies, if anything,
12:42at least to exclude the hypermedia phenotype.
12:44And we also like to of course update the
12:46gene sequencing and the Uncle Genic trimers.
12:49Another trend in our field, this try
12:51to target these strong communications,
12:52but that's not an easy task.
12:54And again,
12:55we're going to talk a little
12:57bit more about that.
12:58But the vast majority of trials right now
13:02is actually trying to find alternative
13:06strategies that address more stable targets.
13:09So the low hanging fruit of stable
13:12targets is actually immuno therapies.
13:14So we do know that blue,
13:17blasphemous do grow in a very
13:19human suppressive microenvironment.
13:20And we have identified several
13:22emergent points that seem to be very
13:25important in this disease.
13:27But on top of identifying
13:29the right even checkpoint,
13:30we have the challenges of the
13:33anatomic location itself.
13:34So you can imagine that it's much harder
13:37to trigger him and logical response.
13:40In the brain,
13:41which is,
13:41you know,
13:42traditionally considered the so-called
13:43sanctuary sites for the immune system.
13:46And we have to get these email
13:48responses to act fast because these
13:51are tumors that grow very rapidly
13:54and they cause symptoms and we
13:56don't have the luxury of waiting
13:59several months or years to react.
14:01The benefits of the email of therapies.
14:04And of course,
14:05if you're triggering inflammatory
14:07responses in the brain,
14:09we have to deal with the risks of
14:11new log symptoms and neurotoxicity.
14:14An another important thing is.
14:16That this information could
14:18potentially mimic some aggression,
14:19so managing these patients can
14:21be challenging because we have
14:24to learn to how to recognize,
14:26see the progression versus real
14:27tumor progression on the MRI.
14:34But we did try and here you're
14:36looking at the very first results
14:39of the very first phase one trial
14:41utilizing image checkpoint inhibitors
14:43in global stoma and this was done in
14:47with VMS and in this trial we treated
14:5040 patients both with nivolumab or
14:53two combinations of Nivola Bintulu
14:55map and what we found is that yes,
14:58the target definitely was present
15:00in the majority of patients,
15:02so 60% of the patients had
15:05PDL one expression.
15:06But we didn't see any brain toxicities
15:08which is good and perhaps bad because
15:10this could potentially reflect the
15:12fact that we are not achieving
15:14much and overall survival was very
15:15similar to historical controls,
15:17although some places it seemed to
15:19Mount more of an email response.
15:21But this went on to be tested in
15:24randomized trials and we are now
15:26reporting the final results of
15:28these shows and one by one they
15:30all failed to improve survival,
15:32both newly diagnose and recurrent disease.
15:37So we're not giving up on immunotherapy,
15:39so I think our task now is to try to
15:42send what is that about the brain?
15:45That in spite of PDL one expression
15:48we're not seeing any help from Anti
15:51PD one or anti PDL one therapies and
15:54I think for for this question I think
15:57it is great to have a helping hand of
16:00people that study the CNS immunology
16:03and in this project what we did is to
16:07partner with Doctor David Hoffer and
16:09I'll also Liliana Luca to look at how
16:12can we actually come up with better.
16:16Image checkpoint inhibition that is
16:18relevant for for this Mike environment
16:21and what the heifers lab came up with
16:24is that this image of point called digit
16:27seems to be much more relevant in the brain.
16:31It was very interesting that in
16:34their studies they found a lot of
16:36teacher expression in DBMS and not
16:38so much digit expression in the
16:41quintessential inflammatory disease
16:43which is multiple sclerosis and.
16:45They went on to perform
16:48several studies utilizing,
16:49so sequencing that sort of confirmed
16:52that T cell dysfunction was being driven
16:55by digit in this particular disease.
16:58So to test this hypothesis in
17:00patients with design,
17:02this clinical trial where we are looking at.
17:06A different cohorts of patients prior
17:09to surgery where they will be treated
17:12with either infected or anti PD one
17:14or the combination or placebo and
17:16then these patients will be brought to
17:19surgery and then we will do a tumor
17:22single cell RNA sequencing with an
17:24axe as well as some studies to produce
17:28some spatial validation of the findings.
17:30And there will also follow these
17:33patients longitudinally to see if we
17:35can monitor what's happening in the tumor.
17:38By analyzing the T cells in the periphery.
17:41So it's a very exciting trial.
17:43So I wish we had started the
17:45development of Inter PD one this way
17:47by understanding the science before
17:49going to more or larger studies that
17:52would end up being negative,
17:53but we're very excited about this mechanism.
17:56Action also is important to emphasize
17:58that this combination of anti teachers
18:01and in fact PD one is very hot in
18:03the fields right now as you know it
18:06is already in phase 3IN.
18:07Non small cell lung cancer.
18:09And we're very excited to bring
18:11this trial here to you.
18:16Also, to understand a little
18:18bit more of the immune system,
18:20we need a good models that are
18:24immunocompetent and one of the.
18:26A richness of our environment
18:28here is Doctor City chains work
18:30producing these jam models of global
18:33stomas where he can pretty much
18:35produce avatars for all of these
18:37phenotypes that I just showed you,
18:40and one of the ideas here is to see
18:42how these different phenotypes respond
18:45to these different immunotherapy's.
