I think it's sort of like.
Consultant in medical oncology and a senior researcher in experimental cancer therapeutics based at the Churchill Hospital.
He came from from Leeds,
having previously trained in Southampton to deal with Agent Harris at the whim and then transferred his number by popular demand,
I'm told, and then was appointed as a consultant here three years ago.
So we're going to hear a bit about the translational research that he's setting up with a breast unit.
Some of you will know that breast, as you said, has moved on significantly over the last five years.
And it's really good to have the science behind that as a collaboration between oncology and surgery.
Simon, thanks very much.
And so as it's just been alluded to, my interest is in drug development, predominantly and particularly in the breast cancer space.
And so today I'm going to present a project that we ran a little while ago,
which is essentially a clinical pharmacodynamic study to try and understand the effects of metformin.
Most of you will have heard of that drug. One of the most commonly prescribed treatments worldwide and treatment for diabetes.
We wanted to understand this pharmacodynamic effects on breast cancer metabolism.
So initially, I'll give you a bit of background about why we were interested in metformin.
So my firm has been around for a bit of history.
I've always been around for a long time, is actually derived from French lilac and mediaeval herbalists in the Middle Ages.
Use it and use this to treat diabetes hundreds of years ago,
but it was first synthesised in the mid-20th century and then licenced for the treatment of type two to type two diabetes in the 1950s in the UK,
later in the US. And now it is the most widely prescribed oral Angie hypoglycaemic drug worldwide
and insisting it reduces all cause mortality for patients with type two diabetes.
So that doesn't just include mortality in the context of diabetic complications.
But there's growing interest in metformin as anti-cancer effects. This was sparked by a series of epidemiological studies,
the first of which was published in The BMJ in 2005 that showed that diabetics
on metformin had a reduced cancer incidence than diabetics on other treatments.
And it's now been over 50 studies worldwide and a number of meta analysis.
Depending on which one you look at. The risk reduction is in the order of 30 percent later.
Metronidazole suggests it may be a bit less than that, but but but it seems to be quite a strong link because of that.
There have now been a number of clinical studies which have tried to understand the effects of metformin on cancer cell proliferation.
Typically, these have been pre surgical in those studies in which patients have been given a small or a short course of metformin two to eight weeks,
depending on the study, and they've had biopsies beforehand and then a sample taken at surgery afterwards.
And these are mainly been in breast, prostate and then mutual cancer.
And and in a number of these studies, they've shown that metformin can reduce Ki 67 expression so well validated marker of cell proliferation.
Some of these studies suggest that the effect may be greater in the obese population, but that is a little controversial still.
But just to show the interest in metformin, the potential of repurposing this drug as a cancer therapy is now over 100 clinical trials worldwide.
Some of those fall into the prevention space. Others are looking at using metformin is an adjuvant therapy following surgery to prevent recurrence.
And then some are also looking at the potential of using metformin advanced disease.
So despite that, despite all this interest in forming and hundreds, if not thousands,
of preclinical publications that have been that have assessed its mechanistic mechanistic effects in cancer,
there's still controversy as to the mechanism of action at clinical doses in tumour cells.
We know that metformin is an inhibitor of complex one, so complex one is a core member of the electron transport chain in mitochondria.
And if you inhibit complex one one it, expect an energy stress.
However, those findings have only been that's only really been observed in in preclinical studies,
typically using doses that are much higher than peak plasma level in patients.
But this is, I guess, the canonical hypothesis that metformin canonical view that metformin inhibits complex one leading to an energy stress,
an increase in the AMP or ATP two ATP ratio that leads to activation of africanised high impact kinases,
the key regulator of energy homeostasis in cells and the downstream effects of activation of AMP
companies to inhibit a number of anabolic pathways that are absolutely key to cell proliferation.
So, for example, fatty acid synthesis or protein synthesis.
However, others take the view that it's probably metformin effects on host metabolism so patients metabolism have the greatest,
greatest influence on on kind of effect on all tumour cells, as it were.
We know that there's good evidence now, Ray, that metformin probably does have an anti mitochondrial effect in hepatocytes leading to MPIC activation.
