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Andrea Miyahira: Hi, everyone. I'm Andrea Miyahira and I'm the senior director of global research and scientific communications at PCF. Today I'm joined by Dr. Akash Patnaik, an assistant professor at the University of Chicago Comprehensive Cancer Center. Dr. Patnaik is joining me today to discuss their recent paper, BET inhibition sensitizes immunologically-cold Rb-deficient prostate cancer to immune checkpoint blockade, published in Molecular Cancer Therapeutics. Dr. Patnaik, thank you for joining us today.

Akash Patnaik: Thank you, Andrea, for inviting me to this UroToday presentation. It's always a lot of fun to be able to talk about our science and be able to share with the wider prostate cancer community.

I'm going to spend the next several minutes talking about a story that we published recently in Molecular Cancer Therapeutics that pertains to a very aggressive variant form of prostate cancer and one of the most frequently mutated genes in human cancer, which is Rb or the retinoblastoma gene. And these cancers typically are found in about 75% of metastatic castrate-resistant prostate cancer. It has become an increasing problem clinically, as we develop more intensified hormonal therapy options for patients with mCRPC or metastatic castrate-resistant prostate cancer patients. They actually develop tumor-emergent neuroendocrine disease, found in about 75%, and then almost all of the neuroendocrine patients, a subset of which have small cell histology, have Rb alterations with or without p53 and often with PTEN loss as well. This is a big problem clinically and one that we don't have solutions for, and this is really going to be the focus of my talk.

This slide just really captures the heterogeneity of advanced prostate cancer with several molecular alterations that have been previously characterized and reported. And Rb loss and CDKN2A are two very frequent alterations, both in advanced lineage plasticity-associated prostate cancer as well as human advanced disease.

One of the first things that we did to try to understand the microenvironment of these Rb-deficient prostate cancers is to do bioinformatic analysis and this particular analysis was done using two sets of samples. We looked at all human cancers across the TCGA, which comprise close to 10,000 samples, as well as metastatic prostate cancer samples from the Stand Up to Cancer cohort. And as you can see here, we found a significant enrichment of Rb1 and/or CDKN2A loss in the subset of patients that had non-inflamed or T cell-excluded cancers, and this was true across the board, from when we looked at all malignancies and also in the metastatic prostate cancer cohort through Stand Up to Cancer, which was a smaller cohort. But nonetheless, we observed a similar enrichment of an immune-excluded or a non-T cell inflamed phenotype in these Rb-deficient cancers. And this was really foundational bioinformatic data that informed a lot of our subsequent mechanistic studies to really understand the basis for why Rb loss drives T cell exclusion, and how we can potentially target this therapeutically.

One of the early experiments that we did when post-doctoral scientist, Brian Olson in my laboratory, who unfortunately passed away recently, really spearheaded in the lab was to try to understand the molecular consequences of Rb loss as it relates to proliferative kinetics, both in vitro as well as in vivo alterations in the tumor microenvironment. What we were struck by early on, when Brian did some of the early experiments, was that he found that there was no change in proliferation when you knock out Rb using CRISPR/Cas9.

However, there was a significant change in immune infiltration observed in these Rb-deficient tumors relative to wild-type tumors. And this was done both in murine models and in collaboration with Sweta Saha, who was a bioinformatics post-doc in my lab, working on doing timer analysis shown in panel B. We found that there was a decrease in Rb loss-associated immune infiltration. So immune infiltration was suppressed both in patients and in the murine model as a consequence of Rb loss.

When we knocked out Rb in vitro and did migration assays, we found that loss of Rb did result in a decreased migration associated with giving CXCL10, which is a well-known chemoattractant cytokine that can drive immune cells in vitro migration assay.
We then did gene expression profiling and did differential gene expression analysis looking at our wild-type versus isogenic CRISPR/Cas9-mediated Rb knockdown.

