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Andrea Miyahira: Hi, everyone. I'm Dr. Andrea Miyahira here at the Prostate Cancer Foundation. Today I'm talking with Dr. Akash Patnaik, an Assistant Professor at the University of Chicago, about his group's latest paper, "Suppression of Tumor Cell Lactate-Generating Signaling Pathways Eradicates Murine PTEN/P53-Deficient Aggressive-Variant Prostate Cancer Via Macrophage Phagocytosis," published in Clinical Cancer Research. Dr. Patnaik, thank you for joining us today.

Akash Patnaik: Thank you, Andrea. Pleasure to be here. Thank you, Andrea and Prostate Cancer Foundation, UroToday, for this opportunity to present some of our recent work, that really gets at this question of how we can harness the metabolic interactions in the prostate cancer microenvironment to develop better therapeutics in this disease.

So these are my disclosures.

So the subset of prostate cancers that has been a big focus of attention in my research laboratory has been PTEN deficient prostate cancers, which comprise approximately 50% of metastatic castrate-resistant prostate cancer patients. And as a medical oncologist, I see these patients in the clinic as well, and they typically carry a very poor prognosis, and very limited responsiveness to standard hormonal therapies, chemotherapy as well as immune checkpoint inhibitors. So this is a tough nut to crack, and certainly one that is highly relevant, just given the high frequency of patients with alterations in either the PTEN or the PI3 kinase pathway.

And there's been an interest in targeting the PTEN deficient cancers, with PTEN loss being one of the most frequently observed alterations in human cancer, and targeting the molecular consequences of PTEN loss, both in aggressive or in prostate cancers as well as in cancer as a whole, it has been largely disappointing with single agent PI3 kinase inhibitors. And this is just a basic schema from a paper that we published a while back, my early postdoctoral training days, where we were interested in understanding the dependencies of PTEN downstream signaling pathways. And this is essentially showing that PTEN loss drives the PI3 kinase pathway. There are several isoforms of PI3 kinase. And PI3 kinase activation has been shown to be reciprocally regulated with androgen receptor signaling in PTEN deficient prostate cancer. That was work done previously by Brett Carver and Charles Sawyers as well, showing that this is indeed a target that is actionable. But even with co-targeting with androgen receptor blockade, the responses have been largely limited.

This is the clinical trial data that looks at co-targeting these two pathways. And that is essentially showing that in the PTEN deficient cohort of patients with metastatic CRPC that were treated with an AKT inhibitor in combination with abiraterone, there was a modest improvement in radiographic progression-free survival, work published by Johann de Bono and colleagues at the Royal Marsden.

We were interested in reverse translating some of this clinical insight in the laboratory, and one of the first things we did was to go back to our genetically engineered mouse model of PTEN/P53 deficient prostate cancer, and do a very simple experiment, where we treated these mice in vivo with a PI3 kinase inhibitor, and essentially measured both antitumor responses, but also what's changing in the microenvironment as it relates to intratumoral signaling pathways.

What we discovered was that, when you block the PI3 kinase pathway with a drug called copanlisib, we found that there was an inhibition of AKT signaling, as you would expect, but there was also a suppression of phospho-ERK signaling in these responsive mice. However, in the resistant mice, we saw an upregulation of phospho-ERK signaling, and that's highlighted with the red text box, red box, just showing that indeed there was a binary readout, where the responders clearly showed suppression of pathway, the resistant tumors had upregulated map kinase signaling.

So we then combined PI3 kinase inhibitors with MEK inhibitors, which affect downstream map kinase signaling, and we observed that we could actually significantly enhance the response rate of these mice that are harboring PTEN deficient and P53 deficient tumors to PI3K inhibitor. So PI3 kinase inhibitor, the single agents, at best, you get a 37% response rate, and that's more than doubled when you give concurrent MEK inhibition with the PI3 kinase inhibitors. And that's shown here in this plot showing both the response rate, which is sum total of partial response as well as stable disease.

What we were struck by was when we took tumor-derived cell lines from these tumors and now treated with the combination, we found that the drug combination had no effect on either the proliferation index of these cell lines or the cell death capability. So the drugs were not impacting tumor cell intrinsic proliferation and/or apoptosis, or cell death. So there was a discordance between what we were observing in vivo and what we found in vitro and treated in the context of these cell lines.

So we then framed a central hypothesis for this line of work, which was that the combination reprograms the immune microenvironment potentially in these PTEN/P53 deficient prostate cancer, resulting in a partial tumor control. And that was sort of the mechanisms that we then decided to pursue, and take a deep dive into the tumor microenvironment.

