Jie Luo: Good afternoon, Andrea. Thank you for having me today to discuss my recent publication, which focuses on disabling the AR neo-enhanceosome by targeting the histone acetyltransferase p300 and CBP. So first, let me do some background introduction. So prostate cancer actually is a prototypical enhancer-driven malignancy, in which the androgen receptor serves as the central driver. AR functions as the master transcription factor, which can bind predominantly to the enhancer regions to regulate the oncogenic transcriptional programs. So this process actually relies on highly coordinated regulatory circuitry that requires the collaborative function of multiple co-activators, including FOXA1, the SWI/SNF chromatin remodeling complex, BRD4, and the mediator complex, as well as p300 and CBP, the acetyltransferases. And more recently, we also, in our lab, identified some unique factors, such as NSD2, which function as AR co-activators. So rather than directly targeting AR, which often leads to the emergence of AR mutations and resistance to AR-targeted therapies, many studies actually support the idea that these co-factors represent compelling therapeutic targets.
Many inhibitors and degraders have been developed to target the co-factors, such as BRD4, the SWI/SNF complex, and NSD2. So in this study, we focus on one of the key co-factors, p300. So p300 and its analog, CBP, are the major paralog enzymes responsible for catalyzing histone acetylation across the core histone proteins. So structurally, the p300/CBP proteins contain several key domains, a central catalytic module composed of the bromodomain, the PHD domain, and the HAT domain, as well as several domains, including the KIX domain and the TAZ2 domains, which mediate the protein-protein interactions. So very importantly, p300/CBP have been identified as AR co-activators. So the [inaudible 00:02:44] study already showed that AR and p300 have a physical interaction. Recently, studies also showed that p300/CBP are the key enzymes responsible for catalyzing the H2B N-terminal acetylation, which have been identified as very good and very important markers for the active enhanceosome. So our story actually starts with the analysis of the p300 and CBP protein levels in the patient-matched prostate cancer tissues versus the adjacent benign prostate tissues. And our results actually show that these enzymes are significantly elevated in the malignant prostate tissues, and consistently, the downstream substrates, like H2B N-terminal acetylation, are also increased in the prostate cancer tissues.
So this is the data. So actually, we're using multiplex immunofluorescence staining, and we can see, in the malignant tissues, the red signals, which represent the p300 levels, are dramatically increased compared to the benign prostate tissues. Consistently, we can see the H2B K20 acetylation signals in the malignant tissues also show dramatic increase compared to the normal prostate epithelial tissue. So this data actually suggests the hyperactivated p300 H2B N-terminal acetylation axis in the malignant-transformed prostate epithelial cells, and also indicating the [inaudible 00:04:18] as potential therapeutic targets for the prostate cancer patient. So in order to dissect the interplay between AR and p300 in the chromatin regions, we classified the AR-bound enhancers into two groups. One is the AR/p300 shared sites, and the other one is the AR-only enhancers. So we compared the epigenetic landscape in these two regions. So based on the ChIP-seq data, we can see while the AR binding intensity in the AR/p300 shared sites versus the AR-only sites doesn't show dramatic difference, however, multiple key co-factors and histone modifications revealed a significant difference. At AR/p300 shared enhancers, we can observe higher enrichment of MED1 and Pol II, H3K27 acetylation, H2B N-terminal acetylations, all of which are associated with active enhancer functions.
Consistently, the ATAC-seq analysis showed increased chromatin accessibility at AR/p300 shared sites compared to the AR-only enhancers. So again, this data highlights the functional importance of the p300 and AR co-occupancy in driving the oncogenic transcriptional programs in prostate cancer. So given the marked acetylation of H2B N-terminal acetylation and p300 levels in the prostate cancer tissues, we developed dual PROTAC degraders which target p300 and CBP proteins to suppress these important histone marks, the H2B N-terminal acetylation. And actually, this kind of suppression cannot be achieved by the classical bromodomain p300/CBP inhibitors alone. So our new degrader, named CBPD-409, it's a cereblon-based, orally available, dual degrader. So we used GNE-049, which is the classical bromodomain inhibitor, as a warhead. So as you can see here, the TMT-based mass spectrometry data demonstrate that CBPD can selectively and potently degrade the p300 and CBP proteins within hours without affecting other bromodomain-containing proteins, such as BRD4, BRD2, BRD3. So compared to the bromodomain inhibitors, we also observed that CBPD-409 potently suppressed the H2B N-terminal acetylation levels, as we can see here, including the H2BK5, K12, K16, K20 acetylations, almost completely eliminated by our CBPD treatment. However, the classical bromodomain inhibitors, like GNE-049, CCS1477, showed very minor or minimal effects in terms of the H2B N-terminal acetylation levels. So we also performed some functional analysis. So the RNA-seq data revealed that, intriguingly, when the prostate cancer cells were pretreated with CBPD-409, the p300 and CBP dual degrader, they almost completely lost their transcriptional response to the androgen, so suggesting that AR signaling is effectively shut down following the p300/CBP degradation.