And what we found was that there were several genes that were upregulated or downregulated, but the important ones to point out are that we found a significant upregulation of immunosuppressive genes, at least five of them that we saw in the immunosuppressive cytokine cohort. And on the flip side, about 20 genes associated with immunostimulation were actually downregulated, consistent with a decreased immune infiltration phenotype that we saw in these Rb-deficient tumors.
However, there was one gene that jumped out as an outlier in that it's known as an immunostimulatory gene called STING, which is actually upregulated in the Rb knockout tumors. So that drew our attention and we zoomed in on STING to understand how Rb loss regulates STING and whether we can target this pathway evolutionarily. And for those of you that don't think about the STING pathway on a daily basis, STING is an evolutionarily conserved innate immune sensing pathway that is typically stimulated in the context of a viral infection with double-stranded DNA in the cytosol that can allosterically activate an enzyme called cGAS, which is an enzyme that generates cyclic dinucleotides. The cyclic dinucleotides can then activate STING, resulting in a downstream signaling axis involving phospho-TBK1 and IF3 that then drives type I interferon generation, which can then secondarily result in an enhanced T cell infiltration. This has all been well studied in the context of pathogenic infections and viral infections.

We hypothesize that perhaps inducing a state of viral mimicry with DNA-damaging agents could potentially enhance cGAS/STING pathway activation within the tumor microenvironment and thereby enhance responsiveness to immune checkpoint inhibitors.
So we actually tested the question of the hypothesis that maybe Rb loss, which is known to have a role in DNA repair, can perhaps create a unique vulnerability that can then be targeted with BET inhibitors, resulting in a DNA damage-induced STING pathway signaling and secondary immune-permissive phenotype. In order to test that question, we went back to the first principles of understanding the biology of Rb versus CDKN2A, and actually interestingly, in patients, Rb loss and CDKN2A are mutually exclusive, so if you have Rb loss, you typically don't have CDKN2A loss and vice versa. And when you look at the pathway biologically, loss of CDKN2A, which regulates the Cyclin D/Cdk4 complex, results in activation of this complex, which can then hyperphosphorylate Rb, inactivate Rb, and allow E2F to then progress through the cell cycle.

On the other hand, Rb loss, as shown in the left panel there, when lost directly can result in E2F-mediated cell cycle progression, not requiring CDKN2A. So it makes complete biological sense that the two genes are mutually exclusive. And there have been several pharmaceutical efforts that have been tailored towards targeting these unique molecular subsets from a tumor cell intrinsic context. CDK inhibitors and CDKN2A-deleted tumors, and these include well-known drugs that have now received FDA approval in breast cancer that's hormone receptor-positive like palbociclib and ribociclib. And in the context of Rb deletion, CHK1 inhibitors, for instance, have been looked at as well as HDAC inhibitors and DNMT inhibitors. And what we were interested in understanding is whether BET inhibitors, which can block the BRD4 complex which is associated with E2F, can create a unique personalized therapeutic vulnerability in these Rb-deficient tumors that are already crippled for DNA repair.

So we tested that hypothesis and initially did some experiments proteomically to look at signaling pathway changes with BET inhibition in wild-type versus Rb knockout cells. And we found that there was, interestingly, an increase in DNA damage associated with treatment with JQ1, a well-known BET inhibitor, that results in an increase in phospho-gamma-H2AX and STING expression. STING is actually more expressed, as I had mentioned before, at the protein level as well in these Rb knockouts.

So the combination of increased STING expression and increased DNA damage specifically in the Rb knockout tumors resulted in a specific increase in NF-kappa B translocation in the nucleus. And that translated to an enhancement in type I interferon production.
We then asked the question of if this is indeed true, can we abrogate this NF-kappa B STING-mediated enhancement of interferon beta release in these Rb-deficient prostate cancer cells by targeting either the STING pathway with a small molecule inhibitor called H-151 or with an NF-kappa B inhibitor that we obtained as a tool compound previously generated by Bristol Myers Squibb.

We did three types of experiments. We tested this in vitro, ex vivo using tumor single cell suspensions from our mouse tumors, as well as then looking at T cell functionality and migration assays with and without these concurrent inhibitory drugs that block STING NF-kappa B signaling.

