Dr Jessica Chitty and Dr Thomas Cox generated and validated a novel selective and irreversible pan-lysyl oxidase inhibitor: PXS-5505. Their experiments in a series of pre-clinical models show that by acting on the tumour microenvironment, this small molecule drug has the ability to significantly enhance chemotherapy efficacy.

Listen to this episode to find out more about the effects that PXS-5505 in combination with gemcetabine has on tumour morphology, behaviour and subject survival in pancreatic cancer.

This study has been published in Nature Cancer 4, pages 1326–1344. DOI: 10.1038/s43018-023-00614-y and was included in the EACR’s Highlights in Cancer Research.

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Our guests in this episode:
  • Jessica Chitty, the Garvan Institute of Medical Research at the Kinghorn Cancer Center at UNSW in Sydney, Australia
  • Thomas Cox, Jessica’s supervisor at the Garvan Institute of Medical Research at the Kinghorn Cancer Center at UNSW in Sydney, Australia

And our host: Alexandra Boitor, EACR Scientific Officer.

Episode transcript

Alexandra: Today we are returning with one of our Research Focus episodes, discussing some of the most interesting and impactful recent papers in cancer research with no other than the authors themselves.

Increasing trends in both incidence and mortality of pancreatic cancer are observed in most countries across the world. Based on Global Cancer Statistics 2020 estimates, pancreatic cancer represents a large part of the global burden of disease, being the 12th most common cancer worldwide, with over 500, 000 new cases being diagnosed every year.

More worrying is its mortality rate, which brings pancreatic cancer as the third leading cancer related cause of death. So pancreatic cancer is one of the most lethal cancers with insidious onset, aggressive nature, and poor prognosis. Whilst the leading cause for the increased mortality of this type of cancer is the late diagnosis, another important reason is the poor treatment response.

Dr. Jessica Chitty and her supervisor, Dr. Thomas Cox from the Garvan Institute of Medical Research at the Kinghorn Cancer Center at UNSW in Sydney, Australia, are some of the researchers looking into improving pancreatic cancer treatments, and they accepted our invitation to discuss their paper “A first in class: pan-lysyl oxidase inhibitor impairs stromal remodeling and enhances gemcitabine response and survival in pancreatic cancer”. The paper published in Nature Cancer last year and featured in the March edition of the EACR’s Highlights in Cancer Research. Hello Jessica and Thomas, and thank you so much for allowing us a glimpse into your research.

So in your paper, you are proposing a new promising therapeutic approach for pancreatic cancer by combining standard of care chemotherapeutics with small molecule drugs that target the tumour microenvironment. What made you direct your attention towards pancreatic cancer in the first place and then towards lysyl oxidases?

Thomas: Perhaps I can begin. I’ve had a longstanding interest in lysyl oxidases, for about 15 or 16 years now, mainly because they are critically involved in the biogenesis of collagen, fibrillar collagens in particular. And this is obviously critically important, not only in health, but in a number of different diseases. So, for example, wound healing, but also in cancer.

So over the years, we’ve been interested in studying not only what they do under normal conditions, but what happens when we see aberrant expression of lysyl oxidases and the implications that this has in things like solid tumours. And given the fact that pancreatic cancer is an incredibly fibrotic tumour, it made sense to start asking the question of, well the major family of enzymes that’s responsible for laying down fibrosis, fibrillar collagens, what might their role be in pancreatic cancer?

Alexandra: I was wondering what is the mechanism of action of this pan-lysyl oxidase drug? I think you call it PXS 5505 in your paper.

Jessica: Yeah so PXS 5505 is a pan-lysyl oxidase inhibitor and it’s a first in class irreversible inhibitor. And what we hypothesise is it binds to the LTQ group of the lysyl oxidase family, and this is a really highly specific group that’s unique to the lysyl oxidases, and is found in their active site.

Thomas: As Jess says, it’s obviously a pan-lysyl oxidase inhibitor. There’re five members of the lysyl oxidase family of enzymes. And what was actually interesting is that when you look at the expression of the different family members in pancreatic cancer, we see that it’s not just one of them that tends to be overexpressed, that in fact many of them, two, three, four, even all five of them, can be overexpressed. And so therefore it makes sense to target the entire family of lysyl oxidases rather than design specific inhibitors to for example, you know, one or two of the members.

