Neuroendocrine Transdifferentiation Research Articles

Abstract 6268: A study of orphan nuclear receptor SHP (NR0B2) in prostate cancer progression

Abstract Prostate cancer (PCa) is a significant male cancer in many countries accounting for around 14% of new cancer cases globally in 2020 and ranked as 3rd common cancer in males in Hong Kong. The major risk factors include old age, high-fat diet, and genetics. Compared to other cancers, PCa is unique as its initial and advanced growth is androgen dependent. As tumor progresses, it accumulates numerous genetic alterations. Main therapeutic options include surgery, radiotherapy and or active surveillance. Upon treatment failure or when metastasis develops, androgen-deprivation therapy (ADT) with the use of potent androgen receptor (AR) antagonists and androgen biosynthesis inhibitors (e.g. Enzalutamide and abiraterone acetate) aiming to suppress AR signaling axis are the standard treatment options. However, the effect of these treatments is short-lived, in which nearly all patients will inevitably relapse to a hormone-independent and metastatic stage known as metastatic castration-resistant prostate cancer (mCRPC) within few years and further drive progression to a more lethal subtype, described as treatment-induced neuroendocrine prostate cancer (NEPC). The treatment options are limited and therefore resulted in high mortality, representing an emerging clinical problem. Recent studies have illustrated that NEPC progression follows a lineage plasticity model with a phenotypic transition to AR loss and neuroendocrine transdifferentiation. Our preliminary study has identified a nuclear receptor NR0B2 (SHP, small heterodimer partner SHP), which is a unique ligand-independent orphan nuclear receptor without a DNA-binding domain, that displays a significant up-regulation in clinical NEPC and prostate cancer cells with NEPC phenotype. Mechanistic studies revealed that SHP could directly interact with AR utilizing its NR Box1 motif while inducing repression on the AR transactivation via its another NR Box2 motif. Based on this, we conducted in vitro growth studies and revealed that NR0B2 overexpression could promote growth differentially in AR-negative PCa cells. However, AR-positive PCa cells were insensitive to AR blockade, providing supporting evidence that NR0B2 may be involved in repressing AR activity in prostate cancer cells. Multiple key NEPC and oncogenic TGFβ signaling markers showed significant upregulation in gene expression. Based on this, we hypothesize that upregulation of NR0B2 could play an important role in driving the advanced progression of CRPC via its direct interference of AR signaling axis and through other AR-independent signaling pathways, including activation of TGFβ signaling. (Study supported by a CUHK Direct Grant for Research 2022-2023, Project No. 2022.081) Citation Format: Hau Tak Daniel Lam, Franky Leung Chan. A study of orphan nuclear receptor SHP (NR0B2) in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6268.

Abstract 2898: Development of the lipid kinase PIKfyve PROTAC degrader against neuroendocrine prostate cancer

Abstract Therapies targeting the androgen receptor (AR) as the main driver of prostate cancer (PCa) can lead to various mechanisms of resistance and promote progression to castration-resistant PCa (CRPC), which has a median survival of only 13-23 months. Amongst recurrent CRPC, 17%-30% of patients develop neuroendocrine prostate cancer (NEPC), which is a PCa subtype characterized by a unique histology. NEPC exhibits a loss of AR signaling during neuroendocrine transdifferentiation which results in resistance to AR-targeted therapies and gain of cell characteristics resembling poorly differentiated neuroendocrine tumors. Despite advances in the understanding of NEPC development, treatment options remain limited, with platinum-based chemotherapy as the first-line treatment for both de novo and treatment-induced NEPC. However, response to first-line chemotherapy in NEPC is short, with a median survival of only seven months. The poor prognosis of NEPC is attributed in part to late diagnosis and a lack of effective therapeutic agents. Our previous work demonstrated that NEPC and AR-negative PCa exhibit higher dependency on the lipid kinase PIKfyve than AR-positive CRPC. Thus, development of PIKfyve inhibitory therapies targeting the emergent vulnerabilities of NEPC or AR-negative forms of PCa is a promising approach. Using a proteolysis targeting chimera (PROTAC) technology, we designed a class of PIKfyve specific degrader. The in vitro degradation efficiency were determined by immunoblotting, and DC50s (degradation concentration at 50%) were calculated for top candidates. Pharmacokinetic and pharmacodynamic were evaluated for in vivo study. In summary, we have developed a class of PIKfyve specific degrader using the E3 ligase von Hippel-Lindau (VHL) as a ligand via a diverse set of linkers. Mechanistic studies revealed that it induced PIKfyve degradation in a VHL- and proteasome-dependent manner. Citation Format: Yuanyuan Qiao, Chungen Li, Yang Zheng, Xia Jiang, Sarah Nicole Yee, Caleb Cheng, Yi Bao, Yuping Zhang, Yuzhuo Wang, Zhen Wang, Ke Ding, Arul Chinnaiyan. Development of the lipid kinase PIKfyve PROTAC degrader against neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2898.

