Abstract Prostate Specific Membrane Antigen (PSMA) is a prominent biomarker in Prostate Cancer (PC) and has evolved into a useful target for both imaging and therapy. But how PSMA becomes overexpressed upon PC development, its modulation by androgen deprivation therapy (ADT), and its absence in neuroendocrine-differentiated PC is not well understood. The exact mechanism by which PSMA is regulated has not been defined. Understanding PSMA regulation in PC patients for diagnostic, prognostic, and treatment purposes would be extremely valuable and could be exploited for PSMA-targeted therapies. To address this, we sought to characterize the epigenetic landscape of FOLH1 (the gene for PSMA) and determine the regulatory factors involved in FOLH1 transcription. Based on histone marker and protein ChIP-Seq data sets alongside ATAC-seq, we found that FOLH1 is a Super Enhancer (SE) and is collectively bound by an array of transcription factors (TFs), including AR, FOXA1, and HOXB13. We developed a hidden Markov model (HMM) with seven chromatin states built on average TF binding in the FOLH1 landscape and trained on the FOLH1 gene and known enhancer regions. Chromatin states were annotated based on features in the FOLH1 gene. With a simple regression model and k-fold cross-validation, mean occupancy of the three TFs and H3K27Ac in each state accurately predicts FOLH1 expression (ρ > 0.9) across PC samples. To assess the functionality of the enhancer regions predicted by our computational HMM, we performed a CRISPR interference (CRISPRi) screen with a nuclease dead-Cas9 protein conjugated to the KRAB transcriptional repressor and directed by a pool of gRNAs that tile across the AR protein-bound regions within the FOLH1 landscape. A sliding window analysis of our CRISPRi screen revealed that the top scoring gRNAs delineate 5 Regulatory Regions (RRs) found within the SE region of FOLH1 that control FOLH1/PSMA expression. Motif identification in these RRs have provided a list of potential TF candidates involved in regulating FOLH1, and knockdown experiments are currently being conducted to evaluate individual and combinations of TF contribution to FOLH1 expression. There is evidence for a constellation of TFs responsible for regulating FOLH1, and context-specific modifications in FOLH1 regulation will need to be evaluated in various models and conditions. These results suggest FOLH1 is regulated by epigenetic control in the SE region and may be altered in progression or with ADT. Characterizing the regulation of FOLH1/PSMA will result in a deeper understanding of the meaning of alterations in uptake on PSMA imaging and may enable some degree of control over expression, thus aiding in PSMA-directed treatments. Citation Format: Margaret E. White, Thomas Pranzatelli, Xavier Moore, Joel Bowman, Jay Chiorini, Peter Choyke, Kathy Kelly. The regulation of FOLH1/PSMA in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 976.

The authors are investigating self-complementary adeno-associated virus (scAAV) as a vector for intra-articular gene-delivery of interleukin-1 receptor antagonist (IL-1Ra), and its therapeutic capacity in the treatment of osteoarthritis (OA). To model gene transfer on a scale proportional to the human knee, a frequent site of OA incidence, studies were focused on the joints of the equine forelimb. Using AAV2.5 capsid and equine IL-1Ra as a homologous transgene, a functional ceiling dose of similar to 5x10(12) viral genomes was previously identified, which elevated the steady state levels of eqIL-1Ra in synovial fluids by >40-fold over endogenous production for at least 6 months. Here, using an osteochondral fragmentation model of early OA, the functional capacity of scAAV.IL-1Ra gene-delivery was examined in equine joints over a period of 12 weeks. In the disease model, transgenic eqIL-1Ra expression was several fold higher than seen previously in healthy joints, and correlated directly with the severity of joint pathology at the time of treatment. Despite wide variation in expression, the steady-state eqIL-1Ra in synovial fluids exceeded that of IL-1 by >400-fold in all animals, and a consistent treatment effect was observed. This included a 30-40% reduction in lameness and similar to 25% improvement in total joint pathology by both magnetic resonance imaging and arthroscopic assessments, which included reduced joint effusion and synovitis, and improved repair of the osteochondral lesion. No vector-related increase in eqIL-1Ra levels in blood or urine was noted. Cumulatively, these studies in the equine model indicate scAAV.IL-1Ra administration is reasonably safe and capable of sustained therapeutic IL-1Ra production intra-articularly in joints of human scale. This profile supports consideration for human testing in OA.

