Arrested Agonist Paradigm For Selective Radiosensitization of Prostate Cancer | bioRxiv
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| Figure 3D A model summarizing these results. DHT acts as a full agonist, activating the entire AR signaling cascade including DSBs and transcriptional programs. OH-FLU acts as an arrested agonist, inducing DSBs through initial AR signaling but not downstream gene expression. |
Arrested Agonist Paradigm For Selective Radiosensitization of Prostate Cancer | bioRxiv
Abstract
As a prototypical nuclear hormone receptor, the androgen receptor (AR) signals via a sequential cascade triggered by binding to androgenic ligands such as testosterone and dihydrotestosterone (DHT). This cascade includes dimerization of the ligand-receptor complex, nuclear translocation, chromatin binding to response elements, recruitment of TOP2B and co-activator complexes, and induction of an effector transcriptional program. In prostate cancers, this AR signaling cascade is an essential driver of growth and survival, yet its activity confers potential vulnerabilities through transient TOP2B-mediated DNA double strand breaks. We investigated the ability of non-steroidal AR ligands to activate initial steps of the AR signaling cascade up to the point of AR- and TOP2B-mediated double strand breaks, with subsequent arrest of the signaling cascade to prevent induction of pro-growth/survival transcriptional programs in prostate cancer cells. We identified hydroxyflutamide (FLU) as such an androgen receptor arrested agonist; in androgen-deprived conditions, FLU induced AR nuclear translocation, chromatin binding, and TOP2B-mediated double strand breaks, but failed to induce AR target gene expression and prostate cancer cell growth. The FLU-mediated arrest in the signaling cascade could be attributed to the inability of FLU to allow association of AR with SMARCD2, a critical component of the BAF chromatin remodeling complex required for androgen induced AR co-activation. Interestingly, the FLU-induced, AR- and TOP2B-mediated double strand breaks could be used to selectively sensitize AR-positive prostate cancer cells to ionizing radiation in vitro and in vivo. These findings support a novel arrested agonist paradigm for selective radiosensitization of prostate cancer cells without inducing AR-mediated pro-growth and survival transcriptional programs.
Competing Interest Statement
The authors have declared no competing interest.
Damaging Cancer Cells to help Radiation Kill Them
This paper describes a novel approach to induce DNA damage in prostate cancer cells by exploiting the androgen receptor signaling pathway. Here are the key findings:
- The authors screened FDA-approved AR antagonists and found that hydroxyflutamide (OH-FLU) can induce initial steps of AR signaling like nuclear translocation and chromatin binding, but fails to activate AR transcriptional programs or promote cell growth. They term this an "arrested agonist" activity.
- OH-FLU was unable to recruit the coactivator SMARCD2, which is required for full AR transcriptional activation. This likely explains its arrested agonist behavior.
- Like AR agonists, OH-FLU induced transient DNA double strand breaks in prostate cancer cells in a TOP2B-dependent manner. However, without SMARCD2 recruitment, it did not activate growth and survival genes.
- Pre-treating prostate cancer cells with OH-FLU led to synergistic DNA damage when combined with ionizing radiation, selectively radiosensitizing AR-positive cells.
- This arrested agonist approach allowed exploitation of AR-induced DNA vulnerability while avoiding growth promotion, representing a potential new prostate cancer treatment strategy.
In summary, this is an innovative approach using non-steroidal AR ligands as arrested agonists to induce DNA damage through initial AR signaling steps, without activating downstream gene expression. The selective radiosensitization has promising therapeutic potential.
androgen receptor (AR) signaling pathway
The androgen receptor (AR) signaling pathway plays a critical role in prostate cancer growth and metastasis. Here is a brief overview:
- Androgens like testosterone and dihydrotestosterone (DHT) bind to the AR, causing it to dimerize and translocate to the nucleus.
- In the nucleus, AR binds to androgen response elements on DNA and recruits coactivators to activate target genes involved in cell proliferation, survival, and metastasis.
- Important AR target genes include PSA, TMPRSS2, and others that drive cancer cell growth and spread.
- In prostate cancer, the AR pathway is often upregulated, fueling metastatic progression. The cells become "addicted" to AR signaling.
- AR signaling can promote key steps of metastasis including epithelial-mesenchymal transition, migration/invasion, stemness, and survival in circulation and at distant sites.