Abstract 554: Determining the role of PROX1 in prostate cancer neuroendocrine trans-differentiation

Abstract Nonbiased mutational screening to identify essential genes and pathways in cancer is a powerful approach for understanding underlying biology as well as the identification of potential therapeutic targets. Epigenetic (EPI) and transcriptional factors (TF) play an important role in the change of phenotype and lineage plasticity from adenocarcinoma to castration resistant prostate cancer (CRPC), double negative (DN) or treatment-induced neuroendocrine prostate cancer (NEPC). When RB1 loss occurs in combination with TP53 alterations, stemness and lineage plasticity increase with one consequence being differentiation to a neuroendocrine (NE) lineage. Here, we are identifying and testing the functional role of epigenetic and transcription factors required for the viability of CRPC, DN and NEPC relative to a matrix of genotypic and phenotypic characteristics. We applied CRISPR-CAS9 dropout screens in prostate organoids and cell lines to identify gene/s associated with CRPC, DN and NEPC viability. We successfully CRISPR-engineered LNCaP clones of single (LNCaP-TP53-SKO, LNCaP-RB1-SKO) and double (LNCaP-TP53-RB1-DKO) gene knockouts, which were subsequently characterized for immuno-phenotype and transcriptome. Our dropout screens, targeting all TFs and EPIs, identified 117 TF and 40 EPI genes responsible for the viability of DN, NEPC and LNCaP models. We found known genes such as FOXA1 and ASCL1, as well as novel genes including PROX1 and KLF3, which were validated through guide RNA competition assays. Our results showed PROX1 is essential for cell viability in a DN organoid model, and its expression is associated with increased aggressiveness, indicating PROX1 has role in stemness/lineage plasticity in treatment resistant adenocarcinoma. RNA-seq analysis following overexpression of PROX1 in the LNCaP-TP53-RB1 null adenocarcinoma model, with and without enzalutamide, identified pathways enriched for cell proliferation, differentiation, neuronal development, and prostate gland development. In summary, our study has suggested an important role for PROX1 in the growth and differentiation of poorly differentiated prostate cancer, which may lead to the identification of potential therapeutic targets. Citation Format: Sonam Raj, Brian Capaldo, Joel Bowman, Margaret White, Xavier Moore, Berkley Gryder, Aian N. Alilin, David Takeda, Kathleen Kelly. Determining the role of PROX1 in prostate cancer neuroendocrine trans-differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 554.

The senescence-associated secretory phenotype induces neuroendocrine transdifferentiation.

The senescence-associated secretory phenotype induces neuroendocrine transdifferentiation.

Abstract 3783: Role of microRNA 28-3p in prostate cancer progression

Abstract Prostate cancer (PCa) is a leading cause of cancer incidence and mortality among men. Prostate cancer is driven by androgens acting via androgen receptor (AR). Therefore, this cancer is treated with androgen deprivation therapy (ADT) initially. However, the cancer rapidly evolves to be resistant to ADT and progresses to a more invasive stage referred to as ‘Castration Resistant prostate cancer’ (CRPC). CRPC is treated with second generation of AR pathway inhibitors such as Enzalutamide and Abiraterone. These treatments are effective initially. However, drug resistance develops in a significant fraction of patients and the cancer undergoes ‘neuroendocrine trans-differentiation (NED) to a lethal variant called ‘neuroendocrine prostate cancer’ (NEPC). As a result of NED, PCa cells express neuronal markers such as synaptophysin, chromogranin A and enolase. The molecular basis of NED is not yet fully defined. With a focus on identifying the role of microRNAs (miRNAs) in NEPC, we performed small RNA sequencing in CRPC tumors with and without NED. This sequencing identified miR-28-3p to be significantly downregulated in CRPC-NE samples as compared to CRPC-adenocarcinomas (CRPC-Adeno). In view of this, we examined the role of this miRNA in NEPC. Analyses of TCGA data of prostate adenocarcinomas suggest that miR 28-3p has an oncogenic role in primary prostate tumors. However, its expression declines in tumors with increasing Gleason score. We profiled the expression of miR-28-3p in patient-derived xenograft models and clinical CRPC-NE and CRPC-Adeno tissues by real time PCR. Our profiling showed a trend towards downregulation with increased NED. Basal expression of miR 28-3p in various prostate cancer cell lines showed an elevated level in androgen dependent cell lines whereas significantly less expression in NEPC cell lines, LASCPC-01 and NCI-H660. In this study, we generated PC3 cell line that stably expresses miR-28 for understanding its functional role in regulating PCa. Preliminary functional assays including cell viability assay, cell cycle studies and invasion and migration assay suggest a tumor suppressor role of miR-28-3p in advanced prostate cancer. In conclusion, our data shows that miR 28-3p potentially has a biphasic role in PCa, where it is oncogenic in early stages and acts as a tumor suppressor in late stage PCa. Citation Format: Amritha Sreekumar, Sharanjot Saini. Role of microRNA 28-3p in prostate cancer progression. [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 3783.