This leads to inhibition of if you Konya Genesis, and this is why you see falling glucose and insulin levels in patients that
you would expect to reduce stimulation of the PI3K pathway in tumour cells,
which is a key regulator of cell proliferation and anabolic metabolism and in all cells.
And is and is already a target for therapy in cancer treatment.
So it's a number of outstanding questions, as I've alluded to, there's still a session.
How does it work in consumer sales? What effect is it having on tumour cells, if any,
as all the preclinical studies relevant where they've used doses of metformin that are typically 100 to a thousand times?
People assume that we're based in in in vitro and in vivo models.
How should we select patients for future clinical trials?
What biomarkers might there be to make sure that clinical trials are properly designed?
What in what state should we be metformin as a clinical therapy in the context of cancer?
Should this be a preventative treatment, perhaps in diabetic patients or obese patients?
Should it be an active treatment that we give following surgery alongside perhaps chemotherapy and other standard treatments?
Well, should we be using an advanced disease?
And lastly, how maybe should we be combining that form in with other treatments to get synthetic lethality or an additive effect, at least?
So this is the these are the sort of the research objectives of the clinical study, such as firstly,
to characterise the effects of metformin on breast cancer metabolism and also to
assess the potential ways that we could select patients for future clinical trials.
This is a design. In total, we recruited 41 patients to the study.
We recruited patients that had an untreated primary breast cancer.
It's essentially pristine, so they haven't been touched by any terapie before just off the diagnosis.
These patients who are going to go on to have neoadjuvant pre surgical chemotherapy.
We gave them a two week window therapeutic window of metformin, and either side of that window, we carried out a series of pharmacy atomic assays,
novel imaging with a dynamic, a pet CT scan, a series of research, biopsies for immunohistochemistry transcriptomics and metabolomics.
And then lastly, we also took research blood samples because we wanted to understand the effects of metformin
on host metabolism so that we could then relate that back to the effects on the tumour.
Once they completed that two week course of metformin, they went on to have that chemotherapy.
And if they chose to continue the metformin alongside before having definitive surgery.
But essentially the study was over after that last month and access following the two week and the initial two run in with with metformin alone.
So one of the first activities we carried out was just to look at the kinetic.
Character, genetic analysis of metformin in our patients, and we saw that there was a relatively wide spread of metformin levels,
peak plasma levels is about two hours per dose, but this was consistent with with the literature from our pharmacokinetic studies in diabetic space.
But interestingly, because we had tumour samples, we were able to therefore also assess the levels of metformin using mass spec in the tumour
samples and then determine whether there was any relationship with serum plasma levels.
And there was there was a significant correlation.
Now you might say, well, that's obvious, but actually a lot of investigators think that the main determinants of the level of metformin,
the tumours, expression of certain key transporters to get metformin into the cell, one of the main one is resolved one.
But here we see actually that at least to a certain extent,
serum level of metformin seems to be important in terms of the amounts you get into the to the tumour.
And that's important because therefore dose escalation studies of metformin in the cancer context might be might be sensible.
We can in cancer patients, I'm afraid we were happy to accept, accept higher levels of toxicity than many other diseases.
So. And there are studies that are going on and looking at that,
particularly in terms of the functional effects of metformin on the cell lung cancer cells.
So moving on to one of the causes of this study,
and that is to understand whether we saw any change in 18F uptake on pet CT imaging following metformin treatment.
And I'll just give you a bit of background as to why we're interested in pets as a modality in this space.
But I'm probably just preaching to people that already understand it, and I'll give a brief overview anyway.
So as you're aware, this is the EFG is essentially a glucose analogue regulating glucose analogue and is used routinely in the clinical setting,
particularly to stage cancer patients. Although there are other applications because tumours will take to take up more glucose than benign tissues,
typically of other certain tissues of his brain and liver also have take up a lot of glucose.
And this is really because tumour cells need a lot of glucose based carbon to proliferate to use for fatty acid synthesis,
amino acid synthesis, nucleus nucleotides insists.