Alexandra: And what is the element of novelty that PXS 5505 brings? How is this attempt different from previous similar drugs that failed after phase two clinical trials?

Jessica: Tom touched on all the family members are very important for activity. And so previous small molecule inhibitors have only really attempted to target one or maybe two of the family members. There’s also been antibodies that have been developed to target one or two of the lysyl oxidase family members. And so really what’s unique about this small molecule approach is that it’s targeting all five family members. The only other compound that is capable of doing that is the naturally occurring compound BAPN. But the drawback to BAPN is that it also inhibits the other amine oxidases and so it can’t be taken into the clinic, just purely down to the fact that it has off target effects.

Alexandra: On that note, given that lysyl oxidases are ubiquitously expressed and are involved in the regeneration potential of many connected tissues, including the skeleton, respiratory tract, and cardiovascular tissue, don’t lysyl oxidase inhibitors affect healthy tissue if their administration is not targeted towards the tumour? Some old studies that investigated pan-lysyl oxidase inhibitors, such as beta aminopropionitrile, reported toxicity.

Thomas: Yes, this is a really good point. The body itself requires lysyl oxidases to synthesize and lay down fibrillar collagen. So essentially if you’re blocking the activity of these enzymes, the body can’t lay down fibrillar collagens. Now actually, under normal conditions, under sort of homeostatic conditions of healthy tissues, there’s actually quite a low level of collagen deposition and turnover. But what we see is that in conditions of, for example, wound healing or repair, then that’s when these get upregulated. Now, what that does mean is that this allows you to closely monitor, you know, patients or individuals where there may be a cause, for example post surgery, these inhibitors would obviously have an effect, but under normal conditions that would be minor. And this is reflected, I think, in the fact that this drug is actually also currently in a phase two clinical trial, for another type of cancer, myelofibrosis, which is a fibrotic bone marrow cancer. And to date, these patients have been taking PXS 5505, or as it’s now being renamed as SYN 5505. They’ve been taking this for, I think it’s around about nine months with no observed adverse effects.

So, we know that these appear to be safe, at least in the current clinical trials, and one more thing I’ll mention is that there’s a relatively short half life to this drug in the body. So, once it’s inhibited its target, the lysyl oxidases, the remainder of the drug is quickly cleared. What then happens is the body re -synthesises lysyl oxidases at a relatively fast rate, and this allows the body to restore activity. So, in the event that you needed to, the therapy could be stopped and, within about three to four days, you’d see a recovery of that lysyl oxidase activity, which would allow you to continue to lay down collagen in a normal manner.

Alexandra: It sounds great from a safety perspective, so what I wanted to ask you is what effects have you noticed following this drug administration in terms of anti-cancer effects and how, and why, did you decide to explore combination therapy?

Jessica: One of the initial findings was that there was a reduction in tumour fibrosis in our genetically engineered mouse models of pancreatic cancer. And what we also saw was there was a reduction in the tumour stiffness, and this was done by bulk compression testing. We then sought to understand some of the more molecular mechanisms that were going on behind here and understand what was going on at the cellular level. And what we saw was there was a reduction in the CAF activation and contractility, as well as a reduction in the STAT3 signaling. And so, we know that there’re a number of signaling pathways that are really important to LOX regulation. And the one that we saw changes in was the STAT3 signaling.

And this was really interesting to us because we know that this pathway is known to respond to tumour stiffness, and it’s also linked to chemoresistance. To your second question, on why it as a combination therapy, we would never set out to use this drug on its own. From the get go, for this project, we were always going to be targeting the underlying fibrosis, as well as administering chemotherapy, to target the cancer cells themselves.

Thomas: One thing that was really interesting when we first started out on this project, was that perhaps at the time, anecdotally, there’d been a lot of reports coming through in the field that chemotherapy and other therapies can actually trigger tumour dysplasia. So, the administration of chemotherapy to some solid tumours leads to actually an acceleration or an increase in the levels of tumour fibrosis present within that tumour.