Abstract 3823: Therapeutic potential of plant-derived nanovesicles for neuroendocrine prostate cancer

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a highly lethal variant of prostate cancer (PCa) that often emerges after androgen receptor (AR)-targeted therapies such as enzalutamide and abiraterone. NEPC arises via trans-differentiation of prostate adenocarcinomas to neuronal lineage, a process referred to as ‘neuroendocrine trans-differentiation’ (NED), wherein prostate cancer cells show an altered expression of lineage markers such as decreased expression of AR and increased expression of neuronal markers. Currently, there is a lack of effective targeted therapies for NEPC. Current treatment options are limited to highly toxic platinum-based drugs. Hence, there is an urgent need to develop novel treatment strategies to treat NEPC. The focused objective of this study is to develop novel, non-toxic therapeutic intervention against NEPC. Towards this, we exploited the potential of plant-derived nanovesicles/extracellular vesicles as a novel therapeutic. Extracellular vesicles (EVs) are lipid-bilayer-delimited particles that are released from almost all types of cells, including plant cells, typically between 30-150 nm in size. Methods: EVs/nanovesicles were isolated from plant-derived sources by ultracentrifugation. Isolated nanovesicles were characterized by Nanoparticle Tracking Analyses and electron microscopy. After characterization, the effects of nanovesicles were tested in NEPC cell lines in vitro. Effects on cell viability and apoptosis were assessed by MTS assay and flow cytometry, respectively. To understand the mechanistic basis of EV induced alterations, next generation RNA sequencing was performed in control vs EV treated NCI-H660 cells on Illumina HiSeq platform. Significant targets were further validated by real time PCR and Western blot analyses. Results: Plant-derived nanovesicles reduced viability and induced apoptosis in NEPC cell lines. Genes involved in ECM-receptor interaction, focal adhesion, proteoglycans in cancer and small cell lung cancer genes were significantly impacted by these vesicles in NEPC cell line. Remarkably, nanovesicles led to significantly attenuated levels of key proneural transcription factors including ASCL1, BRN2 and SOX2. In addition, neuronal markers ENO2 and NCAM1 were downregulated suggesting that treatment with nanovesicles reverse the ‘cardinal’ genetic alterations that drive NEPC. Conclusion: Plant-derived nanovesicles can be employed as a novel treatment modality in neuroendocrine prostate cancer. Citation Format: Amritha Sreekumar, Matthew Simmons, Tae Jin Lee, Ashok Sharma, Sharanjot Saini. Therapeutic potential of plant-derived nanovesicles for neuroendocrine 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 3823.

Abstract 1456: An expression study of circadian clock-associated transcription factors and nuclear receptors in mouse prostate gland and tumor

Abstract The circadian clock lies in the suprachiasmatic nucleus of the hypothalamus and is regulated directly by the daily light/dark cycles, generating rhythmic expression of core circadian-controlled genes (CCCG) some belonging to the nuclear receptors (NR) superfamily. Recent advances show that the disruption of the circadian clock is identified as a risk factor for endocrine cancers, such as prostate cancer which is the most common hormone-related male cancer globally. Whether the prostate gland has its own circadian expression of CCCG and NR, and how the dysregulated circadian clock would influence the fate of the prostate tumors is not well studied. Thus, the present study aims to determine the circadian expression patterns of CCCG and NR in normal mouse prostate and further validate their disrupted circadian expression in a transgenic mouse adenocarcinoma prostate cancer model (TGMAP) with neuroendocrine phenotypes and suppressed androgen gene expression (AR), over the 12 hours light/dark cycle. We establish the rhythmic expression patterns of multiple CCCG and the full class of NR by quantitative polymerase-chain reaction analysis in normal mouse prostate (C57BL/6 strain) and advanced prostate tumors derived from TGMAP in a 12-hours light/dark cycle with 4-hours interval. Our results showed that 30 out of 49 NR and several CCCG follow rhythmic expression patterns, followed by 10 NR with unidentifiable expression patterns and 9 NR showing very low expression in the mouse prostate tissue. The circadian expression of these transcription factors including the AR gene, could be divided into 2 categories with single peak or double peaks at specific time points throughout the light/dark cycle, linking them to the regulating function of the circadian clock in the normal prostate gland. In the advanced prostate tumors derived from TGMAP model that exhibit androgen independence and neuroendocrine trans-differentiation phenotype, the rhythmic cycles of the NR and CCCG showed disruption, and several transcription factors were upregulated in overall circadian expression in comparison to the normal prostate tissue. In summary, this study establishes two categories of rhythmic expression patterns in normal mouse prostate tissue that link the circadian expression of these transcription factors in the peripheral clock to that of androgen signaling axis that is responsible for the maintenance of prostate gland while disruption of such circadian expression patterns was demonstrated in the TGMAP tumors of which the AR signaling was either lost or disrupted. The upregulated circadian expression patterns as revealed in TGMAP model also provide insight into the significance of the dysregulation of circadian rhythm as regulated by CCCG and NR in the advanced prostate cancer. (Study supported by a Direct Grant for Research 2021-2022 Project No. 2021.062, CUHK) Citation Format: Ria Chopra, Franky Leung Chan. An expression study of circadian clock-associated transcription factors and nuclear receptors in mouse prostate gland and tumor [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 1456.

