(P 187) PRECLINICAL VALIDATION OF TARGET ENGAGEMENT ASSAYS AND INVESTIGATION OF MECHANISTIC IMPACTS OF FHD-609, A CLINICAL-STAGE BRD9 DEGRADER BEING DEVELOPED FOR THE TREATMENT OF SYNOVIAL SARCOMA

Objective: Synovial sarcomas express the SSX-SS18 fusion oncoprotein, which drives tumorigenesis by compromising the function of the BAF (BRG/BRM-associated factors) chromatin remodeling complex. Cells with this form of BAF perturbation are heavily dependent on the non-canonical BAF (ncBAF) subunit BRD9 (Bromodomain-containing protein 9) for survival. However, the mechanism by which BRD9 promotes tumor growth is not well understood.
FHD-609 is a heterobifunctional degrader of BRD9 that is currently in clinical development for the treatment of advanced synovial sarcoma. Assays to measure the abundance of BRD9 protein in patient tumors will be important for demonstrating target engagement with FHD-609. Additionally, because tumor biopsies occur infrequently, assays to monitor BRD9 levels in peripheral tissues may permit longitudinal analysis of BRD9 degradation in patients treated with FHD-609. The objectives of the present study were to develop clinically relevant assays to quantify BRD9 protein levels in tumor and peripheral blood mononuclear cells (PBMCs), to profile the pharmacodynamics (PD) of FHD-609 in these tissues, and to explore the downstream impacts of BRD9 degradation in synovial sarcoma.

Methods: Novel IHC and flow cytometry methods were developed to quantify BRD9 protein levels in formalin-fixed, paraffin-embedded (FFPE) tissue and PBMCs. Mice bearing SYO-1 xenografts were treated with either efficacious (3mg/kg) or sub-efficacious (0.1 mg/kg) concentrations of FHD-609, and tumors and plasma were collected 3, 24, 48, 72, 168 and 264 hours after acute or chronic dosing. IHC was used to assess the dynamics of BRD9 suppression in tumors, while plasma and tumor drug concentrations were quantified to estimate FHD-609 pharmacokinetics. In parallel, flow cytometry was used to profile the dynamics of BRD9 suppression in various PBMC populations from naïve, immunocompetent mice receiving equivalent doses to the tumor-bearing animals. In addition to BRD9 IHC, further IHC analyses and RNA sequencing were performed to explore potential mechanistic impacts of BRD9 degradation.

Results: Both the IHC and flow cytometry assays developed for this study exhibited excellent sensitivity and specificity for BRD9. IHC demonstrated robust, dose-dependent BRD9 degradation in tumors within 3 hours of FHD-609 treatment. However, only the 3 mg/kg dose maintained complete degradation for up to 72 hours. Furthermore, multiple doses of either 0.1 or 3 mg/kg were required to achieve homogeneous BRD9 degradation. By flow cytometry, PBMCs showed rapid, dose-dependent depletion of BRD9. This response varied by cell type, with B cells correlating most closely to the degradation seen in tumors.
RNA sequencing of SYO-1 xenografts revealed broad transcriptional impacts in response to FHD-609 treatment. Notably, gene set enrichment analysis identified dose and time-dependent alterations in gene sets associated with oncogenic growth and proliferation. Finally, dose-dependent decreases in cell proliferation markers were also observed by IHC.

Conclusion: Here we report the first use of IHC and flow cytometry to measure target engagement with a BRD9 protein degrader in clinically relevant tissues. Using these methods, we found that higher doses of FHD-609 led to more prolonged suppression of BRD9 in tumors and PBMCs. Our results also suggest that certain PBMC populations are effective surrogates for tumor PD, which may enable longitudinal assessment of target engagement through serial blood collection in the clinic. In exploring the downstream effects of BRD9 degradation in vivo, we found that FHD-609 treatment downregulated gene sets implicated in numerous tumor types, including synovial sarcoma. Finally, these transcriptional changes corresponded to decreases in cellular proliferation markers. Taken together, these results suggest that prolonged degradation of BRD9 impairs synovial sarcoma tumor growth by impinging on transcriptional programs that support cell proliferation.