And that’s some of the work we confirmed in the genetically engineered mouse models that we were using. The standard of care at the time, the gemcitabine, was leading to an increase in tumour associated fibrosis. We’ve actually confirmed that in a number of different chemotherapies now that are front line, gemcitabine plus abraxane, the Folfirinox polychemotherapy. And so, this became really interesting because chemotherapy induces fibrosis, but fibrosis is known to be bad for the progression of the tumour and ultimately can potentially then almost defeat chemotherapy’s efficacy. It reduces the efficacy of that chemotherapy. So then it became sort of an almost, you know, logical next step that if chemotherapy is inducing fibrosis fibrosis and we have a bona fide anti-fibrotic drug, then combining the two, would that result in sort of blunting of that desmoplastic or fibrotic response, which would therefore augment chemotherapy and allow it to continue to act for longer and therefore improve outcome? And essentially, through the mechanisms which Jess described, that’s exactly what we saw.

Alexandra: Earlier in the conversation, you mentioned that this drug has a short half-life and that lysyl oxidase is recovered quite quickly. So, I was wondering, how does this affect the tumour tissue?

Jessica: Because of the short half-life, the drug was administered on a daily basis and so this was really important to ensure that lysyl oxidase activity wasn’t able to recover to a level where, a lot of desmoplasia could occur. In terms of the tumour, what we saw in our genetically engineered mouse models was it took much longer for the fibrosis to occur, and even at end point we were still seeing a reduction in the fibrosis at the end point of the survival study.

Alexandra: You very neatly show that this pan-lysyl oxidase inhibitor decreases pancreatic cancer cell invasion in vitro and metastatic burden in vivo by reducing early metastatic colonization. Do you have any hypothesis how lysyl oxidase signaling promotes cancer cell invasion and metastasis in the first place?

Jessica: What we know is that collagen cross-linking is absolutely important for the cancer cells to be able to invade and metastasise. And so, by having your lysyl oxidases there, for the biosynthesis of new collagen fibers, the CAFs in particular, can produce large amounts of collagen fibers that can then go on to create that framework for the cancer cells to invade and metastasise.

Alexandra: In your paper, you analyse the cancer tissue in quite a bit of detail following the combination treatment. And in addition to the effects on cell invasion and metastatic potential that we just discussed, and the effects on the tumour microenvironment you mentioned earlier, including a decrease in stromal collagen content and subsequent tissue stiffness. Changes in the levels of myCAF, panCAF, and the levels of some immune components of the tumour microenvironment. The decrease in PSTAT3 signaling, a critical mediator of anti-apoptotic pathways and tumour progression. I was wondering if you observed any changes at the tumour vasculature level as well. In a paper from Janine Erler’s lab from 2013, I believe it was, lysyl oxidases have been implicated in tumour angiogenesis and blood vessel formation, both in vivo and in vitro. Did you notice anything like that, anything similar in your models?

Jessica: We did some CD31 staining, to stain for vasculature and we didn’t see changes in total number of vessels. We looked across the whole tumour. We weren’t looking specifically in compartments. The other experiment that we did that’s loosely linked to this is looking at perfusion. So, we used a FITC dextran that is a similar size to that of Gemcitabine and we used this to look at how the FITC dextran can perfuse into the tumours. And what we saw was that there was higher levels of perfusion in our combination therapy versus chemotherapy alone.

Thomas: Just to add on to that, that this is actually quite inciting in the field. People have sort of, traditionally assumed that as tumours become fibrotic, that the perfusion into the tumour becomes limited. there’s not actually too many papers out there that definitively show this. And so, the idea of this, you know, FITC dextran perfusion experiment is that you really can ask that question and say, well, does the presence of tumour fibrosis or the lack of, because of the action of the PXS 5505, does that actually increase the amount of drug that could get into a tumour? And so, this is a nice sort of proof of principle that whilst, as Jessica mentioned, the total number of vessels didn’t appear to change, it’s likely that the patency of these vessels was increased, therefore allowing more chemotherapy in, and that likely then leads to obviously, you know, augmented activity or increased efficacy of the chemotherapy.