Therapeutic potential of plant-derived nanovesicles for neuroendocrine prostate cancer.

186 Background: Neuroendocrine prostate cancer (NEPC) is a highly lethal variant of prostate cancer (PCa) that often emerges after androgen receptor (AR)-targeted therapies such as enzalutamide and abiraterone. NEPC arises via trans-differentiation of prostate adenocarcinomas to neuronal lineage, a process referred to as ‘neuroendocrine trans-differentiation’ (NED), wherein prostate cancer cells show an altered expression of lineage markers such as decreased expression of AR and increased expression of neuronal markers. Currently, there is a lack of effective targeted therapies for NEPC. Current treatment options are limited to highly toxic platinum-based drugs. Hence, there is an urgent need to develop novel treatment strategies to treat NEPC. The focused objective of this study is to develop novel, non-toxic therapeutic intervention against NEPC. Towards this, we exploited the potential of plant-derived nanovesicles/extracellular vesicles as a novel therapeutic. Extracellular vesicles (EVs) are lipid-bilayer-delimited particles that are released from almost all types of cells, including plant cells, typically between 30-150 nm in size. Methods: EVs/nanovesicles were isolated from plant-derived sources by ultracentrifugation. Isolated nanovesicles were characterized by Nanoparticle Tracking Analyses and electron microscopy. After characterization, the effects of nanovesicles were tested in NEPC cell lines in vitro. Effects on cell viability and apoptosis were assessed by MTS assay and flow cytometry, respectively. To understand the mechanistic basis of EV induced alterations, next generation RNA sequencing was performed in control vs EV treated NCI-H660 cells on Illumina HiSeq platform. Significant targets were further validated by real time PCR and Western blot analyses. Results: Plant-derived nanovesicles reduced viability and induced apoptosis in NEPC cell lines. Genes involved in ECM-receptor interaction, focal adhesion, proteoglycans in cancer and small cell lung cancer genes were significantly impacted by these vesicles in NEPC cell line. Remarkably, nanovesicles led to significantly attenuated levels of key proneural transcription factors including ASCL1, BRN2 and SOX2. In addition, neuronal markers ENO2 and NCAM1 were downregulated suggesting that treatment with nanovesicles reverse the ‘cardinal’ genetic alterations that drive NEPC. Conclusions: Plant-derived nanovesicles can be employed as a novel treatment modality in neuroendocrine prostate cancer.

Long-term taxane exposure and transdifferentiation of prostate cancer in vitro.

254 Background: Development of aggressive variants of metastatic castration-resistant prostate cancer (AVPC) is a major challenge in the course of therapy but the underlying mechanisms of aggressive transdifferentiation are not completely understood and appropriate tumor models are missing. Here, we investigated the consequences of long-term taxane exposure on hormone-independent, BRCA2-mutated, AR-V7-positive 22Rv1 cells. Methods: 22Rv1 cells were treated with stepwise increased taxane concentrations for 10 months. Individual clones were picked and further cultured in media containing either docetaxel (Doce) or cabazitaxel (Caba). Passage-matched cells were maintained in culture without treatment. Further characterization was carried out using proliferation, migration, metabolic, and colony formation assays as well as proteomics, RNAseq analyses and xenotransplantation in immunodeficient mice. Results: In total, three single cell 22Rv1-DR clones (50-100-fold resistance to Doce) and three 22Rv1-CR clones (80-150-fold resistance to Caba) were successfully established. All clones showed cross-resistance to either drug. Expectedly, treatment-induced overexpression of ABCB1 was detected and validated. Moreover, alteration of drug resistance related SLC7A5, SLC3A2, and SLC25A24 genes was observed. Additionally, an enrichment analyses identified, among others, neuroendocrine transdifferentiation (GO-term “Neuroendocrine tumors”, p=4.46e-5) to be stimulated in prostate 22Rv1 cells under long-term treatment with Doce or Caba. In line with this, the neuroendocrine features were validated in vitro as well as in xenotransplanted tumors in vivo with upregulation of synaptophysin, chromogranin and neuron specific enolase accompanied by downregulation of the androgen receptor (AR) and upregulation of AR spice variants. Additionally, neuritic morphology, shift to higher nuclear-plasma ratio, partial loss of adherent properties and growth slowdown, along with higher migratory activity were detected. Conclusions: Long-term taxane exposure of 22Rv1 cells resulted in the development of neuroendocrine traits in individual cell clones that have successfully been translated into stable cell lines. Thus, we provide a new cell line model for secondary therapy-induced neuroendocrine transdifferentiation. Further in-depth analysis to identify individual alterations in the course of therapy is currently ongoing.

Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It is characterized by the loss of androgen receptor (AR) signaling in neuroendocrine transdifferentiation, and finally, resistance to AR-targeted therapy. With the application of a new generation of potent AR inhibitors, the incidence of NEPC is gradually increasing. The molecular mechanism of neuroendocrine differentiation (NED) after androgen deprivation therapy (ADT) remains largely unclear. In this study, using NEPC-related genome sequencing database analyses, we screened RACGAP1, a common differentially expressed gene. We investigated RACGAP1 expression in clinical prostate cancer specimens by IHC. Regulated pathways were examined by Western blotting, qRT-PCR, luciferase reporter, chromatin immunoprecipitation, and immunoprecipitation assays. The corresponding function of RACGAP1 in prostate cancer was analyzed by CCK-8 and Transwell assays. The changes of neuroendocrine markers and AR expression in C4-2-R and C4-2B-R cells were detected in vitro. We confirmed that RACGAP1 contributed to NE transdifferentiation of prostate cancer. Patients with high tumor RACGAP1 expression had shorter relapse-free survival time. The expression of RACGAP1 was induced by E2F1. RACGAP1 promoted neuroendocrine transdifferentiation of prostate cancer by stabilizing EZH2 expression in the ubiquitin-proteasome pathway. Moreover, overexpression of RACGAP1 promoted enzalutamide resistance of castration-resistant prostate cancer (CRPC) cells. Our results showed that the upregulation of RACGAP1 by E2F1 increased EZH2 expression, which drove NEPC progression. This study explored the molecular mechanism of NED and may provide novel methods and ideas for targeted therapy of NEPC.

Attractor of a nonlinear hybrid reaction–diffusion model of neuroendocrine transdifferentiation of human prostate cancer cells with time-lags

<abstract><p>Prostate cancer is a serious disease that endangers men's health. The genetic mechanism and treatment of prostate cancer have attracted the attention of scientists. In this paper, we focus on the nonlinear mixed reaction diffusion dynamics model of neuroendocrine transdifferentiation of prostate cancer cells with time delays, and reveal the evolutionary mechanism of cancer cells mathematically. By applying operator semigroup theory and the comparison principle of parabolic equation, we study the global existence, uniqueness and boundedness of the positive solution for the model. Additionally, the global invariant set and compact attractor of the positive solution are obtained by Kuratowski's measure of noncompactness. Finally, we use the Pdepe toolbox of MATLAB to carry out numerical calculations and simulations on an example to check the correctness and effectiveness of our main results. Our results show that the delay has no effect on the existence, uniqueness, boundedness and invariant set of the solution, but will affect the attractor.</p></abstract>

Abstract B029: LSD1-HDAC6 dual inhibitor JBI-802 is an epigenetic modulating agent with a novel mechanism of action that target MYC amplification in multiple neuroendocrine tumor types

