Research led by senior author Nupam P. Mahajan, PhD, Professor of Surgery in the Division of Urologic Surgery at Washington University St. Louis, identified a new modification in androgen receptor (AR) protein that plays a critical role in prostate cancer cells (Sci Transl Med 2022; doi: 10.1126/scitranslmed.abg41).
AR signaling continues to play a dominant role in all stages of prostate cancer, including castration-resistant prostate cancers (CRPC) that have developed resistance to second-generation AR antagonists such as enzalutamide. Realizing the importance of AR signaling, his team has developed the compound, (R)-9b, that knocks down expression of the AR overall, rather than just blocking its ability to bind to testosterone or reducing overall testosterone levels in the body, similar to currently approved drugs.
Earlier, using this compound, his team identified a long noncoding RNA, NXTAR, located next to the AR in the genome. "The discovery of (R)-9b served as an excellent tool that revealed the presence and the role of NXTAR that influences AR expression and/or is affected by it," said Mahajan.
The current therapeutic strategy, including enzalutamide that remains a current treatment-resistant prostate cancer, is designed to block the AR to prevent androgens, in particular testosterone, from fueling the growth of prostate tumors. "Unfortunately, all patients inevitably develop resistance," he noted.
Modification of AR Major Barrier
Building on his previous research, Mahajan's recent study centered on the premise that tumors that become refractory to anti-androgen therapy might modify AR, and this modification could be the major barrier to effective treatment of CRPC. The researchers found a new acetylation event in the androgen receptor at a specific residue that contributes to enzalutamide resistance in this setting.
"This acetylation required the function of the non-receptor tyrosine kinase ACK1," he explained. "Administration of ACK1 inhibitor (R)-9b sensitized mouse models of castration-resistant prostate cancer, which were not responding to enzalutamide treatment, suggesting a possible therapeutic strategy for this recurrent disease."
The AR is a clever player, Mahajan noted. "In our current study, we deduced that for CRPC patients with enzalutamide-resistance, there must be a link to AR. Finding that link led to the possibility of AR modifications."
This thought process became the methodology applied to focus on identifying the modified in AR, which in spite of its binding to enzalutamide, is still able to activate many genes responsible for growth of tumors, which led to the discovery AR K609 acetylation.
Acetylation Crucial for AR
Mahajan's research lab had earlier discovered another AR modification-phosphorylation at Y267. "We found critical evidence; this AR acetylation is dependent on the AR Y267 phosphorylation," he explained. "This 'dual-modified AR' regulates the expression of AR and ACK1 that phosphorylates AR at Y267, thereby creating a self-sustaining circuit that maintains high expression of AR and ACK1."
While the CRPC patients do respond initially to enzalutamide, the clinical benefit lasts for about 2 years, Mahajan noted. "Resistance to enzalutamide, a drug used throughout the world, has seen a significant increase in the number of resistant patients, which is a major issue in the field, as patients with recurrent disease have limited options."
The discovery of this particular acetylation, however, was crucial for AR, which was very interesting, he noted. "We discovered the 609-site present in the DNA-binding region that resembles the fingers holding the DNA, also known as 'zink-finger,'" he said. "This acetylation allowed AR to bind the DNA, even in the presence of enzalutamide. We sought to unravel the mechanistic details of successive interdependent AR modification events that show resistance to AR antagonists. When we realized this acetylation was dependent on AR Y267 phosphorylation, then we could use a compound, (R)-9b that we had discovered earlier in our lab as an inhibitor targeting the ACK1-AR nexus."
Single & Combination Treatments
To begin, the team first injected tumor-bearing mice with the (R)-9b compound, which was highly effective in suppressing the tumor growth.
"Initially, we decided to route the compound subcutaneously; however, we learned a better strategy was to give the compound orally, which worked very well in mouse models," said Mahajan. "Now we have identified a compound that has the ability to suppress enzalutamide-resistant tumors when administered orally."
In a slightly different study, from the human perspective, Mahajan and his team tested 80 human samples of tumor cells from the earliest to the latter stages of cancer, as well as normal human prostate. "We questioned: What is the level of this acetylate in these patients? We were glad to see that those with a normal human prostate barely showed any expression," he noted.
However, as disease progressed, it showed increases in the AR acetylation. "This is an important piece of the evidence that patients with later stage or metastatic disease are more likely to have a harder time responding to enzalutamide," Mahajan shared. "We need to conduct more studies to reach long-term efficacy in the patients, so we understand this disease." He said the team has a clinical trial set for next year.
"We think (R)-9b could be combined to reach a far greater efficacy, as opposed to using one drug," he stated. The goal is to use the drug as a single approach and as a combination of treatments. "We are working diligently and hope that by next year we will accomplish our goal so that these patients have more treatment options."
Amy Gallagher is a contributing writer.