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Andrea Miyahira: Hi, everyone. I am Dr. Andrea Miyahira at the Prostate Cancer Foundation. I'm joined today by Dr. Patrick Pilié, an assistant professor at MD Anderson Cancer Center. Dr. Pilié will discuss his recent paper, "SPOP Mutations Target STING1 Signaling in Prostate Cancer and Create Therapeutic Vulnerabilities to PARP Inhibitor-Induced Growth Suppression," published in Clinical Cancer Research. Dr. Pilié, thank you for joining us today.

Patrick Pilié: Thank you so much for having me. I look forward to a great discussion. Thank you so much for having me today to discuss our recent publication in Clinical Cancer Research focusing on SPOP mutations and how they target STING1 signaling in prostate cancer, creating vulnerabilities to targeted treatments, including PARP inhibition.

This work all centers around a clinical conundrum that we deal with on a daily basis in the clinic, and that is there is significant heterogeneity in the genotype-to-phenotype relationships and outcomes in patients with prostate cancer across the disease spectrum and the treatment course, where the graph at the bottom is highlighting the relationship of prostate cancer in terms of androgen sensitivity, castration resistance, and ultimately androgen indifference. Each step along the way with varying treatment options, there is significant difference in outcomes on an individual patient level.

Briefly, a bit of background. Approximately 25% of metastatic castrate-resistant prostate cancer displays mutations in DNA damage response genes, with the most famous being BRCA2. There are multiple approved PARP inhibitors for these DNA damage response deficient prostate cancers. In addition, we and others have shown well-known intricate interactions between androgen signaling, DNA damage response activity, and immune activation in prostate cancer.

SPOP mutations are frequently found in prostate cancer, with SPOP mutant cancer showing, on the whole, improved responsiveness to anti-androgen therapy. However, even within SPOP mutant prostate cancer, there is heterogeneity, and molecularly targeted therapies in castrate-resistant SPOP mutant cancers are lacking.

The major findings of our current manuscript that we'll be presenting today is that SPOP mutant castrate-resistant prostate cancer does display increased DNA damage, resulting in downstream activation of the STING pathway, with a preferential increase in the non-canonical immunosuppressive pro-tumor STING and NF-κB driven response.

A potent selective PARP inhibitor, though, can shift this balance in STING back towards an anti-tumor interferon-driven canonical STING response in a mutation-specific manner. These findings have potential implications in castrate-sensitive disease and even potentially beyond SPOP mutant cancers to identify patients who may benefit from earlier escalation of therapy with targeted inhibitors such as PARP inhibitors.

Highlighting the first figure of the paper was an attempt to deconvolute this heterogeneity. We previously found in preclinical models that SPOP ubiquitination activity targets STING for degradation. In a castrate-resistant androgen-indifferent patient cohort, we analyzed gene set enrichment, finding that SPOP mutant cancers display increased enrichment of hallmark TNF NF-κB signaling. This was recapitulated in a separate dataset, as seen in panel A in the Robinson Insilico patient data, where SPOP mutations again are showing upregulation of TNF-α NF-κB signaling.

In the heat maps in B, we see a collection of 259 genes involved in STING, both pro-tumor non-canonical NF-κB driven STING, as well as canonical interferon-driven STING. What we find is that in the SPOP mutants, the 29 most upregulated genes are all associated with non-canonical immunosuppressive NF-κB STING pathway.

In C, looking at the Insilico data for the Immunome, we see that SPOP mutant cancers do display an increase in immunosuppressive macrophage signatures.

Then looking in D, this is TCGA data, hormone naive prostate cancer that even within this early disease state we already see a subpopulation of SPOP mutant cancers that are enriched for this non-canonical immunosuppressive NF-κB STING-driven pathway. Moving on to our preclinical data, we see with potent selective PARP inhibition on the left resulting in DNA damage and induction of canonical STING interferon response in a dose-dependent manner in both human prostate cancer cells C4-2B, as well as our mirroring model, RM-1 bone met derivative, with increases in γ-H2AX indicating increased damage, as well as increase in STING activation with phosphorylated STING and downstream phospho IRF3 and interferon beta.

In the panel on the right, we see that with potent selective PARP inhibitors olaparib or talazoparib, that in a mutation-specific manner we see not only an increase in canonical STING signaling with phospho STING1, TBK1, and interferon beta and STAT3, but also a shift away from the non-canonical immunosuppressive NF-κB and IL6, resulting in an increase in apoptosis. Next, we see that there's not only cancer cell-centric impact of PARP inhibition in this mutation-specific manner but also impacts on the tumor microenvironment. Here we have a macrophage co-culture experiment with SPOP mutant prostate cancer cells, showing on the left an anti-tumor efficacy is in part mediated through a paracrine secretory activity, where the application of PARP and its efficacy is abrogated by the GW4869 agent which blocks exosome and secretory products.

In C on the right, we see that from the perspective of the macrophages with the application of a potent PARP-1,2 selective inhibitor like olaparib, in a mutation-specific manner, we see induction in interferon gene transcription. This is also recapitulated in the protein response.