Abstract MYC is considered a master regulator of human cancers by modulating the transcription of numerous cancer-related genes. MYC amplification is reported in about 15% of all human cancers and is generally associated with poor prognosis and resistance to treatments. Focal amplification of MYC together with mutation in RB1 and p53 is an important event in the metastatic process of neuroendocrine tumor development. While limited options are available for direct targeting, transcriptional modulation via epigenetic modulating agent could be an attractive and viable option to target neuroendocrine cancers. JBI-802 inhibits the transcriptional regulator coREST via its component LSD1/HDAC6 therefore blocking neuroendocrine transdifferentiation and inducing cell death resulting in activity against neuroendocrine tumors. At the same time, this molecule has shown a good safety profile in toxicological studies. We have now identified a novel aspect of JBI-802 mechanism of action, the ability to induce downregulation of MYC RNA and degradation of MYC protein both in vitro and in animal models of two neuroendocrine tumors, small cell lung cancer and neuroendocrine prostate cancer JBI-802 showed significant anti-proliferative activity (0.2 to 1 µM) against several cancer cell lines as shown by Alamar blue or CTG assays. Sensitive ones included small cell lung cancer (SCLC), gastric cancer, breast cancer cell lines with RB1 mutation. Interestingly, dual inhibitor JBI-802 was also active in cell lines with MYC over-expression, while LSD1 selective inhibitors have been reported to be inactive in these cell lines. JBI-802 also inhibited MYC at RNA as well as protein level in hematological and solid tumors as assessed by RT-qPCR at RNA level and by Western blotting at protein level, while single agent LSD1 and HDAC6 inhibitors did not show significant modulation. JBI-802 also showed strong tumor growth inhibition of these tumors in mouse xenograft models. MYC levels showed a dose dependent inhibition in these tumors when tested at the end of the study. Only dual inhibition of both LSD1/HDAC6 with JBI-802 as opposed to single target inhibition is able to effectively downregulate MYC level and achieve efficacy in MYC amplified models in vivo and in vitro. This novel mechanism increases the potential population of neuroendocrine patients that could be sensitive to this compound, going beyond the proof of principle already established preclinically and clinically by single target LSD1 inhibitors. Therefore, by targeting 2 major pathways in neuroendocrine tumor development, JBI-802 novel mechanism of action is uniquely suited for the treatment of high unmet neuroendocrine tumors like small cell lung cancer, neuroendocrine prostate cancer and advanced, MYC amplified tumors. Based on this rationale, JBI-802 is being tested in phase 1/2 clinical trial focus on this type of tumors. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Chandru Gajendran, Naveen Sadhu, Mohd Zainuddin, Ramachandraiah Gosu, Luca Rastelli. LSD1-HDAC6 dual inhibitor JBI-802 is an epigenetic modulating agent with a novel mechanism of action that target MYC amplification in multiple neuroendocrine tumor types. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B029.

Neuroendocrine neoplasms of the lung and gastrointestinal system: convergent biology and a path to better therapies.

Neuroendocrine neoplasms (NENs) can develop in almost any organ and span a spectrum from well-differentiated and indolent neuroendocrine tumours(NETs) to poorly differentiated and highly aggressive neuroendocrine carcinomas(NECs), including small-cell lung cancer(SCLC). These neoplasms are thought to primarily derive from neuroendocrine precursor cells located throughout the body and can also arise through neuroendocrine transdifferentiation of organ-specific epithelial cell types. Hence, NENs constitute a group of tumour types that share key genomic and phenotypic characteristics irrespective of their site of origin, albeit with some organ-specific differences. The establishment of representative preclinical models for several of these disease entities together with analyses of human tumour specimens has provided important insights into crucial aspects of their biology with therapeutic implications. In this Review, we provide a comprehensive overview of the current understanding of NENs of the gastrointestinal system and lung from clinical and biological perspectives. Research on NENs has typically been siloed by the tumour site of origin, and a cross-cutting view might enable advances in one area to accelerate research in others. Therefore, we aim to emphasize that a better understanding of the commonalities and differences of NENs arising in different organs might more effectively inform clinical research to define therapeutic targets and ultimately optimize patient care.

Identification of the Regulatory Targets of miR-3687 and miR-4417 in Prostate Cancer Cells Using a Proteomics Approach.

MicroRNAs (miRNA) are ubiquitous non-coding RNAs that have a prominent role in cellular regulation. The expression of many miRNAs is often found deregulated in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Although their expression can be associated with PCa and CRPC, their functions and regulatory activity in cancer development are poorly understood. In this study, we used different proteomics tools to analyze the activity of hsa-miR-3687-3p (miR-3687) and hsa-miR-4417-3p (miR-4417), two miRNAs upregulated in CRPC. PCa and CRPC cell lines were transfected with miR-3687 or miR-4417 to overexpress the miRNAs. Cell lysates were analyzed using 2D gel electrophoresis and proteins were subsequently identified using mass spectrometry (Maldi-MS/MS). A whole cell lysate, without 2D-gel separation, was analyzed by ESI-MS/MS. The expression of deregulated proteins found across both methods was further investigated using Western blotting. Gene ontology and cellular process network analysis determined that miR-3687 and miR-4417 are involved in diverse regulatory mechanisms that support the CRPC phenotype, including metabolism and inflammation. Moreover, both miRNAs are associated with extracellular vesicles, which point toward a secretory mechanism. The tumor protein D52 isoform 1 (TD52-IF1), which regulates neuroendocrine trans-differentiation, was found to be substantially deregulated in androgen-insensitive cells by both miR-3687 and miR-4417. These findings show that these miRNAs potentially support the CRPC by truncating the TD52-IF1 expression after the onset of androgen resistance.