Toward the treatment of osteoarthritis (OA), the authors have been investigating self-complementary adeno-associated virus (scAAV) for intra-articular delivery of therapeutic gene products. As OA frequently affects weight-bearing joints, pharmacokinetic studies of scAAV gene delivery were performed in the joints of the equine forelimb to identify parameters relevant to clinical translation in humans. Using interleukin-1 receptor antagonist (IL-1Ra) as a secreted therapeutic reporter, scAAV vector plasmids containing codon-optimized cDNA for equine IL-1Ra (eqIL-1Ra) were generated, which produced eqIL-1Ra at levels 30- to 50-fold higher than the native sequence. The most efficient cDNA was packaged in AAV2.5 capsid, and following characterization in vitro, the virus was injected into the carpal and metacarpophalangeal joints of horses over a 100-fold dose range. A putative ceiling dose of 5 × 1012 viral genomes was identified that elevated the steady-state eqIL-1Ra in the synovial fluids of injected joints by >40-fold over endogenous levels and was sustained for at least 6 months. No adverse effects were seen, and eqIL-1Ra in serum and urine remained at background levels throughout. Using the 5 × 1012 viral genome dose of scAAV, and green fluorescent protein as a cytologic marker, the local and systemic distribution of vector and transduced cells following intra-articular injection scAAV.GFP were compared in healthy equine joints and in those with late-stage, naturally occurring OA. In both cases, 99.7% of the vector remained within the injected joint. Strikingly, the pathologies characteristic of OA (synovitis, osteophyte formation, and cartilage erosion) were associated with a substantial increase in transgenic expression relative to tissues in healthy joints. This was most notable in regions of articular cartilage with visible damage, where foci of brilliantly fluorescent chondrocytes were observed. Overall, these data suggest that AAV-mediated gene transfer can provide relatively safe, sustained protein drug delivery to joints of human proportions.

Adding tumor specific ligands to enhance vector tumor cell interaction is the conventional concept to generate tumor targeting adeno-associated viral vector (AAV). However, it remains poorly proved whether high AAV tumor cell interaction contributes to high tumor localization in vivo following systemic delivery. Here, we conducted directed evolution selections on patient derived xenograft models using a complex AAV capsid library. Uniquely, we compared the pressure for AAV tumor cell interaction alone (intratumoral library injection) and multi-layer pressure including traveling to the tumor and infection (intravenous library injection). Distinct patterns of AAV capsid motifs were identified after intratumoral and intravenous screenings. Motifs isolated from intratumoral screenings were named tumor specific motifs and those isolated from intravenous screenings were named systematic trafficking motifs. AAVs with tumor specific motifs but not systemic trafficking motifs showed significantly increased tumor cell transduction in vitro, indicating enhanced vector target cell interaction after intratumoral-based selections. Interestingly, following systemic delivery, AAVs with systemic trafficking motifs mediated hundreds of folds higher transgene expression than those with tumor specific motifs and wild type AAV in vivo. The combination of both motifs further increases the tumor tropism but not the transduction efficiency in vivo. When analyzing the AAV genome biodistribution by quantifying the genome copy number, the systemic trafficking motifs greatly reduced the native AAV tropism, which potentiated higher accessibility of AAV to the tumor. In contrast, AAVs only bearing tumor specific motifs maintained native AAV tropism and failed to mediate increased genome localization in tumor. Furthermore, in two independent patient derived xenograft models and two different tumor types, our novel AAV vector armed with combined motifs all showed hundreds of folds increase in transduction efficiency with no detectable off-targeting expression. In conclusion, contradictory to the prevailing theory, our study demonstrated that the AAV tumor cell interaction did not contribute to increased tumor localization but just specificity in vivo. Therefore, targeting is not only depending on AAV tumor cell interaction but also, and more importantly, depending on the accessibility of AAV to the tumor cells following systemic delivery. Future cancer directed AAV vector design should take into account the complex processes during systemic delivery as well as the vector tumor cell interaction.