So when one carries out pet CT or fuses a pet scan, which will image the uptake of EFG into tissues and to a CT scan that delineates the anatomy.
And so you can see here this is a lung cancer patient that you've got a very average tumour in this patient's lung.
But you can also use this technique to monitor response to therapy so you can see post chemotherapy.
They've had an excellent metabolic response.
But why are you interested in this technique in the context of its possible understanding, dynamic response to metformin?
Well, as I mentioned before, we one hypothesis is that metformin inhibits complex one in the mitochondria and it's induces long distress.
You then get AMPK activation.
A key consequence of an activation is that you get upregulation of peak one and click fall to glucose transporters on on cells.
And so you will see increased glucose uptake.
Potentially, you should see increased glucose uptake as a consequence of that.
So if you are seeing a mitochondria effect from metformin that has any sort of cause any sort of significant energy stress,
this is one of the first sequelae that you should see. So firstly, we carried out a static analysis, so you got allies data in different ways.
This would be what you doing standardly in the clinic. So you give a dose of radiation EFG, you take an image forty five, 60 minutes afterwards,
depending on your protocol, and you just get that snapshot of uptake of Asian FTT at that point.
And so in this here, we did not see any change in Asian energy uptake in our primary tumours.
So it's a little disappointing because one of the first notices we carried out,
but thankfully Fergus Gleason suggested we also carried out cinematic imaging and we had a lot.
We had a quartette of medical engineers in the departments here able to help carry out this modelling and analysis for us.
So we thought that iPads, instead of just taking that one, scan forty five 60 minutes after that,
given the dose of SD instead, you take a series of images over that time period,
many more at the beginning,
and you can therefore carry out a kinetic analysis to understand changing or the change in rates of uptake over a period of time.
In this context,
you then have to also integrate an input function that gives you a readout of change in activity of EFG in the bloodstream over that period of time.
Because obviously that will change is going to slow rate each site great any time.
So we in this in our study, we use the left ventricle, which is well described as as a good way of doing this.
Once you carry out the analysis, you can then come up with a variable CFG, which describes the rate of interest EFG phosphorylation.
And this has been shown to be in a number of other studies to be more sensitive
to changes in EFG uptake than just standard SUV max use in the clinical setting.
So this is this is the dynamic. The results of the dynamic nation after picked us is we actually say overall, we saw actually an increase in CFG,
so an increase in the flux of FTE into the primary tumour following metformin treatment.
So this is quite exciting.
You can see in this patient here, we had one of the higher, greater increases in fatigue uptake that the patients pre metformin,
we can't see any axillary nodes two weeks later itself, which may or may not be.
We think it's probably a consequence of the fact that metformin has increased refugee uptake into into the night into those nights.
So potentially that for dynamic ageing fatigue, perhaps it could be a dynamic biomarker of metformin effect on breast cancer metabolism,
although this is not a technique that you can take into the clinic anytime soon.
So besides the imaging essays, we also carried out tissue based assays alongside, which makes this study a lot more powerful.
So first of all, we carried out metabolomic profiling using spent with a collaborator in Cambridge and looked
at a series of different metabolites that we thought might change in the mitochondrial space.
And these. Before metabolites, where there was a significant difference in level following metformin treatment,
so gluconate to short chain short chain kinases are still promising and proportional cleansing and citrulline.
So. This is a bit speculative, but I thought I just sort of show you why these changes might be might be relevant,
so I still can't see an issue and not commenting on both mitochondrial metabolites.
I still saw contain is a key way by which carbons can be pulled from citrate and proportional carnitine
is a breakdown product of a fatty acid option that sits where the meat based upon accounting comes from.
And we saw lower levels of these two metabolites, possibly in the case of mitochondrial interference.
We also saw a decrease in Citrulline. The situation is the only metabolite of the urea cycle that is synthesised within mitochondria.
And so again, this suggests this possibly alludes to a mitochondrial effect of metformin.
And lastly, we saw an increase in gluconate. And this is really speculative.
But this possibly reflects something of glucose carbons away from glycolysis growth going
further down the glycolysis pathway because they can't be utilised by the mitochondria.