Neuropilin-2 promotes lineage plasticity and progression to neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC), a lethal subset of prostate cancer, is characterized by loss of AR signaling and resulting resistance to AR-targeted therapy during neuroendocrine transdifferentiation, for which the molecular mechanisms remain unclear. Here, we report that neuropilin 2 (NRP2) is upregulated in both de novo and therapy-induced NEPC, which induces neuroendocrine markers, neuroendocrine cell morphology, and NEPC cell aggressive behavior. NRP2 silencing restricted NEPC tumor xenograft growth. Mechanistically, NRP2 engages in reciprocal crosstalk with AR, where NRP2 is transcriptionally inhibited by AR, and in turn suppresses AR signaling by downregulating the AR transcriptional program and confers resistance to enzalutamide. Moreover, NRP2 physically interacts with VEGFR2 through the intracellular SEA domain to activate STAT3 phosphorylation and subsequently SOX2, thus driving NEPC differentiation and growth. Collectively, these results characterize NRP2 as a driver of NEPC and suggest NRP2 as a potential therapeutic target in NEPC.

Analysis of neuroendocrine clones in NSCLCs using an immuno-guided laser-capture microdissection-based approach

SummaryClonal evolution and lineage plasticity are key contributors to tumor heterogeneity and response to treatment in cancer. However, capturing signal transduction events in coexisting clones remains challenging from a technical perspective. In this study, we developed and tested a signal-transduction-based workflow to isolate and profile coexisting clones within a complex cellular system like non-small cell lung cancers (NSCLCs). Cooccurring clones were isolated under immunohistochemical guidance using laser-capture microdissection, and cell signaling activation portraits were measured using the reverse-phase protein microarray. To increase the translational potential of this work and capture druggable vulnerabilities within different clones, we measured expression/activation of a panel of key drug targets and downstream substrates of FDA-approved or investigational agents. We isolated intermixed clones, including poorly represented ones (<5% of cells), within the tumor microecology and identified molecular characteristics uniquely attributable to cancer cells that undergo lineage plasticity and neuroendocrine transdifferentiation in NSCLCs.

Abstract 658: AKT pathway as a therapeutic target to constrain lineage plasticity leading to histological transdifferentiation

Abstract Lineage plasticity contributes to therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon drives neuroendocrine (NE) and squamous cell (LUSC) histologic transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with up to 14% and 9% incidences in EGFR-mutant tumors relapsed on EGFR inhibitors, respectively. Notably, survival of patients with NE- or LUSC-transdifferentiated tumors is lower than that of either LUAD or de novo LUSC patients. To date, little is known about the molecular effectors enhancing lineage plasticity and driving histological transdifferentiation due to the paucity of well annotated pre- and post-transdifferentiation clinical samples amenable for molecular analyses. Currently no specific therapies for LUSC or NE transdifferentiation prevention are available for patients at high risk of transformation. We performed multi-omic profiling of transdifferentiating clinical samples, as well as control never-transformed LUAD and de novo LUSC and small cell carcinomas, including comprehensive and integrative genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and protein (antibody arrays) characterization. Findings were validated in preclinical models including cell lines as well as LUSC- and NE-transdifferentiation patient-derived xenograft models. Our data suggest that histological transdifferentiation is driven by epigenetic -rather than mutational- events, and indicate that transdifferentiated tumors retain molecular features of their previous LUAD state. Integrative analysis revealed biological pathways dysregulated specifically for distinct histological outcomes, including downregulation of RTK signaling and Notch-related genes in NE-transformed tumors, and upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses revealed commonly dysregulated pathways for transdifferentiated tumors, including marked downregulation of a variety of immune-related pathways and upregulation of genes involved in AKT signaling and in the PRC2 epigenetic remodeling complex. Concurrent activation of AKT and MYC overexpression induced a squamous phenotype in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacological targeting of AKT in combination with osimertinib delayed both squamous and NE transformation in EGFR-mutant patient-derived xenograft transdifferentiation models. These results identify common and histology-specific drivers and dysregulated pathways in NE and LUSC transdifferentiation, and nominate AKT as a therapeutic target to constrain lineage plasticity and prevent the acquisition of resistance to EGFR-targeted therapies through histological transdifferentiation. Citation Format: Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yingqian A. Zhan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S. Shah, Umesh K. Bhanot, Jacklynn Egger, Juan Qiu, Elisa de Stanchina, Natasha Rekhtman, Brian Houck-Loomis, Richard P. Koche, Helena A. Yu, Triparna Sen, Charles M. Rudin. AKT pathway as a therapeutic target to constrain lineage plasticity leading to histological transdifferentiation [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 658.

NF-κB-dependent secretome of senescent cells can trigger neuroendocrine transdifferentiation of breast cancer cells.