Alexandra: This sounds very promising. I was wondering whether you foresee any mechanism of resistance or reoccurrence and aggressiveness occurring in tumours following this combinatorial approach.

Jessica: We are inhibiting an enzyme by irreversibly binding to their active site. So, we don’t foresee any problems in terms of resistance of PXS 5505 and there aren’t any other enzymes that carry out this critical function. So, I couldn’t see how the tumour could become more aggressive using this drug.

Thomas: I think one of the exciting things about using an antifibrotic in this case is that it has a number of roles, and Jess touched on those briefly, you know, the decrease in collagen content and so decreasing fibrosis leads to a decrease in stiffness within that primary tumour.

Stiffness is known to be associated with more aggressive tumours and spread. As Jess mentioned, it leads to a decrease in the phosphoro STAT3 signaling within the tumour cell compartment. But what she didn’t mention was that it also leads to a deactivation of the stromal compartment, so the cancer associated fibroblasts.

So, we actually see a decrease in the activation of that compartment as well. And then combined with, as we mentioned, the increased perfusion of the chemotherapy, and you’ve sort of got a number of mechanisms by which you’ve seen to be affecting the tumour as a whole. And in that sense, we believe that the emergence of resistance would be probably quite minimal.

What would be more likely is just the emergence of resistance to your standard of care chemotherapy that you might be using, such as the Gemcitabine or the Gemcitabine/Abraxane. And those are well known and well documented pathways of chemoresistance. That’s not something we did look into for this paper, but something that we’re actively pursuing at the moment.

Alexandra: So, you think that potentially this drug could be used with a range of different chemotherapeutics and improve different sorts of treatment? And on the same note, I was wondering how much do you think these results could be generalised? Even though pancreatic cancer is probably one of the best examples for fibrotic cancer, it’s not the only type of cancer that’s associated with increased fibrosis. So, I was wondering whether you think that a similar approach could be applicable to other types of cancer?

Thomas: Absolutely. So, you really hit on that there, Alexandra, which was that a large number of solid tumours have either tumour fibrosis within them, or develop this desmoplastic response during progression, and many of them also during response to chemotherapy. It’s also likely, and there has been some work looking at lysyl oxidases and their importance in the response to radiotherapy.

Radiotherapy is also known to trigger fibrosis in and around tumours that are being treated. So, we would definitely hypothesize that any solid tumour that has a high level of desmoplasia, would likely benefit from a combination therapy. What we haven’t tested, but I think is the next step and the next obvious question is, would an antifibrotic improve the efficacy of immunotherapy through facilitating increased access into the tumour? And again, that’s something that we’re now starting to look into a bit more closely. So we can’t really say definitively yet.

Alexandra: That all sounds very interesting. In addition to all these promising results for the treatment of pancreatic cancer we just discussed, there was one other thing that grabbed my attention in your paper. Maybe I’m going off on a tangent here, but I noticed in your paper that you mentioned a single molecule array-based detection assay that you used to determine LOX concentrations in human plasma.

And this comes in addition to the data you showed that LOX expression correlates with patient survival. So do you think that a liquid biopsy assay could be developed to predict patient response to such a combinatorial therapy as the one you’re suggesting?

Jessica: Syntara has actually published a bio probe, that they have developed. And this is something that can measure the LOX activity from blood. And so this can look at your LOX activity, but also confirms that the 5505 is inhibiting human lysyl oxidases. So yeah, absolutely, developing a higher probe is something that’s being done already.

Alexandra: Thank you very much for this very stimulating conversation. I really enjoyed reading your paper and I appreciate having this opportunity to discuss the results with the authors of the paper themselves, with the people that did the experiments and know best, what’s behind the publication. So, thank you very much for accepting our invitation and for allowing us a glimpse into your research.

Thomas: Thank you very much for the invitation to join you and tell you a little bit more and also your listeners about the work that we’re doing in the lab.

Jessica: Thank you for having us!


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