Neuroendocrine Tumor Cell Lines Research Articles

Opposing effects of cannabidiol (CBD) on pheochromocytoma/paraganglioma and neuroendocrine tumor cell lines and individual patient-derived primary cultures

Opposing effects of cannabidiol (CBD) on pheochromocytoma/paraganglioma and neuroendocrine tumor cell lines and individual patient-derived primary cultures

Disulfiram mediated anti-tumour effect in pituitary neuroendocrine tumours by inducing cuproptosis

ContextMedical treatment plays a critical role in pituitary neuroendocrine tumour (PitNET) treatment. Dopamine agonists and somatostatin receptor agonists are the only known drugs for effectively treating PitNET. Thus, the identification of potential therapeutic targets and drugs is urgently needed. ObjectiveTo discover potential drugs that can suppress PitNET growth and to further investigate the underlying mechanism involved. MethodsHigh-throughput drug screening of primary cultures of 17 patient-derived PitNETs was performed to identify potential therapeutic compounds. Cell viability assays, Western blot analysis and flow cytometry were used to investigate pituitary neuroendocrine tumour cell lines and patient-derived PitNET cultures in vitro. In vivo drug efficacy was examined in a mouse xenograft model. ResultsSeventeen primary PitNET samples were collected for high-throughput drug screening, and a class of copper ionophores that can effectively inhibit cell growth, such as zinc pyrithione, elesclomol, and disulfiram (DSF), was identified. Subsequent experiments initially validated the dose-dependent cell growth-suppressing effect of these copper ionophores on AtT20, GH3, and MMQ cells and several primary PitNET cell lines. Moreover, we confirmed that the cytotoxic effect of DSF depends on the presence of copper. Additionally, we determined that cell death occurs via cuproptosis, with events such as Fe-S cluster protein loss, dihydrolipoyl transacetylase oligomerization and heat shock protein 70 upregulation. Finally, we verified the cytotoxic effects of DSF in vivo. ConclusionThe present study revealed copper ionophores as a potential class of drugs for PitNET treatment. DSF induced PitNET cell death via cuproptosis and might be a promising option for PitNET therapy.

Abstract 5953: DUSP6 is a novel target in neuroendocrine tumors

Abstract Background: Neuroendocrine tumors (NETs) are rare malignancies derived from enterochromaffin cells of the diffuse neuroendocrine system and most frequently observed in the gastrointestinal tract and bronchopulmonary system. Surgery is the major treatment for localized NETs. In high-grade NETs, treatment opportunities include chemotherapeutic drugs such as temozolomide (TEM), and capecitabine and small-molecule targeted therapies such as everolimus or sunitinib. Despite promising initial responses, most patients develop therapy resistance by unknown mechanisms. Therefore, there is a pressing need to apprehend the mechanisms of resistance to improve the therapeutic efficacy of the current treatments. Phosphatases are emerging and novel targets in human malignancies and their modulation yields significant anti-tumor activities. Dual specificity phosphatase 6 (DUSP6) is a MAPK phosphatase and mainly binds to and dephosphorylates extracellular signal-regulated kinase (ERK)1/2. DUSP6 expression is increased in several malignancies, correlating with distant metastases and a worse clinical outcome. Moreover, DUSP6 mediates resistance to certain chemotherapeutics and small molecular tyrosine kinase inhibitors and its blockade increases therapeutic sensitivity. The current study aimed to investigate the contribution of DUSP6 to therapy resistance in the NET models. Material and Methods: Using Western blot analysis, the expression of DUSP6 was measured in 8 clinical samples of small bowel neuroendocrine tumors, the NET cell lines BON-1, H69, IMR-32, QGP and H524 and two PDX models, 1452 (rectal neuroendocrine) and 913 (pancreatic neuroendocrine). The effects of DUSP6 depletion on cell viability were explored using a pharmacological inhibitor, BCI, and its siRNA-mediated knockdown. Moreover, BCI-mediated apoptotic cell death was measured by an annexin V/PI staining assay. Results: DUSP6 expression was positive in 5 out of 8 clinical samples, 4 NET cell lines and both the PDX models. BCI treatment induced an apoptotic response in the NET models and synergistically increased sensitivity to TMZ and cisplatin in 1452 cells., RNAi-mediated depletion of DUSP6 also increased sensitivity to sunitinib and triggered TMZ-induced apoptotic cell death in 1452 cells, which was associated with changes in (O6-Methylguanine-DNA-methyltransferase) MGMT expression. These findings suggest that DUSP6 is an important therapeutic target in neuroendocrine tumors and its blockade is a promising therapeutic approach to enhance the therapeutic index of current treatments. Citation Format: Majid Momeny, Vahid Khalaj, Solmaz AghaAmiri, Po Hien Ear, Servando Hernandez Vargas, Sukhen Ghosh, Ali Azhdarinia. DUSP6 is a novel target in neuroendocrine tumors [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 5953.