As I say, speculation read bit, but but did that change in the metabolite levels linked to our imaging assay?
So yes, they did.
There was actually a strong correlation between a positive correlation between changing as technology intervals and changing its energy flux.
Moving on to the transcriptomic analysis, we carry it out.
Transcript chairman, I seek. And when we carried out an initial pathway analysis,
one of the striking things was that there is a large number of pathways associated with metabolism that were either upregulated or downregulated,
as you can see in the circles on the left in the left panel.
Even more striking was that there were a great number of mitochondrial pathways associated with
mitochondrial metabolism that were significantly upregulated following metformin treatment.
And many of these were the most significantly upregulated pathways that we saw on pathway analysis.
Who then look to integrate the transcriptome of data back to other all their other assays.
The first thing we do was carry out hierarchical clustering, looking at four key pathways or clustering genes,
looking at four key pathways that we might expect to be modulated in the context of of mitochondrial interference,
oxidative phosphorylation, TCA cycle glycolysis and Spartacus and glutamate metabolism and aspartate and glutamate are a key amino acids.
This importance in providing all their synthesis and utilisation are key with
regards to certain resistance mechanisms related to mitochondrial interference,
and it has been shown in metformin and in preclinical studies. But anyway, what was what was he?
There is a group of eight patients who had global activation of number of genes to annotate to these pathways,
and we called this group the Oxytocin Transcriptional Response Group.
We then went on to see if there was to relate that back to our other assets, and here we saw that for the experts transcriptional response group.
If anything, actually we didn't see any change in EFG flux,
but rather it was the other patients where we were more likely to see an increase in EFG Flux.
So we term these guys the AFG Response Group.
When we looked at some of our metabolites, we also saw that there was a discrepancy picture here between the two groups,
so the OK for the Oxford transcription was not sure.
We saw a marked decrease in this.
We saw a decrease in our style casting that was following that forward.
But this was not really observed on a general level for the desktop, for the FG Response Group.
So we just looked at sort of a key set of genes at this point.
Annotated to mitochondrial metabolic pathways.
We then went on to look at the whole transcriptome. And when we carried out hierarchical clustering of the whole transcriptome,
we again saw that the focus was transcriptional response group clustered together.
When we looked at clustering of genes, the fold change of clustering, of change of genes that are encoded by mitochondrial DNA.
Once again, the oxytocin transcriptional response we've clustered together, giving giving us greater evidence that we were seeing a real effect here.
But how might this reflect back on possible therapeutic effects?
So we used a messaging approach. And observe that for the Oxford Transcriptional Response Group, if anything,
there was an increase in expression of genes that made up a well validated proliferation messaging and suggesting,
if anything, that these guys were resistant to treatment.
Whereas there are a number of patients in the EFG response group who had a decrease in expression of this proliferation,
Mataji suggesting they this they may well be more sensitive to metformin.
I alluded to the importance of glutamine and the spotlight on previous slides,
so we know this glutamine utilisation of glutamine carbons because they can avoid the TCA
cycle of our process of reductive oxidation and thereby be used for a spot in System B.
Fatty Acids insists that utilisation of glutamine is a key resistance pathway to mitochondrial interference hypoxia as it happens and also metformin.
And so we wanted to to hone down on on some of the genes involved in glutamine metabolism and then also a aspartate metabolism,
particularly with a view to possibly targeting these genes to see if we see them.
So in the future clinical studies or targeting these pathways in future
clinical studies to try and get additive benefit alongside metformin treatment.
And we saw that firstly, there were a number of genes with increased expression that a key checkpoints,
these pathways in particular are citrate dehydrogenase of rejected car selection pathway and
then a large number of genes had increased expression that regulate the aspartate male shuttle.
But also it seemed that there was a markedly increased expression, particularly in the air group versus the EFG Response Group,
suggesting that certainly these pathways were activated, at least in some patients.
And therefore there may be an opportunity to target these pathways in combination with metformin and
there their already drugs out there that target our citrate dehydrogenase in coming into the clinic.
And there are those being developed to target target aspartate metabolism.