Again, in contrast, the bromodomain inhibitor, GNE-049, only can partially suppress the AR signaling upon androgen stimulation. So this data, again, highlights the important role of p300 for the AR signaling, and also emphasizes the necessity to degrade p300/CBP, but not just suppress the p300/CBP bromodomain activity. So we then tested the efficacy of CBPD-409 across a panel of cell lines, including various cancer lineages, as well as normal cells. So as we can see here, CBPD-409 selectively suppressed growth of specific cancer types, such as AR-positive prostate cancer, which is highlighted as the red, and also neuroblastoma and multiple myeloma. So all these cell lines actually have been proved as enhancer-driven malignancies. So a lot of the other cancer lineages showed minimum or no response. So this time, all this data actually suggests that CBPD-409 has very good selectivity. And also here, I want to emphasize, importantly, all the tested normal cell lines weren't affected by CBPD-409, again highlighting the selective inhibitory effects of the p300/CBP degradation. Consistently, we also observed a strong elevation of the H2B N-terminal acetylation in CBPD-409-sensitive cancer cells compared to the resistant cancer cells or the normal cells. So this consistent trend was not seen with other histone marks, such as H3K27 acetylation or H3K27 trimethylation. So this data suggests that H2B N-terminal acetylation levels can serve as a predictive biomarker for cellular sensitivity to the p300/CBP-targeted therapies, particularly for the p300/CBP degraders.
And more importantly, we also did some in vivo efficacy studies. So the in vivo studies revealed that CBPD-409 exhibits greater tumor inhibition efficacy in multiple CRPC preclinical models without inducing any evident toxicity in animals, supporting its potential for clinical translation in the future. So overall, our study proved the p300 H2B N-terminal acetylation axis is critical in the AR neo-enhanceosome complex activity, and CBPD-409 is a potent, selective, and orally available PROTAC degrader of p300/CBP, with no evident toxicity in all different kinds of animals we have tested. So the degradation of p300/CBP exhibits a distinct mechanism of action compared to the classical bromodomain inhibition, which the degrader can suppress the H2B N-terminal acetylation, which cannot be achieved by the bromodomain inhibitors. So again, the p300/CBP degraders can eliminate the H2B N-terminal acetylation, which is a key enhancer mark, which cannot be suppressed by the bromodomain inhibitors. So the H2B N-terminal acetylation levels also can serve as a predictive biomarker for the response to the p300/CBP degradation. So the take-home message from our study is selective degradation of p300/CBP rewires the AR neo-enhanceosome function by eliminating the H2B N-terminal acetylation enhancer mark. So this effect cannot be achieved by the conventional inhibition, so establishing protein degradation as a superior strategy, and identifying H2B N-terminal acetylation as a predictive biomarker for patient stratification. So for the future plan of our project, recently, we just developed a new modality, called DALTAC, which can achieve tissue-selective targeting of p300 in AR-positive prostate cancer cells.
So we believe this new modality is going to provide some benefit, especially we can prevent some unwanted side effects for targeting p300/CBP, and this is going to favor and also help the future clinical translation of targeting p300/CBP in prostate cancer. Here are some acknowledgements. So I want to thank my supervisor and mentor, Dr. Arul Chinnaiyan and Dr. Abhijit Parolia, and all the members in MCTP. I also want to thank PCF to fund my studies through the Challenge Award and the PCFYI, the Young Investigator Award. I also want to thank the University of Michigan and HHMI. Thank you.
Andrea Miyahira: Well, thank you so much, Dr. Luo, for sharing this super interesting paper. So do you know if this p300/CBP axis plays any role in progression to or the biology of lineage-plastic prostate cancer subtypes, such as NEPC, or more generally in prostate cancer heterogeneity?