Abstract 5957: Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer

Abstract Introduction Transcription regulators, such as cyclin-dependent kinase 9 (CDK9), have been implicated in the super-enhancer driven transcriptional control of oncogenes in multiple cancers including pancreatic cancer. CDK9 has been shown to be employed by cancer cells for the constant production of short-lived oncoproteins such as c-Myc to maintain their survival. Due to its critical role in regulating transcription in cancer cells, CDK9 has emerged as a potential therapeutic target. Here, we report the antitumor activity of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, in preclinical models for pancreatic cancer. Experimental Procedures Fragment-based MolecuLern driven AI workflow methods were used to design a series of ATP-competitive CDK9 specific kinase inhibitors. BLX-3030 was selected as one of the most potent leads with an IC50 of 1.1 nM in a cell-free CDK9 kinase assay. A Sulforhodamine B (SRB) assay was used to evaluate the cell growth inhibitory activity of BLX-3030 in a panel of 10 pancreatic ductal adenocarcinoma (PDAC) and 2 pancreatic neuroendocrine tumor (pNET) cell lines. Western blotting was used to determine the effects of BLX-3030 treatment on c-Myc expression in cancer cells. The combination effects between BLX-3030 and standard of care regimen, gemcitabine (GEM) and nab-paclitaxel (NP), was assessed using the SRB assay. Finally, the in vivo antitumor activity of BLX-3030 alone or in combination with GEM and NP was evaluated in a mouse cell line xenograft model for pancreatic cancer. Results BLX-3030 exhibited potent cell growth inhibitory activity in the panel of pancreatic cancer cell lines with IC50 values ranging from 133.5 nM to 508.1 nM. The potency of BLX-3030 in the cell lines appears to correlate with the expression level of c-Myc in the cell lines. BLX-3030 showed similar activities in PDAC and pNET cell lines with mean IC50 values of 244.5 nM and 194.6 nM, respectively. However, treatment with BLX-3030 for 48 hours did not seem to significantly reduce c-Myc expression in PSN-1 PDAC cells. When combined with GEM and NP, BLX-3030 demonstrated mostly additive effects in PSN-1 cells. Preliminary data from the in vivo study show that BLX-3030 improves the antitumor activity of GEM and NP. Conclusion In summary, BLX-3030 potently inhibited the growth of both PDAC and pNET cells in vitro. The growth inhibitory activity of BLX-3030 in PDAC cell lines correlates with their c-Myc expression levels. BLX-3030 showed an additive effect when combined with standard of care drugs (GEM and NP) in PDAC cell lines. Overall, the novel CDK9 inhibitor, BLX-3030, demonstrated potent activity in pancreatic cancer cell line models and presents a promising preclinical lead for pancreatic cancer. (This work was supported in part by Biolexis Therapeutics and the Seena Magowitz Foundation). Citation Format: Yesenia Barrera-Millan, Zhaoliang Li, Hariprasad Vankayalapati, David J. Bearss, Daniel D. Von Hoff, Haiyong Han. Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic 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 5957.

Abstract 3585: DNA-PK inhibition to enhance radiation sensitivity in metastatic pancreatic neuroendocrine cancer