18:47So this is very exciting data
18:49which again illustrates how we can
18:52concomitantly to the development in
18:54the clinic to also try to understand.
18:57Are these treatments in parallel in the lab?
19:02Now another barrier for.
19:07For the development of effective
19:09even responses is the work
19:12being done by the Iwasaki slab.
19:14So Akiko has been working with
19:17Eric Song and General Thomas,
19:20and she has recently had this nature
19:24paper where they showed that.
19:27There is a defective lymphatic
19:29drainage from the brain that you
19:32can correct utilizing the GFC.
19:34So in her models that the combination
19:36of Veg FC and Anti PD one actually
19:40improves survival and was also
19:42interesting that they also produce
19:44some experiments by injecting anti
19:47PD one directly into the CSF and also
19:51the results seem to be better than
19:54systemic administration of anti PD one.
19:57So this is all giving rise to another
20:00generation of characterizing and
20:02some new compounds that we hope to
20:05bring to clinic in the mid term.
20:10Now also again another important
20:12barrier in Spanish solid tumors,
20:14but particularly in glomus,
20:16is the role of tumor associated macrophages
20:19and how they produce these emails.
20:22Suppressive tumor convergence
20:23and one of the ways that we could
20:27potentially intervene in this was
20:29discovered by an item here at go,
20:32where she's looking at the role of
20:35this little Robo one, signaling
20:37which seems to attract and polarize.
20:40Save Microfridge is in in the
20:42brain microenvironment and
20:43Livingstone my confirming.
20:45And when she did experiments to knock down,
20:48slid to, or to block this pathway,
20:51she achieved better immune responses
20:53and inflammation of anti PD one.
20:55She had a really significant improvement
20:57in survival or in this tumor bearing mice.
21:00So the idea here is now to generate
21:03enter Robo Nanobodies one of the
21:06barriers project would be then
21:08how can we get this number?
21:10At least to penetrate into the brain.
21:13And since she's very resourceful,
21:15she has the answer.
21:17It looks like.
21:18If you block antibodies,
21:20if you use antibodies blocking
21:22this receptor called UNC 5B,
21:24you conserve produce an on demand
21:26blood brain barrier opening,
21:28so this is less a few hours and it's
21:31great for drugs up to 40 kilodaltons.
21:34So the idea here is that if this is
21:37successful, we could combine this.
21:39These agents with many of the
21:41chemotherapies in order target therapies
21:44that we are trying to use to treat
21:47these patients in a more efficient way.
21:50And overcome the problem of living
21:51there countries so very exciting
21:53work that we hope to see more of.
21:57No moving on into.
21:59It's still sticking to the Mockingbird,
22:02but moving on to partnerships with pharma.
22:05One of the our partnerships
22:08is with this drug.
22:11This company called In Pharmaceuticals
22:13and these folks have discovered
22:16a novel receptor with within the
22:18Alpha V beta three integrin that
22:20is started by this FP PMT drug
22:23that seems to have an amazing
22:25activity in their mouse models.
22:27Really melting the mice.
22:29And this was the first.
22:31Now we're now designing the 1st
22:33in human trial here GAIL,
22:36that will start in a couple of months.
22:39But to understand this better we
22:41did bring Yellow Labs into the mix
22:44to better define how is this rug
22:46really working and who are the
22:48best candidates by understand a
22:50little bit more about the effects
22:53on cell invasion signaling networks
22:55and gene expression.
22:56So one of the assets that.
22:59We're realizing in partnership with
23:02under left ankle is looking at these.
23:05The use of his integrated platform,
23:07which is the so called race essay
23:10which is a disrupted analysis of cell
23:13phenotype extremes where he uses
23:15the cell migration as a surrogate
23:18marker of tumor aggressiveness and
23:21and then you can test the multiple
23:24drugs utilizing this essay as
23:26a form of drug screening,
23:28and he's applying this rug.
23:31Your days were very interesting results
23:33and we hope to then identify partners.
23:37Which are the best genomic candidates
23:39and then see if we can optimize
23:42the trial as we go by in reaching
23:45with either best candidates or
23:48potentially novel combinations.
23:50So again,
23:51that's just to illustrate that
23:53it's very important to really
23:56involve our laboratories.
23:57Even in trials that are
24:01being conducted by pharma.
24:03Now sticking again,
24:04not now moving on to other
24:07more stable targets,
24:08and one of them is ideas with patient
24:11and this story came out of Doctor
24:14Kendra's lab where he found it ideas.
24:17Mutations change DNA repair through the
24:19production of two hydroxy obliterate,
24:21which is the byproduct of this mutation,
24:24and this results in sort of brokenness
24:26that then can be targeted by PARP inhibitors.
24:29So he has several clinical
24:32trials of these park inhibitors.
24:34And we are now hoping to see
24:37if this will actually improve
24:39outcomes for these patients.
24:44Also, again sticking to the DNA repair thing,
24:48we recently submitted a United team led
24:51by Mayo Clinic and John Jennifer Correa
24:54in partnership with even colonies.
24:56Doctor Bindra and I.
24:58So we have two projects.
25:00One is trying to optimize MDM two
25:03inhibition for these patients and 80
25:06Rd in ambition for these patients.