We also related the expression of certain genes back to our imaging data.
And so, for example,
here we we saw that expression of glued to one key glucose transporter had a strong positive correlation with changing 18 EFG flux,
which is what you might expect. So earlier, I mentioned that we also there's also a separate hypothesis that suggests that the.
Considers this possibly metformin as effects on host metabolism,
rather than the direct effects on the tumour cells that are most important for driving any final chemical effects of metformin in the cancer context.
And certainly so we measured we took blood samples pre and post metformin in our Windows study.
And in these non-diabetic, they had to be had to have a fasting glucose to get into the study.
That's a term that they were not diabetic.
We saw in this group of patients that there was a decrease in a number of metabolic host metabolic markers for metformin treatment,
including glucose, insulin, C-peptide, which is an excellent marker of insulin secretion and then higher,
which is a function of education, it's good that determines of insulin sensitivity.
There were dramatic decreases you might see in a diabetic patients, but there was they were strongly significant.
They were small but strongly significant, consistent. So we then used our transcriptomic data to try and see if there was a relationship here.
And we wanted to understand whether the change in expression. Of genes correlated with the different.
The change in our different assays, including 18 G.G. Flux C peptides,
says good measure of circulating insulin levels and one of the key metabolites we measured in the tumour style carnitine.
And what we observed was that there were a great deal, a large number of genes that whose changing expression significantly.
Correlated with change in both Asian refugee flux and also stock policy.
But this was not the case when we realised when we looked at C-peptide,
and this is highly significant, suggesting that the change in these two things are linked.
From this data, we think we think this basic data gives a good indication that we are seeing a direct effect of metformin
on tumours have this and it's not related to the key driver is not the effects on circulating insulin.
Of course, we can't determine mechanism. Absolutely.
In a study like this, which is that we have to make certain inferences from from the data.
But I think this was a very intriguing finding. So in conclusion, for him, it increases refugee flux into pouring breast tumours.
This is consistent with the upregulation of glycolysis or at least glucose transport into cells, tumour cells.
And this would be consistent with a metformin having a direct mitochondrial effect.
Metformin reduces short chain cell carnitine levels and regulates multiple mitochondrial pathways that the transcriptome level in forms of cancer.
And we describe two metabolic response patterns that may define sensitivity to metformin.
So if you want to read it about this a bit more, it's came out in cell metabolism actually about four weeks ago,
and it's open access, so you have to pay anything to look at next steps.
So we there's some intriguing data published by a number of groups.
This is the the the seminal paper from about four years ago that shows that if you knock out certain genes in complex one,
there's certain subjects of complex one. You can markedly change or reduce the sensitivity of tumours to two metformin.
And then there's quite a lot of data to suggest that mutations in complex one
really do significantly alter sensitivity to various mitochondrial insults,
such as hypoxia or low glucose levels in vitro and in vivo.
And so we've now got some funding, a whole exome sequencing, so that we will be able to assay the mitochondrial mutational burden,
but particularly focus on complex mutations using tiny samples from the study and
then relate back to that back to the metabolic response that I've just shown.
And that might give us some baseline biomarkers that we can then test in future.
Trials are actually working with a group that has completed an achievement study in assessing breast cancer,
which the five years of metformin to three and a half thousand patients will often receive metformin, the other half randomised to placebo.
And so they have very good long term outcome data in that setting.
And potentially if we can show that our metabolic response patterns are relevant related to the
mitochondrial mutational burden using their banks translational samples that they took from it in.
In that study, we could then potentially look at that assay in context or relevant clinical context.
So I just like to thank everyone that was involved, particularly Agent Harris,
who was instrumental in setting up the city, the Oxford Cancer Imaging Centre.
You and Joel Finit is now moved to Liverpool, carried out with the analysis apprenticeship, offer science and reach and carry out match analysis,
mainly for the transcriptome data that also help with some of the metabolomics analysis as well.
And then Christine and Eduardo carried out the next step.
And lastly, these are our funders who is quite a lot of fun, and you can imagine a study like this, and they are very grateful.
So they're not making. OK, any questions?