And so, the first thing we did was did some immune profiling studies in mice that were treated with this combination. And we found that it actually enhanced the activation of tumor-associated macrophages, which are really the predominant cell type in the tumor microenvironment in these genetically engineered mice. And this is very consistent with data that we've observed in patients, in metastatic biopsy samples, that the predominant cell type, or I should say, the most enriched cell type in the immune microenvironment are these myeloid suppressive cells, and particularly tumor-associated macrophages. And we found that this drug combination was actually turning on or activating these macrophages. As you can see here on the right side with MHC Class II, which is a marker for macrophage activation, we're able to see a clear increase in the MHC Class II activation relative to the single agents or the untreated control.

We had recently published, several months ago, that the PI3 kinase inhibitor can actually suppress lactate production, because it can abolish the Warburg effect downstream of PTEN loss. And that release of lactate can actually epigenetically transform macrophages through a post-translational modification called histone lactylation. And this histone lactylation can drive macrophage immunosuppression. So when you block PI3 kinase, you can actually suppress the lactate production, suppress histone lactylation, and thereby activate these macrophages. But this resulted in a very, very modest tumor control in the context of these single agent PI3K inhibitors, which could explain in part why these drugs have not been so successful in the clinic.

So we then asked the question of, if we give the combination with the MEK inhibitors, what happens to lactate production? So we took our tumor-derived, essentially took our tumors from these genetically engineered mice, and generated single cell suspensions from these tumors, and then treated exogenously with these drugs, and demonstrated that there was indeed a decrease in lactate production that was additive when you give the combination of these two drugs.

So we then asked the question of, when you give this combination, what happens to the ability of the suppressed lactate to then reprogram macrophages through this epigenetic modification, histone lactylation? So we first looked at essentially the tumor-derived essentially conditioned media from single cell suspensions of these PTEN/P53 deficient tumors that were treated with these drugs. So this was all done ex vivo. And then we were able to take that conditioned media from these tumors that were treated exogenously, and then overlay those onto tumor-associated macrophages that were also harvested separately from these tumors, and essentially do co-culture studies. And what we discovered, interestingly, was that we were seeing a profound suppression of histone lactylation in these tumors that were treated with the combination even more pronounced than what we saw with the single agents. Single agents were having a fairly significant suppression, but even more drastically seen with the combination.

But importantly, what we observed is not just that we were suppressing histone lactylation, but we were also altering the functionality of these macrophages, and we measured their capability to eat cancer cells through a process called phagocytosis. And we found that with the combination there was a significantly increased phagocytosis observed in these MHC Class II high or activated macrophages. So they're just much more able to eat tumor cells in an assay that we did ex vivo. So that was interesting and could, in part, explain some of the more profound antitumor extrinsic responses that we were seeing in the mouse model.

So we then asked the question of, we saw a response rate of about 80% in these mice that were treated with the PI3 kinase MEK inhibitor, what accounts for resistance in those 20%? So we did a simple experiment where we took, again, cell lines derived from these tumors, and we treated them with the combination of PI3K inhibition/MEK inhibition at different time points, at 24 versus 72 hours. And what we discovered was that in the context of 24-hour treatments, we see a suppression of lactate production, but then over time, at 72 hours, there's a restoration of lactate that we observed when we treated these cell lines for longer periods of time.

And then to try to understand what's restoring lactate in these cell lines that were treated for three days, as opposed to just one day, we did signaling proteomics using western blotting, and we discovered that there was an upregulation of the beta-catenin signaling pathway in these cell lines that were treated for longer periods of time, suggesting the possibility, or an association between increased lactate and restoration of Wnt/beta-catenin signaling.

So we then went back to our in vivo models, and essentially looked at the single agent and the double combination with the PI3K/MEK inhibition, and correlated response versus resistance with the status of beta-catenin and histone lactylation. And we observed something that was really interesting, where we found that in the combination of mice where there was a response, we found a suppression of beta-catenin and a suppression of histone lactylation. So histone lactylation was adequately suppressed, and that translated to an antitumor response. However, in the resistant tumor, as shown in the last lane of this western blot, we found a profound increase in histone lactylation, and that corresponded to an upregulation of beta-catenin signaling.

So connecting the dots, we hypothesized that perhaps activation of beta-catenin signaling is indeed driving restoration of lactate and histone lactylation in these tumors. So we then actually treated with the triple combination, and we found that we were able to suppress lactate ex vivo, and I don't, in the interest of time, have the time to show you the data, but it's certainly in the paper. And we were able to demonstrate that indeed we were able to suppress lactate and histone lactylation ex vivo and in vivo, and that translated to 100% antitumor response in vivo.

