Drug resistance in cancer is often linked to changes in tumor cell state or lineage, but the molecular mechanisms driving this plasticity remain unclear. Using murine organoid and genetically engineered mouse models, we investigated the causes of lineage plasticity in prostate cancer and its relationship to antiandrogen resistance. We found that plasticity initiates in an epithelial population defined by mixed luminal-basal phenotype and that it depends on elevated JAK and FGFR activity. Organoid cultures from patients with castration-resistant disease harboring mixed-lineage cells reproduce the dependency observed in mice, by upregulating luminal gene expression upon JAK and FGFR inhibitor treatment. Single-cell analysis confirms the presence of mixed lineage cells with elevated JAK/STAT and FGFR signaling in a subset of patients with metastatic disease, with implications for stratifying patients for clinical trials.

The inherent plasticity of tumor cells provides a mechanism of resistance to many molecularly targeted therapies, exemplified by adeno-to-neuroendocrine lineage transitions seen in prostate and lung cancer. Here we investigate the root cause of this lineage plasticity in a primary murine prostate organoid model that mirrors the lineage transition seen in patients. These cells lose luminal identity within weeks following deletion of Trp53 and Rb1, ultimately acquiring an Ar-negative, Syp+ phenotype after orthotopic in vivo transplantation. Single-cell transcriptomic analysis revealed progressive mixing of luminal-basal lineage features after tumor suppressor gene deletion, accompanied by activation of Jak/Stat and Fgfr pathway signaling and interferon-a and -g gene expression programs prior to any morphologic changes. Genetic or pharmacologic inhibition of Jak1/2 in combination with FGFR blockade restored luminal differentiation and sensitivity to antiandrogen therapy in models with residual AR expression. Collectively, we show lineage plasticity initiates quickly as a largely cell-autonomous process and, through newly developed computational approaches, identify a pharmacological strategy that restores lineage identity using clinical grade inhibitors.

227 Background: Despite the remarkable successes of targeted cancer therapies, certain cancers, including lung, breast, and prostate cancer and melanoma, invariably become resistant to therapy. One mechanism of secondary resistance—lineage plasticity—arises when cells alter their identity and transition into aggressive states. In the case of prostate cancer, cells can acquire a neuroendocrine histology. This is often associated with a loss of tumor suppressor genes, such as TP53, RB1, and PTEN. However, while these genomic events initiate plasticity, tumor progression is not always associated with successive genomic alterations. This, in essence, not only poses a clinical challenge, but also confronts us with a wide-open biological question—what are the molecular underpinnings of lineage plasticity, and importantly, can the process be reversed? Methods: To study the temporal evolution of lineage plasticity and its relationship to androgen receptor signaling inhibitor (ARSI) resistance, we utilized genetically engineered mouse models (GEMMs) and murine organoids that were deleted for Tp53, Rb1, and/or Pten. Single cell RNA analyses were utilized to dissect which genes and pathways were up-regulated and most associated with the progression of plasticity. Plasticity-associated genes and pathways were perturbed using FDA-approved inhibitors or genetic editing tools. The presence of these pathways was confirmed in a subset of metastatic index lesions obtained by radiologically guided biopsies and as visualized by metabolic imaging. Furthermore, relevant findings were functionally validated in human tumor derived organoids (“tumoroids”). Results: Using GEMMs and organoid models, we found the lineage plasticity depended on increased Janus Kinase (JAK) and fibroblast growth factor receptor (FGFR) activity. Pharmacologic inhibition using FDA–approved inhibitors of JAK/STAT (ruxolitinib) and FGFR (erdafinitib), or through genetic knockdown, demonstrated increased androgen receptor (AR) signaling and restored ARSI sensitivity. These findings were further validated in a subset of ARSI-insensitive human tumoroids. By performing single cell RNA sequencing on mCRPC tumors biopsies, the presence of highly plastic JAK/STAT- and FGFR-high cells were confirmed, with implications for stratifying patients for clinical trials. Conclusions: JAK/STAT and FGFR signaling pathways promote lineage plasticity and result in complete insensitivity to androgen receptor signaling inhibitors (ARSIs). FDA-approved inhibitors of JAK/STAT (ruxolitinib) and FGFR (erdafitinib) synergize to reverse lineage plasticity and restore ARSI sensitivity.

The inherent plasticity of tumor cells provides a mechanism of resistance to many molecularly targeted therapies, exemplified by adeno-to-neuroendocrine lineage transitions seen in prostate and lung cancer. Here we investigate the root cause of this lineage plasticity in a primary murine prostate organoid model that mirrors the lineage transition seen in patients. These cells lose luminal identity within weeks following deletion of Trp53 and Rb1, ultimately acquiring an Ar-negative, Syp+ phenotype after orthotopic in vivo transplantation. We performed single-cell transcriptomic analysis of a time-course experiment on the prostate organoid following Trp53 and Rb1 deletion. Critical to this study, we developed SEACells, a method that enumerates distinct, highly granular cell states, allowing for robust transcriptomic quantification. Leveraging the SEACell platform, we developed several graph-based computational approaches based on Markov absorption, diffusion maps, and attributed stochastic block models to quantify dynamic changes in plasticity. These quantitative models independently confirmed rapid collapse of cell-type fidelity in the form of a mixed luminal-basal phenotype following tumor suppressor gene deletion. These methods compute metrics for plasticity that we correlated to candidate driver gene programs. Among the strongest plasticity correlates, Jak-Stat and Fgfr signaling stood out as gene programs activated early in the time-course prior to any corresponding morphological changes. We further developed a regression-based approach to nominate ligand-receptor interactions that activate downstream Jak-Stat signaling, which identified Fgf-Fgfr interactions that were functionally validated with growth factor addition and pharmacological inhibition. Most strikingly, genetic or pharmacologic inhibition of Jak1/2 in combination with Fgfr blockade not only reversed the plastic state and restored organoids to their wild-type morphology, but also re-sensitized drug-resistant cells to antiandrogen therapy in models with residual AR expression. We additionally confirm early activation of Jak/Stat transcriptional programs in an Rb1/Trp53/Pten-deleted genetically engineered mouse model undergoing substantial cell-type diversification under plasticity in the context of the tumor microenvironment. Collectively, we show that lineage plasticity initiates quickly as a largely cell-autonomous process that is further increased in the in vivo setting, and through newly developed computational approaches, we identify a pharmacological strategy that restores lineage identity using clinical grade inhibitors. Citation Format: Joseph M. Chan, Wouter R. Karthaus, Manu Setty, Jillian R. Love, Samir Zaidi, Jimmy Zhao, Zi-ning Choo, Sitara Persad, Justin LaClair, Kayla E. Lawrence, Ojasvi Chaudhary, Ignas Masilionis, Linas Mazutis, Ronan Chaligne, Dana Pe'er, Charles Sawyers. Reversal of lineage plasticity in RB1/TP53-deleted prostate cancer through FGFR and Janus kinase inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1594.