Jie Luo: So yeah, actually, this is a very good question. So in our hands, we did test the p300/CBP degrader in different kinds of prostate cancer, including NEPC. However, our data showed that p300/CBP degradation is potent in AR-positive prostate cancer cells, but has very minor or minimal impact on the growth of the AR-negative NEPC models. So this data actually suggests the p300/CBP-H2B N-terminal acetylation axis is not a core survival dependency in the established NEPC, at least for the NEPC subtypes we tested. So more broadly, we view p300/CBP-H2B N-terminal acetylation as an AR-enhancer-associated enhancer mark, so that helps to explain the prostate cancer heterogeneity. So highly predictive in AR-driven disease, but less informative for the AR-indifferent or lineage-plastic states.
Andrea Miyahira: Okay, thanks. And what other non-prostate roles does the p300/CBP axis play, and what side effects might you anticipate by targeting it with the PROTAC or the DALTAC, which you've shown inhibits more completely than the prior p300 inhibitors that have been tested clinically?
Jie Luo: Yeah. So beyond prostate cancer, actually, we did some cross-cancer lineage drug screening. We do see p300/CBP play critical roles in multiple enhancer-driven malignancies, including neuroblastoma, acute myeloid leukemia, AML, and multiple myeloma. So consistent with this data, our present screening data also shows our degrader, CBPD, can potently suppress the proliferation across these cancer lineages, supporting the broad relevance of targeting p300/CBP in enhancer-addicted tumors. So regarding the potential side effects, previous studies of p300/CBP dual inhibition have reported some hematologic toxicity, particularly thrombocytopenia, likely reflecting the on-target effects on normal hematopoietic components. So because the PROTAC-mediated degradation can achieve more complete target suppression than the earlier clinical inhibitors, this toxicity remains an important consideration. So to mitigate these kinds of risks, we are developing a next-generation tissue-selective modality, DALTAC. So based on this modality, we can selectively target p300/CBP in AR-positive prostate cancer cells and prevent unwanted side effects in other normal tissues.
Andrea Miyahira: Okay. Well, that sounds really exciting. Just tell us more about your team's translational next steps for targeting p300/CBP and the use of the H2B N-terminal acetylation as a predictive biomarker. For instance, what patient population, selective strategy, and therapeutic strategies, such as combination with enzalutamide, are you anticipating?
Jie Luo: Yeah. So our immediate translational priority for the p300/CBP degrader is IND-enabling studies in preparation for first-in-human, a Phase I clinical trial. So we anticipate initially enrolling patients with CRPC, castration-resistant prostate cancer, where AR is the key driver and AR should be positive, of course, and also the clinical data indicate the strongest dependency on p300/CBP H2B N-terminal acetylation in these kinds of patients. So from the patient selection standpoint, we envision using tumor H2B N-terminal acetylation levels as a predictive biomarker to identify patients with enhancer-addicted, AR-active disease, most likely to benefit from p300/CBP degradation. So this biomarker-guided approach may help stratify responders and inform early pharmacodynamic readouts in the clinic. So therapeutically, we plan to evaluate the p300/CBP degrader both as a single agent or in [inaudible 00:17:21] combination with AR-pathway inhibitors, such as enzalutamide. So given the complementary mechanisms, direct AR blockade and elimination of the p300-dependent enhancer activity, we hypothesize that dual targeting will achieve a deeper and more durable suppression of AR signaling, potentially delaying the overcoming of resistance observed in the current AR-directed therapies.
Andrea Miyahira: Okay. Well, that's really exciting. And what major questions remain about the epigenetics of prostate cancer that you plan to pursue next?
Jie Luo: So one major question actually I plan to pursue in the future is how to selectively target epigenetic dependencies in AR-positive prostate cancer cells without interrupting or inhibiting global chromatin regulators, which are required for normal tissues. So to address this, as I just mentioned, we're developing DALTAC-based strategies which can exploit oncogenic protein-protein interactions to achieve context-restricted epigenetic inhibition. So this modality can minimize the systemic toxicities associated with broad co-factor inhibition. So in parallel, I'm also very interested in understanding some epigenetic basis of AR-negative and lineage-plastic prostate cancers, particularly in neuroendocrine prostate cancer, NEPC cells. So a key question is how the epigenetic landscape is reprogrammed during lineage plasticity, and which chromatin regulators become essential for NEPC cell survival and maintenance. So to address this, I plan to do systematic profiling of the epigenetic state of NEPC, and identify the subtype-specific epigenetic co-factors, which eventually we can develop targeted therapies to target these specific co-factors and eventually overcome NEPC.
Andrea Miyahira: Okay. Well, it all sounds really exciting, so congratulations, and I look forward to hearing about your progress. Thanks.
Jie Luo: Thank you.