25:08And this will again bring two other
25:11Phase 0 slash 1 clinical trials.
25:14To our portfolio, hopefully soon.
25:21Now we don't have time to
25:23review all of our portfolio,
25:25but we do have partnerships with
25:27industry for opening other tries
25:29to fill in gaps in our portfolio.
25:31Doctor Blundin has activated the
25:33Agile trial which is a multi drug
25:36multi arm clinical trial that is
25:39happening worldwide so we have access
25:41to these drugs for our patients
25:43and have a bunch of other choice.
25:45But the theme here is really to focus on
25:48early therapeutic development and then
25:50participating inside cooperative groups.
25:52Please, for those extremely rare phenotypes,
25:55for example,
25:56byref mutant craniopharyngioma switch,
25:58again very difficult to find patience
26:01and for those we do need to partner
26:05with other places around the country.
26:09And I could go on and on talking
26:11about all of the years signs
26:14that is going in brain tumors.
26:16I select a few stories that
26:18are closest to clinic,
26:20but all these people in this picture
26:22and many others that I'm not even
26:25mentioning today are producing amazing
26:27size that we can actually use into
26:30our portfolio and bring it in anymore.
26:32Let's say intelligent trials
26:34ranging from data science,
26:35junior imaging and all sorts
26:37of therapeutic approaches.
26:39So in conclusion,
26:40so we're lucky enough to have this
26:42unique breath of scientific expertise.
26:45Our focus is really on investigating
26:47shaded trials that are home grown
26:49and our other focuses on early stage
26:52development with former partners,
26:54but also bringing along our
26:56own labs email collagen,
26:57a repair have emerged as leading teams,
27:00but here there we have many patents about,
27:03although many are not ready
27:05for complication and need a
27:08lot of help for development.
27:10We certainly need more work
27:12on existing available drugs,
27:13for example coming from Seatac
27:15and Pharma and a lot of work in
27:17functional genomics so that we
27:19can figure out finally how to
27:22target these undruggable targets.
27:23So that I would like to finish
27:26by thanking all of the people.
27:28So when we talk clinical trials,
27:30really the merit is all of others of the
27:34labs of the all of the infrastructure.
27:37I would also like to acknowledge
27:40our division attendings and aips,
27:42or actually managing treating
27:43these patients in the trials.
27:45I would like to thank again the Cito staff.
27:49They're going through rough times,
27:50but right Decker is navigating and it's
27:53going to get us out of this situation.
27:57A big thanks to the PRC reviewers
27:59'cause one of my hats is actually as
28:02the Pearcey chair and we we acknowledge
28:04along with Barbara Burtness that there
28:07was a lot of work that goes into
28:09this and that I would like to thank
28:12them publicly at this opportunity.
28:14Lots of things.
28:15So why CCI that help us with
28:17investigating share clinical trials.
28:20All of the people that have been
28:22enabling this research and finally
28:24a big thank to the YCC and Smile
28:26leadership with more Pickens.
28:28Kevin versus Kevin Beans loosely,
28:30and neither will all understand importance
28:33of our clinical trials portfolio.
28:35Last but not least, again,
28:37I would like to thank the show her
28:39family for their generous gift.
28:41In fact, then I'm not going to
28:43talk about this today because we're
28:45still working on the details,
28:47but the word is out of the receive a
28:49generous gift from that foundation,
28:51and we're hoping to put together a
28:54nice program that will again enable
28:55and expand on our research efforts.
28:58Thank you very much and I'll take
29:00some points if you have time.
29:03OK, thank you very much Antonio.
29:05Very interesting work.
29:06Are there any questions that people
29:09want to enter into the chat?
29:11While we're waiting,
29:12I have a quick question.
29:13You mentioned this idea
29:14of opening up the bread.
29:16The blood brain barrier
29:18by targeting a molecule.
29:20Is it worth going back to some
29:21of the earlier drugs that
29:23weren't terribly effective to
29:24see that whether or not that
29:26might help them work better?
29:28Yes, I think there is a whole list of drugs
29:33that perhaps will need to be revisited.
29:37Although most of these drugs would
29:40actually be again in rare phenotypes,
29:42because I think those are,
29:44we still need to select them
29:47by those specific mutations.
29:48The problem of copy number remains
29:51regardless of flipping connectors.
29:52I don't think blood brain barrier penetration
29:55was the reason why we couldn't target
29:58EGFR amplification or Pete and loss.
30:01I think that is a different question,
30:03but if we are to even answer
30:06those we still need.
30:08This kind of approach,
30:09'cause it makes our life so much easier.
30:14Are there any questions from the audience?
30:23I was also struck by the lots of different
30:26mutations upon recurrence. He showed.
30:31What is that thought to
30:33be due to is just so high
30:36perforation rate of these tumors.
30:38Yeah, well, I think so.
30:40First of all, these tumors are very
30:42heterogeneous to begin with, right?
30:44So these are guns that are
30:46were there to begin with,
30:48but it looks like the treatment process
30:51ends up eliminating a lot of this so
30:54called cancer associated mutations.
30:56Another unknown mutations emerge and also
30:58a lot of these are actually epigenetic.
31:01Changes.