To cut through the chase of this very busy slide, we discovered that giving concurrent STING inhibitor or NF-kappa B inhibitor resulted in a suppression of the interferon beta production as observed with the BET inhibitor treatment in Rb-deficient tumors. So this was suggestive of the idea that these pathways induced by DNA damage downstream of BET inhibition is actually driving NF kappa B and STING signaling.

We also then went back to our in vivo mouse tumors and tried to understand how BET inhibition might be increasing immune infiltration and delaying tumor growth in these Rb-deficient prostate cancers. So we wanted to test this in both wild-type as well as Rb-deficient tumors. As you can see here, the wild-type tumors, there was no impact of JQ1 on tumor growth in the wild-type context. However, in the Rb knockout context, we clearly did see a suppression of JQ1-mediated tumor growth inhibition. This was partially rescued by giving either the STING inhibitor or the NF-kappa B inhibitor consistent with our in vitro data suggesting a suppression of type I interferon signaling.

It's a little bit easier to see in this format since some of these curves make the panels busy. But as you can see here, JQ1 partially suppresses and this is rescued with concurrent H-151 and BMS treatments.

We also then were interested in asking the question, does this type I interferon production in vivo result in an enhancement of T cell trafficking as well as macrophage-mediated trafficking? So we looked at our T cell frequency in these in vivo tumors and, consistent with the data showing that tumor growth inhibition was suppressed with JQ1, we saw an increase in T cell frequency as well as an increase in macrophage infiltration as well. And when we treated with concurrent clodronate, which suppresses macrophages and/or CD4 CD8 depletion, we found that we could rescue the effects observed on tumor growth inhibition elicited by JQ1.

So this data collectively suggested that JQ1, or the BET inhibitor, was actually working through initially a tumor cell intrinsic DNA damage-induced STING pathway activation. That STING pathway activation was extrinsically enhancing T cell infiltration and macrophage infiltration. And this was then driving an antitumor immune response. When we block the trafficking of these immune cells, both T cells and macrophages, we can actually partially abrogate the antitumor response, and if we treat it with drugs that perturb the STING signaling pathway, we also saw an abrogation, so this clearly demonstrated an important immunological consequence of giving BET inhibition, both in terms of STING pathway activation and enhanced immune-mediated antitumor response.

We then asked the question of can this effect of BET inhibition sensitize these cancers to PD-L1 blockade? In order to test that hypothesis, we first asked the question of whether isogenic knockdown of Rb resulted in any change in PD-L1 expression, both in the tumor cells and macrophages and other myeloid cells including GR-MDSCs.

What we discovered was that there was a clear increase in PD-L1 expression in these tumor-associated macrophages, tumor-promoting macrophages as well as the GR-MDSCs, and observed specifically in these myeloid cells, we did not see a change in the PD-L1 frequency in the tumor cells, which are CD45-negative. And then when we treated the combination of PD-L1 with JQ1 in the presence of androgen deprivation therapy, which is the standard of care for prostate cancer therapy, we did see an enhancement of antitumor response shown in the red curve here with the triple combination of degarelix JQ1 and PD-L1, and this was rescued by giving concurrent STING inhibitor or the NF-kappa B inhibitor. So this again underscoring the importance of the innate immune signaling as the major stimulus to drive a T cell and macrophage-mediated infiltrative and activation response that ultimately suppresses growth in these very aggressive tumors.

These rescue studies then were done across all of the different cohorts, as shown here, again, a busy panel but demonstrating the importance of concurrent combination treatments and the abrogation from STING NF-kappa B inhibition.

Putting this all together, what we have demonstrated in this paper is that Rb loss creates a unique therapeutic vulnerability for innate immune activation elicited by BET inhibitors. This is a very aggressive form of prostate cancer with limited treatment options. And if we can turn off a DNA damage program in these tumor cells, turn on STING with BET inhibition, and this is a non-canonical NF-kappa B-mediated STING pathway activation, that results in a potent type I interferon response and then drives both macrophage and T cell infiltration. From our rescue studies, we've demonstrated that both macrophages and T cells are important for this antitumor immune response. When combined with conventional androgen deprivation therapy and PD-L1 blockade, we see a significant tumor control elicited by this combination therapy.