And now, as you can see here with the triple combination of the PI3K inhibitor, the MEK inhibitor, and a porcupine inhibitor, which inhibits the Wnt/beta-catenin signaling pathway, we see a 100% response rate. What was even more striking, was that when we depleted the phagocytic macrophages in vivo, using a small molecule agent called clodronate, which can actually systemically deplete activated or phagocytic macrophages, we completely abolish the antitumor response. We're not even talking about a partial response, but a complete abrogation of an antitumor response, suggesting that these tumors, as a consequence of treating with these signaling targeted therapies, are actually working largely through macrophage-mediated innate immunity. We've also gone on and done experiments where we've depleted immune subsets, including T-cells, and shown that this response is entirely independent of T-cells and is purely a macrophage-mediated response. At least in the context of this model.

There's been concerns about combining kinase inhibitors, understandably in the clinic, going back several years. There's data demonstrating that these kinase inhibitor cocktails can be toxic to patients. And so, most of the studies that I showed you were done in mice that were treated for up to a month, and we see these very striking antitumor responses that are immune-mediated. So we were then interested in essentially carrying out a Phase I clinical trial in mice to really address the toxicity question. Because if we give these agents continuously, we will run into toxicity issues. And in fact, we did when we treated these mice long-term, with these combination cocktails given continuously.

So the postdoc in my lab, Kiranj Chaudagar, who's the first author on this paper, actually devised a very creative intermittent dosing schedule, where he was able to give the drug nine weeks on and three weeks off. And he was able to demonstrate that in the context of this intermittent dosing schedule, he was able to keep these mice alive for longer than a year and a half, which is amazing for a mouse model as aggressive as the PTEN/P53 deficient model. These mice typically die within four to six months, and these mice were living longer than a year and a half without toxicity, no tumor growth. And actually, we could have kept them longer if we chose to, but we had sort of met our survival endpoint based on pre-specified statistical endpoints. So this was pretty promising, suggesting that if we can develop an optimal pharmacologic strategy to give these agents intermittently, we might be able to mitigate toxicity while preserving efficacy. And I think that was a proof of concept in this particular experiment.

We also did our histopathologic analysis, in collaboration with a close collaborator and friend, Dr. Massimo Loda at Weill Cornell. And we were pleased to see that we were seeing near-complete histopathologic responses in these mice that were treated with the triple combination, both at four weeks and then at much later time points using the intermittent dosing schedule.

So putting this all together, what we have essentially discovered is that lactate, which is an important metabolite released from cancer cells that have hyperactivated PI3 kinase signaling, can be profoundly immunosuppressive in the tumor microenvironment vis-a-vis the crosstalk between tumor cells and macrophages, histone lactylation, and immunosuppression. If we can suppress lactate, we might be able to make these macrophages more activated, and thereby more phagocytic. However, cancer cells are hardwired to preserve aerobic glycolysis. And in order to effectively treat these tumors in patients, we're going to need a co-targeting strategy, because we demonstrated that blocking with just the PI3 kinase inhibitor would be insufficient, because the signaling pathways like map kinase signaling and Wnt/beta-catenin signaling can restore lactate production. And so, using a combinatorial approach, we were able to demonstrate 100% tumor control, and particularly in the context of intermittent dosing, we could achieve long-term durable control in an androgen-independent context in an aggressive variant prostate cancer model. And this was able to preserve efficacy without toxicity.

And so, taking this all together, this is essentially our conclusions. That co-targeting these pathways can result in complete tumor control and a significant prolongation of survival without significant long-term toxicity, and abrogation of lactate-mediated crosstalk between cancer cells and macrophages results in a durable ADT-independent tumor control in PTEN/P53 deficient aggressive variant prostate cancers.

And the reason I would like to emphasize ADT-independent is in our first story, which we published a few months ago, we demonstrated that ADT, in combination with PI3 kinase inhibition and PD-1 blockade, can result in antitumor responses, but 40% of the mice were still resistant. And so, we came up with this strategy, where we can suppress lactate, without even requiring androgen deprivation therapy and PD-1 checkpoint blockade, to turn on innate immunity. So this begs the question of, if we can move this forward in a clinic, and we can develop an intermittent dosing approach, can we sensitize immune checkpoint prostate cancers, especially aggressive variant subtypes, to immune checkpoint blockade? And that's really where we're headed next.