31:01So there's a whole line of research
31:04trying to then understand this and
31:07more canals are interested in in that
31:10line of research and other labs to
31:12see how we can target these tumors
31:15at recurrence that are sort of,
31:17you know,
31:18very simple from a genomic standpoint,
31:20but not so simple at the epigenetic level.
31:23Well, thank you very
31:25much. Very interesting.
31:26We have to move on to the second speaker.
31:31In our second stewartii climb down
31:34from the Hill from Science Hill,
31:36is Seth hairs on who's the Milton
31:39Harris professor of Chemistry
31:41received his PhD at Harvard and then
31:44post Doc at University of Illinois,
31:46and he's interested in natural
31:49product's particular products that
31:52affect the synthesis or damaged DNA.
31:55And he's received numerous
31:56multiple Young Investigator awards
31:57and working with Jason Crawford
31:59is a terrific collaboration.
32:01Looking at them,
32:02the metabolites made by the human
32:04microbiota and identified some of
32:06them that actually damaged DNA and
32:08therefore contribute to cancer.
32:09So Seth, we're looking forward
32:11to hearing about your work.
32:13Thank you.
32:15OK, thanks Dan,
32:17thanks for the introduction.
32:19And thanks to all to
32:21everyone for the invocation.
32:22Comment for attending the lecture.
32:26I will talk today about work we've
32:27been doing in the human microbiome,
32:29but actually ignore it.
32:33Just calling on ever. Snap because.
32:38Cave against drug resistant TMZ resistant.
32:40GBM that we're very excited about but
32:43that will be a story for another day.
32:49And so right. So today I'll talk
32:52about a project that's been ongoing
32:54in my group for about 6 years.
32:56And we've been looking to understand
33:00the molecular basis of a carcinogen
33:03carcinogenic phenotype that was
33:06observed from certain gut bacteria so.
33:09I'll go through sort of the sequence
33:12of events to kind of outline sequence
33:15of discoveries to outline the problem,
33:17and so in 2006 this was the paper that
33:21set off a lot of interest in this area.
33:25Eric Oswald and coworkers identified
33:28certain strains of commensal and
33:31pathogenic E coli that had a biosynthetic
33:35gene cluster known as the CLB cluster.
33:38So by that I mean that gene genetic
33:42locus contains the coding for enzymes
33:46that make a secondary metabolite and
33:49he took these CLB containing bacteria
33:53and did a transient infection.
33:56HeLa cells with them and then
33:58looked at the effect on the cells,
34:02and he found that they underwent
34:04cell cycle arrest.
34:05Meglos cytosis and using a comet assay.
34:09Another Gamage to XD he saw that
34:12they accumulated double strand
34:15breaks in their DNA.
34:17And so this is a very interesting phenotype.
34:19It's not the first time.
34:22Microbes, have, you know,
34:23produced Gina toxins,
34:24but it was was was a very interesting
34:28example and I'll come to in a second wait.
34:31Why it's attracting so much attention?
34:34Subsequent to that report,
34:37there's been numerous studies
34:40trying to ascertain whether or not
34:43there is a role for these bacteria
34:47in colorectal cancer formation
34:49and from the same group in 2010.
34:54It was shown that in in
34:58intestinal loop models of.
35:01My step or infected with CLB bacteria
35:04they observe DNA damage in vivo.
35:07They observed gamma H2 X they observed
35:11increased mutations in the HP RT&TK
35:14loci and then also hyperproliferation
35:16following exposure to the bacteria.
35:19So they seem to be driving tumorigenesis
35:22and then there were subsequent
35:24studies following up looking at
35:27similar types of in vivo effects.
35:30So using IL.
35:32Knockout mice,
35:33it was shown that infection with
35:36these bacteria leads to a higher
35:39rate of tumor formation and then
35:42there were three groups that did.
35:45Meta analysis of of ***** samples
35:48from from CRC patients and what
35:51we find is that about 60 to 70% of
35:55CRC patients have these bacteria
35:58and that's and that's versus about
36:0120% in the healthy population.
36:02And the other sort of bit is that
36:05the preponderance of these bacteria
36:07tracks with the severity of the cancer,
36:10so people with more advanced CRC were
36:12at the high end of that correlation,
36:15whereas people with early stage
36:17CRC were more at the lower end.
36:21And so it wasn't really until last
36:23year that a causal relationship
36:26was unequivocally established.
36:28There were two studies from mayor
36:31and then Boxtel and Cleavers an
36:34in the Cleavers study.
36:36They generated an organoid and
36:40infected that organoid chronically for
36:43about three or four months with the
36:48CLB positive bacteria and what they
36:52showed is that you get the mutational
36:56signature transformation and proliferation.
36:59We also find that that mutational
37:04signature is found.
37:06Enriched in in CRC patients as well,
37:08and so the mayor study came
37:11to similar conclusions,
37:12and essentially these two papers you know,
37:15this is a rare example in the microbiome
37:18where you actually establish causation.
37:20So these two papers brought this
37:23phenotype to the two sort of
37:26a causal level and what my lab
37:28has been trying to do of course,
37:31is understand the molecular
37:32basis for all of this OK and so.
37:39Oswald, in his initial paper,
37:42had done a series of very nice and you know,
37:48robust control experiments to establish
37:51that this genotoxic phenotype.