Abstract Osteosarcoma (OS) is a pediatric cancer with 40% mortality despite aggressive treatments. Major treatment challenges are that some tumors do not respond to therapies or develop resistance over time, processes partially attributed to cellular heterogeneity within a tumor. We have previously demonstrated such heterogeneity using a reporter consisting of an Oct4 promoter driving expression GFP. Cells capable of activating the reporter, the GFP+ cells, are 100-fold more tumorigenic than the GFP- cells. Pure populations of GFP+ cells injected into mice produces heterogeneous tumors, indicating that the GFP- cells arise from the GFP+ cells. The two populations display distinct gene expression profiles, which are reproducible between tumors and representative of global changes. These characteristics suggest that the emergence of a GFP- cell population is due to epigenetic changes. The objective of this study was to investigate to what extent tumorigenic potential in OS is governed at the epigenetic level and can be manipulated exogenously by agents that modify or re-pattern the epigenome. To test this, sorted GFP+ OS cells were treated with the histone deacetylase inhibitor Trichostatin A (TSA) in vitro at a dose that increased histone acetylation without inducing cell death. Although TSA treatment showed promising antitumoral effects in vitro by inhibiting proliferation and inducing cell cycle arrest, however, global interrogation of the gene expression changes induced by TSA treatment revealed an activation of many cancer associated pathways. In fact, TSA treated Oct4-GFP+ cells injected into NSG mice generated tumors that displayed enhanced metastatic dissemination, a reduced GFP+ population, and histologic changes. Differential expression analyses between the GFP+ cells isolated from TSA tumors or tumors generated from DMSO treated cells showed that the new phenotype was associated with enrichment for cell cycle pathways and a decrease in extracellular matrix pathways. This was confirmed at the protein level and with proliferation assays. As rapidly proliferating cells are generally more sensitive to chemotherapy, we investigated the sensitivity of the GFP+ cells from TSA and Control tumors to Doxorubicin (Dox). Cells isolated from TSA tumors were significantly more receptive to Dox-induced cell death. Our results suggest that OS malignancy is partially governed at the epigenetic level and that it is possible to reduce intratumoral heterogeneity. A reduction in intraturmoral heterogeneity can be exploited in cases of tumors that are refractory to treatment or have acquired epigenetic resistance. Intermittent treatment with epigenetic modifiers could potentially sensitize the tumors to the other treatments, as have been demonstrated for other cancer types. Citation Format: Emma V. Hyddmark, Padraic Levings, Margaret White, Maria Guijarro, Elham Nasri, Ali Zarezadeh, Glyn Palmer, Steve Ghivizzani, Charles P. Gibbs. Epigenetic reprogramming of osteosarcoma tumor initiating cells alters histologic and metastatic phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2432.

Introduction: Osteosarcoma (OS) is the most common primary bone tumor in childhood and adolescence. Despite numerous treatment approaches, OS remains difficult to treat and often reoccurs after primary treatment. This failure may be explained by the intra-tumoral heterogeneity; an individual tumor is composed by cells with varying sensitivities to commonly used treatments. We have shown previously that we can identify and fractionate two distinct cell populations in OS, based on the cells’ ability to activate an exogenous human Oct4/GFP reporter (hOct4/GFP). We found that tumorigenic potential is almost entirely restricted to the GFP+ population. Based on differential analysis of global gene expression, we identified the proteasomal degradation pathway as a potential target to be considered in treatment of OS. The aim of this study is to investigate the effects of Bortezomib (an FDA approved Proteasome Inhibitor) on osteosarcoma cells and to compare it to commonly used drugs such as Doxorubicin.