Lastly, in the preclinical data in our in vivo models, we see in an SPOP mutation-specific manner on the top left anti-tumor efficacy with the application of PARP inhibitor olaparib. Or this was recapitulated with talazoparib. On the right, we see in the tumor staining of the xenografts, significant increase in γ-H2AX, representing DNA damage, as well as significant increase in, again, interferon response in the figure seen in F.

At the bottom is mapping out the pathway changes that are occurring with both an SPOP mutation leading to an imbalance in STING towards a pro-tumor immunosuppressive NF-κB signaling, and that this imbalance can be overcome with the application of PARP inhibition that has both tumor cell intrinsic effects, as well as impacts on the tumor microenvironment and macrophage activity.

Here we see preliminary data from ongoing clinical trials where patients have tumor biopsies prior to treatment and on AR signaling inhibitor treatment. In this patient with SPOP mutant prostate cancer, in their pre versus post AR signaling inhibition tumor staining, we see increase in DNA damage, as noted by phospho γ-H2AX, increase in PARP1, increase in replication stress as noted by phospho ATM, and increase in this NF-κB activity with phosphorylated NF-κB in the surviving cancer cell populations.

Thus, we have an ongoing trial looking at prostate tumor biopsies at screening and three months on potent androgen-signaling inhibition to look at the cellular and molecular level to see indeed if patients with early evidence of androgen indifference are displaying upregulation of PARP1 activity, as well as this immunosuppressive STING NF-κB driven pathway in a mutation-specific manner.

The information from these biopsies is then guiding an adjuvant approach of continued anti-androgen therapy or combining anti-androgen therapy with potent PARP inhibition. This is a correlative rich study focusing on these intersections of AR signaling, DNA damage response, and this balance of innate immunity in the tumor microenvironment in patients with high-risk localized prostate cancer.

Thank you for your time and for the opportunity to speak with you today about this important work. I'd like to acknowledge my mentor and bench collaborator, Dr. Timothy Thompson, as well as Dr. Chuandong Geng who did a majority of the preclinical work in this manuscript, Ganiraju Ranyam who did bioinformatic work related to this manuscript, as well as all our co-authors and collaborators, including from Cornell, Dr. Christopher Barbieri and Dr. Massimo Loda. Of course, would always like to thank the patients and their families who participate in this research. Lastly, a big thank you to the Prostate Cancer Foundation and the YIA program, who allowed me the time and resources to support this work and now carrying forward to an NIH grant and these investigator-initiated clinical trials.

Andrea Miyahira: Thank you so much Dr. Pilié for sharing this wonderful study with us.

Do you think the interaction between SPOP mutations and the STING pathway might impact responses to AR-targeted therapy or to certain immunotherapies?

Patrick Pilié: Great question and absolutely. That is part of our follow-up and ongoing work is are these changes in a mutation-specific manner, including SPOP, occurring early on in response to androgen and anti-androgen therapies?

Before the clinical definition of castration resistance and androgen indifference develops, are we already seeing changes in DDR function, DNA damage, and this balance of immunosuppressive STING versus anti-tumor STING response downstream of that damage? Ongoing studies looking at both patient tissue, as well as preclinical models, will help to determine is this pathway really central in androgen response and the need to escalate therapy earlier on?

Andrea Miyahira: Thank you. Do SPOP mutant tumors have an altered immune tumor microenvironment compared with other prostate cancer molecular phenotypes?

Patrick Pilié: Yeah, really good question. Work from others and other DNA damage response gene mutations have shown there are differences in the immune makeup of DDR-deficient tumors. SPOP is central in maintaining genomic stability, and specifically in DNA damage response. Prospective studies that are able to look at tissue, both SPOP mutant, SPOP wild type, and other DDR mutants, and do immune profiling are needed and ongoing.

Andrea Miyahira: Thanks. Have patients with SPOP mutant prostate cancer been treated with PARP inhibitors in trials? And if so, what have the results been?

Patrick Pilié: There has been preclinical data in models to suggest that SPOP mutant prostate cancers do display sensitivity to potent PARP inhibition. There has not yet been, to my knowledge, published data looking prospectively at SPOP mutants specifically and their response to PARP inhibition. However, retrospective data in these PARP inhibitor studies, including most recently large phase three studies combining PARP inhibition and androgen signaling inhibition, would be wonderful to assess, retrospectively to start, for responses mediated, in part at least, by SPOP mutations, and also allow us to look at what might be some modifying factors in patients that would dictate a response to PARP inhibition outside of just SPOP mutations.

Andrea Miyahira: Thanks. Does this interaction between DNA damage response and innate immunity have impacts beyond SPOP?

Patrick Pilié: Absolutely. I feel that there is known interactions between AR signaling, DNA damage response, and innate immunity, as already mentioned, and that there is likely a convergence on a mechanism that allows cancer cells to accumulate damage, yet survive that instability and evade the immune system. So with alternate DNA damage response defects, including BRCA, TP53, and others, may there be this subpopulation within those groups that displays this STING imbalance and may benefit from PARP-based therapies?

Andrea Miyahira: Okay. Well thank you so much for taking the time to share this with us today.

Patrick Pilié: Thank you.