37:54Is due to the final biosynthetic
37:56product product of the CLB cluster.
37:59In other words, if one modifies any
38:01of the enzymes in the CLB pathway,
38:04you lose this genotoxic phenotype OK,
38:07and so the implication then is that
38:10it's the fully elaborated molecule.
38:13That is the active toxin,
38:15not something in route to
38:18another another product.
38:20And we call that molecule Coley bactine.
38:23And. So the field basically
38:26set out to do what we do best,
38:30which is isolate compounds and the
38:33classic way of isolating natural
38:35product secondary metabolites is to
38:38culture the Organism of interest.
38:40In the case of bacterial
38:43secondary metabolite,
38:44you might grow it in liquid culture,
38:47growing on scale.
38:50Extract start to fractionate by
38:52HPLC and then we typically do.
38:55It's known as activity guided fractionation,
38:57where you're essentially testing each of
38:59these fractions for a particular phenotype.
39:02And then you keep purifying and testing
39:05and purifying testing until you get to
39:08a single compound and you characterize it.
39:11The problem is that this
39:13approach does not work for Kohli.
39:15Bakhtin, OK,
39:16so the molecule is very unstable.
39:19It is very difficult to get the bacteria
39:24to express the CLB pathway ex vivo.
39:27And what we find is that because of the.
39:32You know,
39:33primarily anaerobic environment of the gut.
39:35The molecule actually undergoes oxidative
39:38degradation when you attempt to isolate it.
39:41Sort of on the bench under air,
39:44and just to to.
39:47Give you an example of how
39:48challenging this is.
39:49This is not work from our own laboratory.
39:51This is a group at at Berkeley and scripts.
39:55They've been pursuing Cali,
39:57backed in in the isolated this.
39:59Molecule here in 2019 they obtained 50
40:03micrograms from a 2000 liter fermentation.
40:07If anyone can imagine that,
40:10so we're talking about literally
40:13vanishingly small quantities.
40:17And they they advanced this molecule
40:19as a candidate calling back,
40:21and unfortunately this was derived from a
40:23triple mutant Frankenstein like bacteria,
40:25and I wrote a commentary.
40:26If you're interested on this at
40:28the general thinking in the field
40:30is this is probably not relevant
40:32to the genotoxic phenotype.
40:34But the point is,
40:35these are the links that people
40:37are willing to go to to try
40:39and isolate these molecules.
40:44And so, how do we approach this?
40:47So, as Dan mentioned,
40:48we've been collaborating with Jason Crawford.
40:51Jason is one of the leaders
40:54in understanding Kohli,
40:55backed in biosynthesis.
40:56And So what we've been doing is really
40:59taking knowledge from the biosynthetic
41:02pathway and trying to infer what types
41:05of substructures might be within Kohli.
41:08Backed in itself and how
41:10those might interact.
41:12With DNA and so one of the sort
41:15of models that came out of these
41:18biosynthetic studies is that you
41:20have these fully linear products
41:23offloaded from the PKS assembly line.
41:26There's a searing protease that
41:29removes this residue and blew this ACL.
41:32Asparagine residue.
41:34That generates a primary amine,
41:36and once you format that can start
41:39to wrap up and ultimately lead to
41:42this compound on the bottom here,
41:44which has a cyclopropane ring in
41:47conjugation with with it with the Alpha,
41:50beta unsaturated imming and for
41:52those in the audience that have
41:54worked with Gina toxins,
41:56you know that these electrophilic
41:58cyclopropane's are not uncommon.
41:59This is a sort of a pharmacophore that's
42:02found in a variety of genotoxic natural.
42:05Products and so this was,
42:07you know,
42:08sort of very logically following
42:09from that type of precedent.
42:11The problem is that the problem is this.
42:14No one had isolated these imines.
42:16No one had any spectroscopic data on them.
42:19All we had was this.
42:21This kind of this mechanistic hypothesis.
42:24And so we set out to make it,
42:26and I'm not going to have time to go
42:29through all of the synthetic work that
42:31that went into developing these roots.
42:34But the key steps are shown here.
42:36So we start from this linear precursor
42:38and what we find is that if we
42:41concentrate this down from dilute acid,
42:43we can get this.
42:45Carbon and nitrogen to condense
42:47onto the ketone. You found this.
42:49Finally this image.
42:50We then do a bond formation
42:52deprotect the Bach route to get
42:54to this compound on the left,
42:56we isolate this as as it's TFA salt.
43:00But if you neutralize this,
43:02it's snapshot and so this
43:04carbon attacks this ketone,
43:05you lose water, any formatting.
43:08And.
43:11The assay that we use, that's 'cause
43:13it's nice to give us a lot of detail.
43:15It's inexpensive, it's fast, is is.
43:19Taking linearized plasmid DNA
43:21incubating with the molecule,
43:23running a denaturing gel,
43:24and basically if you look at
43:27the right hand lanes here,
43:29you see these streaks on the gel
43:32going down to about 100 animal or what
43:35that tells us is that at 100 animal
43:38or concentration of this compound,
43:41we're getting extensive
43:42degradation of the DNA.
43:44These are smaller fragments that
43:46have higher mobility on the gel.
43:49And so that was very exciting to us.