- Standard treatments like ADT aim to suppress AR signaling systemically, but advanced prostate cancers often become castration-resistant through mechanisms like AR overexpression/mutations.
- New drugs like enzalutamide try to more fully shut down AR signaling, but resistance still develops.
- Targeting the AR pathway remains critical to treating metastatic prostate cancer, but new approaches are needed to exploit vulnerabilities like the DNA damage effects seen with arrested AR agonists.
The AR signaling cascade is a major driver of prostate cancer metastases. This study provides a novel way to induce DNA damage through the AR pathway while avoiding growth promotion, representing an innovative therapeutic approach.
Hydroxyflutamide (OH-FLU)
Hydroxyflutamide (OH-FLU) is the active metabolite of the anti-androgen drug flutamide. Here are some key points about OH-FLU:
- Flutamide is an older non-steroidal anti-androgen drug that was previously used to treat prostate cancer by blocking AR signaling. It is metabolized into OH-FLU in the body.
- OH-FLU can competitively bind to the AR, but acts as an "arrested agonist" rather than a full antagonist.
- While OH-FLU binds AR and induces initial steps like nuclear translocation and DNA binding, it fails to fully activate AR transcriptional programs or promote growth.
- This is likely because OH-FLU induces a unique AR conformational change that prevents proper recruitment of essential coactivators like SMARCD2.
- Without being able to properly assemble the full transcription complex, OH-FLU-bound AR cannot fully activate target gene expression, despite initiating early signaling steps.
- So OH-FLU exploits the vulnerability of AR-induced DNA damage, but avoids switching on the pro-growth and survival genes that are the normal downstream effects of AR signaling.
- While flutamide itself is no longer commonly used, the researchers propose optimizing its active metabolite OH-FLU as an innovative arrested agonist approach for prostate cancer treatment.
In summary, OH-FLU can initiate upstream AR signaling to induce DNA damage, but fails to activate the full signaling cascade and growth programs due to impaired coactivator recruitment. This selective mechanism explains its unique arrested agonist behavior.
How could it treat mCRPC
The arrested agonist approach using OH-FLU or optimized derivatives could potentially help treat metastatic castrate-resistant prostate cancer (mCRPC). Here are a few reasons why:
- mCRPC remains dependent on AR signaling for growth and survival, even though the pathway is altered to resist traditional hormone therapies.
- Drugs like OH-FLU that exploit DNA damage through partial AR agonism could circumvent resistance mechanisms that rely on intact transcriptional activation.
- The arrested agonist activity selectively damages AR-positive cancer cells while avoiding growth promotion in mCRPC.
- Combining these AR-DNA damaging effects with radiation could give synergistic anti-tumor activity, as the study showed in models of castration-resistant disease.
- Since mCRPC continues to rely on AR signaling, targeting the altered pathway with this innovative approach could provide clinical benefit.
- If resistance eventually develops, it may further alter the AR pathway so that arrested agonists are no longer effective. But they could still help treat tumors before this occurs.
- Additionally, combining arrested agonists with next-gen AR-targeted drugs may prevent or overcome resistance mechanisms.
In summary, there is strong rationale that this novel treatment paradigm could be effective in mCRPC, particularly in rationally designed combinations to prevent resistance. Clinical trials would be needed to thoroughly evaluate the therapeutic potential in advanced prostate cancer patients.
How arrested AR agonists work
Figure 3 demonstrates that both full AR agonists like DHT and arrested AR agonists like OH-FLU can induce DNA double strand breaks (DSBs) in prostate cancer cells, while antagonists like enzalutamide do not.
The key findings shown in Figure 3 are:
- A) Immunofluorescent staining shows increased γH2A.X foci (indicating DSBs) in cells treated with DHT or OH-FLU, but not enzalutamide. This is quantified by the percentage of cells with >5 foci.
- B) The neutral comet assay directly measures DSBs by electrophoresis of broken DNA. DHT and OH-FLU significantly increased comet tail moment compared to untreated cells, confirming DSB induction.
- C) Knockdown of AR or TOP2B prevented DSB induction by both DHT and OH-FLU, implicating AR and TOP2B in the DNA damage caused by both full and arrested agonists.
- D) A model summarizing these results. DHT acts as a full agonist, activating the entire AR signaling cascade including DSBs and transcriptional programs. OH-FLU acts as an arrested agonist, inducing DSBs through initial AR signaling but not downstream gene expression.