So I'd like to end by just thanking my team at the University of Chicago, which was obviously a fantastic team effort. Kiranj Chaudagar was the first author, several other members of the lab contributed to this story, also in the photograph here. Massimo Loda, who is an amazing pathologist, worked with us very closely to analyze some of the histopathologic data. Importantly, Prostate Cancer Foundation's support of this work. This was our first Challenge Award that we received after I started my independent research program at the University of Chicago, and that got a lot of this work off the ground, and so, we're really grateful for that support. As well as the National Cancer Institute of Prostate SPORE, which has subsequently funded some of the more recent work, which I didn't get a chance to talk about today.

So with that, I'll stop and take questions. Thank you.

Andrea Miyahira: Well, thanks for sharing that with us. So what side effects might you anticipate in humans with this combination therapy?

Akash Patnaik: Yeah. So that's a great question, Andrea. Borrowing a page from the trials that were done with PI3 kinase inhibitors, and also there's been some data with MEK inhibitor combinations, hyperglycemia is a very common on-target toxicity, because PI3 kinase blockade affects glucose metabolism in these tumors. And so, you do see an increase, essentially drug-induced diabetes. It's an on-target toxicity. And we have a clinical trial right now with abiraterone and an AKT inhibitor in hormone-sensitive metastatic prostate cancers. And certainly, hyperglycemia has been a concern, but something that's manageable. We often will treat these patients with metformin, which has also been shown to lower insulin and IGF-1 levels and lower glucose levels. So I think it's a manageable toxicity.

The combinations of the MEK inhibitors, you would expect to see a lot of the common toxicities associated with kinase inhibitor cocktails, which include rash, diarrhea, GI side effects, and loss of appetite. These PI3K/MEK inhibitors have been previously tested in the context of continuous dosing paradigms and toxicities have been a concern. Having said that, I think what makes our study attractive, from a translational perspective, is that we can now think about intermittent metronomic dosing, where we're not continuously hitting the target and co-targeting, but essentially alternating these agents, and coming up with a creative pharmacologic strategy. That would need to be tested in patients given the PKPD in patients is going to be different from mice. So these are ideas that we're interested in pursuing.

The Wnt/beta-catenin pathway and porcupine inhibitors have been associated with enhanced bone resorption as a toxicity. And that is another area of concern in prostate cancer, given that our patients often will have bone metastases and are at higher risk for skeletal-related events. And they're also on androgen deprivation therapy, which will also increase bone resorption. So that is a concern with these agents, but again, with the intermittent approaches, and even with concurrent blockade of other molecules, like DKK1 for example, which can actually increase osteoblast activity. Because Wnt/beta-catenin biology is very complex and different, depending on what cell types in the tumor microenvironment you're looking to target. So you could envision a strategy where you could give a porcupine inhibitor, but now come in with a DKK1 blockade strategy, and then be able to turn on osteoblast activity and bone formation, even though the Wnt/beta-catenin signaling could potentially abrogate bone formation and enhance bone resorption.

So those are ideas that we're certainly interested in, and hopefully, that answers your question.

Andrea Miyahira: Yeah. Thanks. So I guess my next question is, would targeting lactate production alone be sufficient to mediate the tumor control with TAMs? And are there other strategies for targeting lactate production by tumor cells?

Akash Patnaik: Yep. Yeah, that's an excellent question. So we have been thinking hard about this since we've published this paper. Instead of doing this as a Phase I trial, looking at three different agents and figuring out the complex intermittent dosing strategy, what if we can come up with a very targeted metabolic approach to suppress lactate?

And so, one of the ideas that we've been very interested in is targeting the ability of lactate to be transported to macrophages. There are transporter molecules called monocarboxylate transporters, it's a family of transporters, that can enable lactate to be taken up by macrophages or tumor cells. And these transporters have different biology. So MCT1, for instance, can kick out lactate from the tumor cell, MCT4 can import it into macrophages.

And there are drugs that have now entered clinical trials. AstraZeneca has an MCT1 inhibitor, for instance. So there are now strategies where you can potentially block lactate's transport, and maybe that could have some antitumor efficacy in this context. And those are ideas that we're pursuing going forward.

Andrea Miyahira: Thank you. Do you have any translational plans for these findings?

Akash Patnaik: Yeah. So as I sort of alluded to in my responses to the other questions, we definitely would be interested in thinking about Phase I design strategies, with either intermittent dosing approaches or directly targeting lactate cargo transport through the MCT inhibitors.

And so those are ideas that are in evolution and development in the conceptual stage. And hopefully, we can partner with our industry colleagues to sort of move this forward. And of course, with the support of PCF and the NCI, we'll hopefully be able to move some of this forward down the road.

Andrea Miyahira: Okay. Well, thanks again for coming on and sharing this with us today.

Akash Patnaik: Thank you. Pleasure to be here.