43:52And you know,
43:54we hypothesized again that it
43:56was this nucleotide addition to
43:58the cyclopropane that was leading
44:00to this degradation of the DNA.
44:03And so to probe that in a little
44:06bit more detail,
44:07we made a couple of control compounds.
44:10So the first one.
44:12Was this dimeric structure up
44:14top here and so the hypothesis is
44:18that if this is alkylating DNA,
44:21perhaps we can induce two fold
44:24alkylation and perhaps we can then
44:27detect and interstrand crosslink?
44:30And when you incubate with that compound,
44:33indeed you can see down here Crosslink Band.
44:36This corresponds to our positive
44:39control for crosslinking cisplatin.
44:41And then the other thing we did was
44:43we made a negative control where we
44:46took that cyclopropane and converted
44:49it to the gem dimethyl substituent.
44:51The hypothesis being if the
44:53cyclopropane is truly involved,
44:54this compound should be inactive
44:56and going up to half millimolar.
44:59We don't detect any damage in our
45:01assay and so without characterizing
45:03the product without even having
45:05isolated the natural products,
45:07we were able to sort of formulate this
45:10proposal for how these things might be.
45:13Might be alkylating DNA.
45:17And. We are, you know,
45:20we sort of at that point.
45:22Got stocks so that was around
45:232018 when we had identified this.
45:25You know, this DNA reactive
45:27substructure in the molecule.
45:28We knew that it was incomplete.
45:30In other words,
45:31there were other functional groups,
45:33other rings and things with in Cali bactine.
45:35But we didn't know what they were.
45:38And as I mentioned in the beginning,
45:41the you know the classical
45:42isolation approach is not very
45:44successful in this in this context,
45:46and so we were stuck.
45:49Until this paper came out,
45:51and so this is also from the Oswald Group.
45:55They did a beautiful experiment
45:57where they took the collie,
45:59backed in producing bacteria,
46:01grew them up in liquid media,
46:04added exogeneous DNA,
46:05isolated that DNA following incubation,
46:07and ran a denaturing gel and the point
46:10is that they observed interstrand
46:13crosslinks in that DNA that was
46:16exposed to these bacteria OK and so.
46:19I have a note here at the bottom to remind
46:22me you know if you're paying attention.
46:25The original female phenotype
46:26was double strand breaks.
46:28Now I'm talking about cross links.
46:30Those two lesions are are intimately related,
46:32and I'll talk about that at
46:34the end if there's time.
46:36But basically we're very excited about
46:38this paper because you can imagine that
46:40Kohli bactine is entrained in that crosslink,
46:43right?
46:43You know,
46:44if that's what's causing the crossing at that
46:46point wasn't completely certain that either,
46:49but.
46:49Assuming that it is all we have to do,
46:52all we have to do is isolate that
46:54crosslink can characterize it.
46:57And so at this point in time, you know.
47:0290% of the biosynthetic
47:03pathway had been mapped out.
47:05We had a very good understanding of what
47:09went in of the amino acids that went into
47:13the pathway and where they ended up.
47:16Following sort of offloading
47:18of the biosynthetic products,
47:20and so for example, it was known through
47:24work that Jason did very early on,
47:27and then others that this amino
47:30cyclopropane comes from methionine,
47:32and these thiazole rings
47:34derived from cysteine OK.
47:37And So what?
47:38This allowed us to do is conduct
47:41essentially isotope labeling experiments
47:44where we generated auxotrophic strains,
47:48either deficient in methionine
47:50or cysteine biosynthesis,
47:52and then supplemented those cultures
47:55with C13 labeled amino acid OK.
47:59And so we can take the wild
48:02type strain the oxygen riffs
48:04with their amino acids incubate.
48:06Here we're using linearized puck 19 DNA.
48:09We can run a gel to verify
48:11that we got crosslinking,
48:13and then we can try and isolate that
48:16cross link and characterize it,
48:18and one of the things that's worth
48:21pointing out is that to do these assays
48:24were talking about 250 microliters
48:26of culture versus 2000 liters.
48:28You know using the old?
48:30The sort of the old fashioned method,
48:33and so to give you an idea of
48:35what the data looks like and
48:37why we do this isotope labeling.
48:39I'll show you this slide.
48:41So for example,
48:42we can spot these ions that I've
48:44marked in colored boxes here,
48:46and the top chromatogram is
48:48the wild type strain.
48:50And what you can see in the Sistine
48:53Auxotroph the middle graph is that
48:55those ions are shifted by three units,
48:58and so that's very useful to us
49:00because it tells us two things.
49:03One is that that Ion is probably
49:05contains Poly back in or the vestiges
49:08of Cali Bactine and then two.
49:10It contains one thiazole residue,
49:12there was one cysteine incorporated
49:15into that unit.
49:17And we can play the same game
49:18with the methionine auxotroph.
49:20So here we get a shift by plus four.
49:22So that tells us there's 11 amino
49:24cyclopropane and tells us it's
49:26also related to Cali Bactine.
49:27OK, so this was the initial work that we did.
49:31We had to carry out a lot more
49:34labeling in order to get the full
49:38structure assignment and So what we
49:41did was we generated a series of we
49:44had our cysteine and methionine auxotroph.
