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Radioligand therapy (RLT) with prostate-specific membrane antigen (PSMA)-targeting ligands is effective in ~50% of patients with advanced prostate cancer (PC). Causes for RLT failure are not well understood. The prevalent PSMA heterogeneity in PC might contribute to RLT failure. Here we investigate the relationship between RLT efficacy and PSMA levels per cell, and PSMA heterogeneity. PC cells expressing different levels of PSMA (RM1-PSMA low, medium, high, or RM1-YFP that do not express PSMA), or a mix of PSMA− and PSMA+ cells (RM1-YFP/ RM1-PSMA-high; PC3/ PC3-PIP) at various ratios were subcutaneously injected into NSG mice. Mice were treated with 177Lu- or 225Ac-PSMA617 (RLT) and tumor growth was monitored. In a subset of mice, radioligand uptake (γ-counting), DNA damage and PSMA expression (anti-53BP1 and -PSMA immunohistochemistry, respectively) were quantified in tumors resected 2 days after RLT. Increasing PSMA levels and fractions of PSMA+ cells improved RLT efficacy in both, the RM1 and PC3 model. PSMA expression correlated with radioligand uptake into the tumor and the degree of DNA damage and. Treatment with 225Ac-PSMA617 (vs. 177Lu-PSMA617) improved RLT outcomes and tended to enhance the differences in therapeutic efficacy between experimental groups. Taken together, we demonstrate in mouse models of PC that optimal anti-tumor efficacy of RLT hinges on homogenously high target expression. Although PSMA-RLT is effective even in tumors with low PSMA levels or with a small number of PSMA+ cells, low or heterogeneous PSMA expression might result in undertreatment and selection of treatment resistant clones. Systematic assessment of intra- and inter-lesion PSMA heterogeneity is currently not feasible clinically; however, this issue might be addressed by individual patient dosimetry to optimize safely delivered maximal tumor doses. Clinical studies designed to determine intra- and inter-lesion PSMA heterogeneity and to optimize PSMA-RLT for each patient are highly warranted. Citation Format: Katharina Lueckerath, Kyle Current, Catherine Meyer, Clara Magyar, Christine E. Mona, Joel Almajano, Roger Slavik, Andreea D. Stuparu, Chloe Cheng, David Dawson, Caius Radu, Johannes Czernin. Heterogeneous tumor PSMA expression represents a resistance mechanism to PSMA-targeted radioligand therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5345.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States, with an overall survival of less than one year. An improved knowledge of PDAC biology, to uncover vulnerabilities specific to cancer cells, is needed to develop more effective therapeutic options. We are investigating the intersection between three aspects of PDAC biology that can, ultimately, be developed for therapeutics: (i) cytokine signaling, with a particular focus on the metabolic effects of interferons (IFNs), which are present in the highly inflamed and dense PDAC stromal microenvironment; (ii) nucleotide metabolism, a network of tightly regulated biochemical pathways that produce deoxyribonucleotide triphosphates (dNTPs), which are required for DNA replication; and (iii) the replication stress response pathway, an intracellular signaling mechanism that is activated by perturbations in DNA replication, and has been recently shown to govern key aspects of nucleotide metabolism. We hypothesize that IFN signaling reduces the levels of already limited dNTP pools in PDAC cancer cells, which respond by initiating metabolic and signaling mechanisms that coordinately function to increase dNTP recycling and biosynthetic mechanisms while simultaneously reducing their consumption. Our results, which include integrated metabolic, transcriptomic, and proteomic analyses, indicate that IFN signaling in PDAC cells induces a switch in nucleotide metabolism from a biosynthetic to a predominantly dNTP catabolic phenotype. This switch appears to be mediated by dNTP phosphohydrolysis catalyzed by the Sterile Alpha Motif and Histidine/aspartic acid Domain-containing protein (SAMHD1). Furthermore, we have investigated the effects of IFN signaling on dNTP phosphohydrolysis to deoxyribonucleosides across a panel of PDAC cancer cells, resembling the spectrum of the human disease. We also examined the biochemical fates of the deoxyribonucleoside products of SAMHD1 using targeted LC-MS/MS metabolic tracing experiments. We have also demonstrated a role for the replication stress response kinase Ataxia Telangiectasia and Rad3-related protein (ATR) in regulating dNTP levels in PDAC cells exposed to IFN. Furthermore, we have demonstrated that ATR activity is an actionable co-dependency of IFN-exposed cells and that pharmacologic inhibition of ATR eradicates PDAC cells exposed to IFN. Collectively, these studies increase our understanding of the interplay between cell extrinsic (IFN signaling) and intrinsic (replication stress) signal transduction networks, and the regulation of nucleotide metabolism in PDAC, and uncovered critical vulnerabilities to be exploited by new therapeutic approaches against this extremely aggressive and difficult to treat malignancy. Citation Format: Evan R. Abt, Amanda Dann, Thuc M. Le, Joe R. Capri, Chloe M. Cheng, Juna Yi, Soumya Poddar, Woosuk Kim, Timothy R. Donahue, Caius G. Radu. Identification of new modulators of nucleotide metabolism and replication stress in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4971.