I'd like to end by acknowledging Brian Olson. This work is really a tribute to Brian, who spearheaded this work while he was a postdoctoral scientist in my lab, and then when he start continued it while he was at Emory University as an independent assistant professor. Brian will be dearly missed. He is in our hearts and we hope to continue to work on this tirelessly as Brian was an amazing scientist who really did some seminal work to move forward the importance of targeting the Rb-deficient cancers for which we have very limited treatment options.

I'd also like to thank some of the other members of my laboratory team, postdoctoral fellows who worked with Brian to bring this paper to closure. And importantly, I wanted to thank our funding sources, the Prostate Cancer Foundation. This work was funded through a Challenge Award back in 2016, and then a Young Investigator Award as well given to Brian for developing some of his own research projects. And then the NCI prostate SPORE as well, that supported some of this work, and American Cancer Society institutional grant.

I'll stop there and take questions.

Andrea Miyahira: Thank you for presenting that, Akash. And I'm also just so sorry about Brian's recent passing. He was a PCF Young Investigator and we've gotten to know him pretty well over the years. He was an expert in cancer immunotherapy and just a wonderful person who we will dearly, dearly miss.

As for questions about your study, you show that Rb loss decreased expression of multiple immune-related genes, yet it enhanced STING activation. It appears that JQ1 is activating STING now by eliciting DNA damage. Have you looked at other types of treatments that also elicit DNA damage, for instance PARP inhibitors or radiation? Would they be effective alternatives?

Akash Patnaik: Yep. So that's a great question, Andrea. Based on all of the work that we've done, I think the BET inhibitor has a unique precision-oriented vulnerability here in these Rb-deficient cancers, given the pathway of regulation of E2F. We think that PARP inhibitors may be less effective in inducing a DNA damage-induced response in Rb-deficient cancers. Having said that, there's reports that have been published previously demonstrating co-loss or co-deletion of BRCA2 and Rb. And it could be that in that context, PARP inhibitors may actually work well, just given the known synthetic lethality and STING pathway activation induced with PARP inhibitors in the BRCA2 context. So in the purely Rb-deficient context, we think that BET inhibitors are likely to induce more DNA damage for the mechanistic reasons that I had described previously. Hopefully, that offers patients a more precision medicine-based therapeutic strategy to treat their aggressive disease.

Andrea Miyahira: Okay. Yeah, that it does imply that there's a precision medicine immunotherapy strategy for patients with Rb loss. Do you think this could be expanded to other patients?

Akash Patnaik: Yes. So we're interested in this concept of using precision medicine to sensitize cancers to immunotherapy. As we had discussed a few weeks ago, related to our other manuscript that recently was published in PTEN-deficient cancers, which is yet another aggressive form of prostate cancer, where we demonstrated that targeting the PI3 kinase pathway there can result in immunomodulation of macrophages through an entirely different mechanism.

I think each of these molecular subsets will likely have unique vulnerabilities that can be exploited to reprogram the immune microenvironment and thereby sensitize these cancers to checkpoint inhibitors, and that's certainly been a major focus for my own group here at the University of Chicago.

Andrea Miyahira: Okay, awesome. And do you have translational plans for these findings?

Akash Patnaik: Yes, absolutely. So we have a clinical trial actually that's ongoing, that is really based on parallel studies that we did co-clinically looking at the mechanisms and mouse models and then the development of the trial. And this is a BET inhibitor from Zenith, in combination with enzalutamide and pembrolizumab in mCRPC patients. This trial is open, it's an investigator-initiated trial. It's open nationally, including at our site, and we're excited to enroll patients to test the hypothesis in these subsets of patients that have what we call neuroendocrine or treatment-emergent neuroendocrine disease, where they're treated with intensified androgen deprivation therapy and undergo lineage plasticity and the tumor becomes much more aggressive with Rb alterations, can this strategy work for these patients to prolong survival and overall clinical outcomes. We'll see how the results turn out.

Andrea Miyahira: Well, I look forward to it, and thank you again for joining me today.

Akash Patnaik: Thank you, Andrea.