49:46We generated steering and glaci Knox
49:49Atros because those are also incorporated
49:52into the into the natural product.
49:54And then we also did Universal labeling C
49:5813 labeling with glucose an 15 labeling.
50:01With ammonium chloride.
50:03And we can run the same experiment
50:06where we incubate with the DNA,
50:09isolate the crosslink,
50:10digest it,
50:11analyze it by 10MM S and we can then
50:14see different shifts in those ions.
50:17And this data turned out to
50:19be very powerful for us,
50:21because without isolating the compound
50:23without getting any spectroscopic data,
50:25we can,
50:26we can glean an incredible amount of insight
50:29into the molecule structure.
50:30So from the glucose labeling,
50:32we get a shift by 37 units.
50:35That tells us, of course,
50:37that it has 37 carbons.
50:39Ammonia shifts by 8 units,
50:41we have eight nitrogens we can see
50:43that in the methionine auxotroph,
50:45and I'm talking about a higher
50:47molecular weight ion here.
50:49At the top we get a shift by 8
50:51carbons and so that told us that
50:53we had two of these cyclopropane
50:55residues or what was left of them.
50:58Two thiazole rings based on A6
51:00carbon shift in this in the
51:02Sistine Extra if you get the idea,
51:04and so we can basically tease out a
51:07lot of structural data to sort of
51:09see what pieces are need to be put
51:12together here to make the molecule.
51:14And so at any rate we found this
51:18higher molecular weight ion at 956.
51:21Using all that data,
51:22we were able to fit it to this structure
51:25here and so it contains one adenine
51:28residue and have explicitly drawn the
51:31Adenine without connectivity to the base,
51:34because at the time that we did this,
51:37we couldn't specify where
51:39it was bonded to adenine.
51:41We now know that that's in three,
51:44but had one adenine on the right hand side.
51:48You have a cyclopropane that's still intact,
51:51OK.
51:51And then you've got the rest of the core
51:54molecule sort of linking it together,
51:56and so it's it's almost C2 symmetric,
51:58it's it's a hetero dimer.
52:00It's not quite C2 symmetric.
52:01If you look carefully at these
52:03thiazole rings they have different
52:04appendages in different connectivity,
52:06but it's very close OK,
52:08and this structure fit RMS
52:09data within one PPM,
52:11so we're very excited about that.
52:15And so if that is simply a mono
52:18adenine addict and we're getting icy,
52:21else, presumably there's a
52:22dinucleotide add up and we went,
52:25and we were able to find the dyad an addict.
52:29OK, and this fits, fits within 1/2 PPM error.
52:33OK, and so working backwards,
52:35if that's the dyad, and in an act,
52:39then this is the structure of Kohli,
52:42bactine on the bottom here.
52:44OK, and so we've got two cyclopropane's.
52:48And in the middle we have this
52:521/2 dicarbonyl residue OK.
52:55There's a detail here
52:56which is worth mentioning,
52:58which is that this is this kind of compound.
53:02On the bottom is what we characterized what
53:05we expect based on the biosynthetic pathway.
53:08Is the self amino ketone at the top,
53:11but we've done work that shown that
53:14this thing is unstable towards aerobic
53:16oxidation to an Alpha keto imine,
53:19and then hydrolysis 212 die ketone and
53:22so working under air on the bench.
53:25This is this is the compound that
53:27you would have expected to get.
53:29And still so no ones isolated
53:32calling back in yet.
53:33And so how do you prove
53:35the structure assignment?
53:36We can go back and try and make it,
53:39and so we spent some time developing a
53:42synthesis of the molecule and it was.
53:45It was not straightforward because of
53:47its instability, but we could make it.
53:50And then we can do an LCMS coinjection and
53:54we see that has the same retention time.
53:58It has the same tandem Ms as
54:01the as the natural material,
54:04and then finally we did a crosslinking
54:07assay where we basically ran that same
54:10experiment that we ran with the bacteria,
54:13except replace the bacteria
54:16with our compound.
54:17And so this thing will crosslink
54:19add up to about, you know,
54:22down to about 500 nanomolar.
54:24And then we can do the tandem Ms
54:26analysis of those cross links.
54:28And so let me explain what's on this slide.
54:32So when we do the bacterial experiment
54:34where we treat the DNA with the bacteria,
54:37we can isolate the crosslink.
54:39You then run tandem Ms,
54:41you get a whole list of ions,
54:43primary and secondary and tertiary ions
54:46that you see from those crosslinks and so.
54:49You know the argument is.
54:51If we're making the same molecule
54:53that the bugs are making.
54:55Are synthetic compound oughta interact
54:57with DNA in the same way and it ought to
55:01blow apart in a mass spec in the same way?
55:05And So what this plot shows on the
55:07X axis or all of the ions that
55:10we found in the tenant and Ms of
55:13the bacteria derived crosslinks,
55:16we see all of those ions with
55:19our synthetic material.
55:20And the Y axis is simply the experimental
55:23minus theoretical error for those
55:25ions using this synthetic material.
55:27And so the point is we get all the same
55:30ions that we get when we use the bacteria.
55:33They're all within with the
55:35exception of 1 within two PPM OK,
55:38and so we don't have an NMR of Cali bactine
55:41to compare to.