In conclusion, this figure demonstrates that AR signaling is associated with TOP2B-mediated DSBs, and that both full agonists like DHT and arrested agonists like OH-FLU can exploit this vulnerability to damage DNA as part of their distinct mechanisms of action. This highlights a novel therapeutic opportunity.
DNA damage radiosensitizes Cancer Cells
Figure 4 shows that the DNA damage caused by arrested AR agonists like OH-FLU can selectively radiosensitize prostate cancer cells in vitro and in vivo.
Key findings in Figure 4:
- A-B) Pretreating cells with OH-FLU synergistically increased DNA double strand breaks when combined with low doses of ionizing radiation (IR). This shows the DNA damaging effects are synergistic.
- C) In clonogenic survival assays, OH-FLU pretreatment sensitized cells to IR, reducing the fraction of surviving colonies. This effect was most prominent at lower IR doses.
- D) In mice with prostate cancer xenografts, OH-FLU plus IR significantly delayed tumor growth compared to either treatment alone.
- E-F) Immunostaining of tumor biopsies showed increased γH2A.X foci at 12 hours after OH-FLU treatment, indicating DNA damage occurred in vivo before IR.
- G) A model summarizing the use of arrested AR agonists to selectively radiosensitize prostate cancer cells by inducing synergistic DSBs along with IR-induced damage.
In summary, this figure shows arrested AR agonist-induced DNA damage can synergize with IR to selectively radiosensitize prostate cancer cells in both cell and animal models. This provides preclinical proof of concept for this new treatment approach.
Treatment
Based on the preclinical data presented in this study, here are some suggestions for the potential clinical use and ideal patients for this arrested AR agonist treatment approach:
Treatment course:
- Administer an optimized arrested AR agonist like OH-FLU to induce DNA damage through partial AR agonism.
- Short-term or pulsed schedules may be preferred to induce transient DNA damage without prolonged AR pathway manipulation.
- Combine arrested agonist with localized radiation to achieve synergistic DNA damage and cancer cell death.
- Cycle treatment to exploit DNA vulnerability while avoiding constant AR pathway exposure.
- Monitor PSA levels but not as sole measure of response due to arrested agonist mechanism.
Ideal patients:
- Metastatic castration-sensitive or castration-resistant prostate cancer.
- Tumors positive for AR expression and intact initial activation pathway.
- Low levels of AR alterations or mutations that might confer resistance.
- Few or no neuroendocrine features, which could indicate AR independence.
- Early in disease course before extensive AR-dependent resistance develops.
- Reasonable performance status to tolerate multi-modal therapy.
In summary, the treatment would leverage the radiosensitizing ability of arrested AR agonists in a rationally designed, intermittent schedule in combination with radiation in well-selected prostate cancer patients.
Authors
Here are some key details about the authors' background and institutional affiliations:
- The authors are based at Johns Hopkins University School of Medicine and its affiliated centers like the Sidney Kimmel Comprehensive Cancer Center.
- Senior authors Srinivasan Yegnasubramanian and Theodore L. DeWeese are professors and researchers in the departments of Pathology and Radiation Oncology, respectively.
- They have been long-time collaborators on studies related to DNA damage and the androgen receptor signaling pathway in prostate cancer.
- First author Jonathan B. Coulter is an instructor in Radiation Oncology and Urology at Johns Hopkins.
- Other Hopkins authors come from departments like Urology, Pharmacology, and Oncology.
- This group has made seminal contributions to understanding how androgen receptor signaling can induce DNA double strand breaks and how this can be exploited therapeutically.
Relevant previous papers from this group:
- Haffner et al, 2010 Nature Genetics - Showed AR signaling causes TOP2B-mediated DNA breaks.
- Hedayati et al, 2016 Clinical Cancer Research - Demonstrated androgen cycling radiosensitizes prostate cancer cells through DNA damage.
- Schweizer et al, 2015 Science Translational Medicine - Clinical trial supporting bipolar androgen therapy.
This is an experienced collaborative group of researchers and clinicians at a top cancer center who have laid the groundwork for this current study on exploiting AR signaling vulnerabilities through the arrested agonist approach. Their expertise and previous work in this specific area lend credibility to the findings.
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