Cellular senescence is characterized by a stable proliferation arrest in response to stresses and the acquisition of a senescence‐associated secretory phenotype, called SASP, composed of numerous factors including pro‐inflammatory molecules, proteases, and growth factors. The SASP affects the environment of senescent cells, especially during aging, by inducing and modulating various phenotypes such as paracrine senescence, immune cell activity, and extracellular matrix deposition and organization, which critically impact various pathophysiological situations, including fibrosis and cancer. Here, we uncover a novel paracrine effect of the SASP: the neuroendocrine transdifferentiation (NED) of some epithelial cancer cells, evidenced both in the breast and prostate. Mechanistically, this effect is mediated by NF‐κB‐dependent SASP factors, and leads to an increase in intracellular Ca2+ levels. Consistently, buffering Ca2+ by overexpressing the CALB1 buffering protein partly reverts SASP‐induced NED, suggesting that the SASP promotes NED through a SASP‐induced Ca2+ signaling. Human breast cancer dataset analyses support that NED occurs mainly in p53 WT tumors and in older patients, in line with a role of senescent cells and its secretome, as they are increasing during aging. In conclusion, our work, uncovering SASP‐induced NED in some cancer cells, paves the way for future studies aiming at better understanding the functional link between senescent cell accumulation during aging, NED and clinical patient outcome.

AKT inhibition as a therapeutic strategy to constrain histological transdifferentiation in EGFR-mutant lung adenocarcinoma.

e21166 Background: In lung adenocarcinomas (LUADs), lineage plasticity drives neuroendocrine (NE) and squamous cell (LUSC) transdifferentiation in the context of acquired resistance to targeted inhibition of driver mutations, with up to 14% and 9% incidences in EGFR-mutant tumors relapsed on EGFR inhibitors, respectively. Notably, survival of patients with NE- or LUSC-transdifferentiated tumors is remarkably lower than those of LUAD or de novo LUSC patients. The paucity of transforming clinical specimens amenable for molecular analyses has hindered the identification of histological transformation drivers, and to date no specific therapies aimed to prevent or delay transdifferentiation-led therapy relapse are available for patients at high risk of transformation. Methods: We performed multi-omic profiling of LUAD-to-LUSC and LUAD-to-NE transdifferentiating clinical samples, including comprehensive and integrative genomic (whole exome sequencing), epigenomic (bisulfite sequencing), transcriptomic (RNAseq) and protein (antibody arrays) characterization. Clinical findings were validated in preclinical models including cell lines as well as LUSC- and NE-transdifferentiation patient-derived xenograft models. Results: Our data supports that histological transdifferentiation from LUAD to LUSC or NE tumors is driven by epigenetic remodeling rather than by mutational events, and indicate that transdifferentiated tumors retain epigenomic features of their previous LUAD state. Integrative epigenomic, transcriptomic and protein analysis revealed divergent biological pathways dysregulated for each histological outcome, such as downregulation of RTK signaling and Notch-related genes in NE-transformed tumors, and upregulation of genes involved in Hedgehog and Notch signaling and MYC targets in LUSC-transdifferentiated tumors. Most interestingly, these analyses identified commonly dysregulated pathways in both NE- and LUSC-transdifferentiating tumors, including remarkable downregulation of a variety of immune-related pathways and upregulation of genes involved in AKT signaling and in the PRC2 epigenetic remodeling complex. Concurrent activation of AKT and MYC overexpression induced a squamous phenotype in EGFR-mutant LUAD preclinical models, further accentuated by EGFR inhibition. Pharmacological targeting of AKT in combination with osimertinib delayed both squamous and NE transformation in different EGFR-mutant patient-derived xenograft transdifferentiation models. Conclusions: These results identify common and divergent dysregulated pathways in NE and LUSC transdifferentiation, and nominate AKT as a therapeutic target to prevent the acquisition of resistance to EGFR-targeted therapies through histological transdifferentiation.

Monoamine oxidase A drives neuroendocrine differentiation in prostate cancer

Neuroendocrine transdifferentiation (NED) of prostate cancer (PCa) is the main cause of failure of androgen receptor inhibitor treatment. However, the molecular mechanisms underlying the development of NEPC, especially treatment-induced NEPC, remain unclear. Emerging evidence indicates that elevated monoamine oxidase A (MAOA) contribute to the proliferation, cell stemness, and bone metastasis in PCa. Here, we generated an enzalutamide-induced NED cell model to assess the role of MAOA during NED. Overall, MAOA expression was significantly increased upon Enz long-term exposure and was required for neuroendocrine marker expression. In particular, Enz was found to induce NED via the MAOA/mTOR/HIF-1α signaling axis. Further analyses revealed that the MAOA inhibitor clorgyline(CLG) may bring multiple benefits to CRPC patients, including better therapeutic effect and delays NED. These findings suggest that MAOA may be an important target for the development of anti-NED therapies, thereby providing a novel strategy for the combined application of CLG and AR inhibitors in the clinic.