55:42But we can say that structure
55:45that we made interacts with DNA.
55:47It crosslinks DNA and then it blows
55:49apart in attend imeson exactly.
55:51In indistinguishable fashion.
55:53And So what about this?
55:56I see LDS be, you know,
55:58apparent contradiction,
55:59so there's been a lot of debate in
56:02the literature between you know,
56:04debating the mechanism of action
56:06because Oswald had originally observed
56:09DNA double strand breaks using a comet
56:11assay and then came along and said,
56:13no wait, it's cross links,
56:15and for any of you that are familiar
56:18with these repair pathways,
56:20you know that these two phenotypes
56:22are intimately linked, right? And so.
56:24When you start to repair an ICL,
56:27you actually form a DSP that
56:29leads to activation of HR and so
56:31you're going to see gamma, H2, X.
56:34You're going to see streaking
56:35in your comment essay,
56:37and so the two phenotypes
56:39are entirely consistent.
56:40And we actually identified another pathway,
56:43which is just a spontaneous pathway.
56:46So it's well known in the old sort
56:49of Gina toxin literature that N
56:513 adenine addicts are unstable
56:54towards depurination.
56:56And if we run our crosslinking assay,
56:59we sort of.
57:00We modify the assay to be able
57:03to sort of get at this data,
57:05but this is the conclusion is
57:08essentially that these these Icl's
57:10undergo a slow, deep urination.
57:12And then there's a second elimination
57:14of the phosphate that occurs to
57:16lead to a single strand break.
57:18And you can imagine then you know,
57:20in tandem with the repair pathways
57:22and also other alkylation lesions.
57:24Eventually you're going to get those
57:27single strand breaks close enough to
57:29each other to get a double strand break.
57:32And so this brings us to where
57:34we're at in the project, and so.
57:39What we're currently doing is working
57:41with this molecule on the bottom here.
57:44And this is not Kohli backed in itself.
57:47It is a analog of Kohli Bakhtin.
57:50The differences are highlighted in green,
57:52and so the dye ketonen Kohli backed
57:54in on the top here is very unstable.
57:58You can't work with this compound,
58:00it would be, you know Suffiecient
58:02asked to try and use this in a
58:05series of essays to examine it.
58:07Sort of cellular activity.
58:09And so we made what we call the
58:12Dez di ketone analog on the bottom
58:14and working with Christian Jobin
58:16at the University of Florida.
58:18Christians been basically
58:20taking this compound.
58:21Through all of the essays that have
58:24been run by by Oswald and Box Tone
58:27Cleavers and others looking at the
58:30genotoxic phenotype of the bacteria and
58:33seeing if the molecule recapitulates
58:35it and up until now, it seems too
58:38so we see we get activation again.
58:41My page 2X.
58:43Fancy D2.
58:45What he's doing right now is basically
58:47looking to see if this induces the same
58:50type of mutational signature that one
58:52gets with the bacteria and that will be,
58:55you know, sort of the end.
58:57The end point.
58:58Hopefully you know it will
58:59be a positive result,
59:01but whatever it is that will be
59:04sort of the endpoint for this.
59:07And so I just acknowledge all
59:08the people that did the work.
59:10I'll just go through this
59:12quickly to save time.
59:13But this is my collaborator Jason.
59:15Many of you know him.
59:17Many people from my group contributed
59:19to this project over the years,
59:20acknowledged the NCI and Yale for funding.
59:23Thanks again for the invitation
59:24and I'm happy to stay on and
59:27take any questions you have.
59:32Thank you Seth, very interesting.
59:34It makes me appreciate
59:35that I'm a microbiologist.
59:36It's not nowhere near as hard
59:38as being a chemist, I think.
59:40Are there other questions for process?
59:49I want question of course,
59:51DNA damaging agents can cause cancer,
59:54but there also used to treat cancer.
59:58Is there any thought? Is there
60:00possibility of using these compounds
01:00:01therapeutically? Absolutely yeah, that's
01:00:03something we're very excited about,
01:00:04so I didn't get into it.
01:00:07You know, one of the challenges that
01:00:09we in my group is always the chemistry.
01:00:12You know, the chemistry work is
01:00:14a general chemistry to make these
01:00:15molecules is very robust and we started
01:00:18to characterize them with Ranjeet,
01:00:20Bindra slab and he's found
01:00:22that in bracket two mutants,
01:00:23these things are hyperactive and so that's
01:00:26the immediate direction we're going in.
01:00:28Long term, we're looking to see if we can,
01:00:31you know, optimize the properties of
01:00:33these molecules a little bit more.
01:00:34You know it's more than just another
01:00:37crosslinker because I didn't get into it,
01:00:39but there's a mechanism by which
01:00:40we can gauge the activity of the
01:00:42molecule and potentially target it,
01:00:44and so there's a lot.
01:00:46Yeah, there's a lot that we can do,
01:00:48and that's sort of the phase
01:00:50that we're entering into with
01:00:51the project traffic we are after
01:00:531:00 o'clock, so if people
01:00:55have other questions for Seth,
01:00:56just email him I'm sure. Be happy too.
01:00:59Talking thank both speakers for
01:01:01really stimulating talk today. Thank
01:01:04you thanks everyone.