Abstract Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) are neoplasms originating from the gastrointestinal track and pancreas. Unfortunately, most patients at the time of diagnosis have extensive metastatic disease and are not candidates for curative surgical resection of metastatic tumors. In such cases, targeted radiation therapy (RT) can slow the progression of metastatic disease, but eventually tumors develop resistance to radiation therapy. In this study, we examined whether DNA-PK inhibition could sensitize pancreatic NETs to radiation therapy and enhance the radiation therapy effect in a preclinical model of pNET metastasis. Methods. We used 96-well clonogenic assays to evaluate the efficacy of the DNA-PK inhibitor peposertib in combination with RT in vitro in QGP-1 and BON pancreatic NET cell lines. Double-strand break-induced DNA repair was visualized with confocal laser scanning microscopy using γ-H2AX immunofluorescence after peposertib treatment alone or in combination with RT. Western blot analysis was used to confirm inhibition of DNA-PKcs (pDNA-PKcs S2056) combination RT. The efficacy of peposertib in combination with RT was evaluated in QGP-1 and BON human xenograft models in athymic nude mice and a BON preclinical lung metastasis model. Results. Clonogenic assays demonstrated absence of cytotoxicity from peposertib alone at doses below 1000 nM and a strong radiosensitizing effect at 200 nM in both QGP-1 and BON cell lines. Pretreatment with peposertib or treatment within 2 h after RT suppressed colony formation; treatment at later timepoints did not enhance radiotherapy. Immunofluorescence analysis revealed high numbers of γH2AX foci with peposertib therapy alone at 96 h or when given 2 h after RT. Western blot analysis confirmed inhibition of DNA-PK Ser2056 after RT in combination with peposertib. DNA-PK inhibition in combination with RT led to remarkable repression of pNET growth both in subcutaneous xenografts (>90%) and in the preclinical lung metastasis model (>90%). Conclusions. Our results showed several advantages of targeting DNA-PK to enhance radiation sensitivity in high-grade pNETs: (1) DNA-PK inhibition alone disrupts DNA damage response in pNETs as demonstrated by strong H2AX phosphorylation. (2) DNA-PK inhibition is most effective at enhancing radiotherapy either right before or after radiation therapy administration. This demonstrates that extended DNA-PK inhibition is not necessary to achieve marked responses during pNET radiotherapy. (3) DNA-PK inhibition 30 min before radiation therapy is sufficient to greatly reduce pNET tumor growth or metastasis progression in pNET preclinical models. In summary, selective DNA-PK inhibition provides a potent therapeutic strategy for disruption of non-homologous end joining DNA double-strand break repair and may offer a novel therapeutic approach in advanced NET radiotherapy. Citation Format: Piotr G. Rychahou, Aman Chauhan, Zeta Chow, Tadahide Izumi, Quan Chen, Eun Y. Lee, Dana Napier, Lowell Brian Anthony, Michael J. Cavnar, Courtney M. Townsend, B. Mark Evers. DNA-PK inhibition to enhance radiation sensitivity in metastatic pancreatic neuroendocrine 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 3585.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

Abstract 4039: RET overexpression is frequent in lung neuroendocrine tumors (NET) and associates with response to RET tyrosine kinase inhibitors (RET TKIs) in NET cell lines

Abstract Background: RET (rearranged during transfection) is a proto-oncogene encoding for a tyrosine kinase membrane receptor that promotes protein synthesis and cell proliferation. A small percentage of human tumors harbor RET fusions or mutations as oncogenic drivers and RET-TKIs have been developed. Two of them, selpercatinib and pralsetinib, have recently been approved. However, their effect on tumors overexpressing wild-type RET (wt-RET) has not been investigated. A large-scale mRNA analysis performed by our group revealed that ~2% of lung tumors present high expression of wt-RET in absence of fusions. Interestingly, ~60% of high wt-RET tumors presented neuroendocrine characteristics. Methods: Twenty large cell neuroendocrine (LCNEC) and 19 small cell (SCLC) lung carcinomas from two additional institutions were analyzed by nCounter® to validate the previous results. For in vitro experiments, a panel of cell lines derived from lung NETs (DMS-53, NCI-H82, NCI-H727) was used, together with a lung adenocarcinoma cell line (LC2/ad, a positive control harboring the CDCC6-RET translocation). RET fusions and mRNA expression were analyzed by nCounter®, RET protein expression by Western blotting and immunohistochemistry (IHC) and RET mutations by next generation sequencing. The inhibitory effects of selpercatinib and pralsetinib were analyzed in vitro by MTT in 2D and spheroid/3D cultures. Finally, in vivo experiments implanting tumor cells into athymic nude mice are ongoing. Results: Among the 20 LCNECs and 19 SCLCs analyzed, 3 (15%) and 3 (15%) presented very high levels of wt-RET mRNA by nCounter, respectively. Fusions were absent in all cases. The NCI-H82 and NCI-H727 NET cell lines showed very low RET mRNA and protein expression by nCounter, Western blotting and IHC. In contrast, DMS-53 cells presented levels of RET mRNA and protein higher than the observed in the RET-fusion positive LC2/ad. In 2D models, the three NET cell lines tested showed moderate resistance to pralsetinib and selpercatinib, with IC50s of 1.5 - 9.7 µM for pralsetinib and 4.6 - 6.1 µM for selpercatinib; while the RET-fusion positive LC2/ad cells showed an IC50 in the nM range, as expected. In 3D cultures, DMS-53 was found to be sensitive to RET TKIs, with IC50s of and 0.3 µM for praseltinib and 2.5 µM for selpercatinib. In contrast, spheroids of the two RET low cell lines showed complete resistance to both drugs (IC50>10 µM). Experiments in animal models are currently ongoing, final results will be presented at the meeting. Conclusions: Overexpression of RET is frequent in lung tumors with neuroendocrine features (LCNEC, SCLC). Selpercatinib and Pralsetinib show antitumor activity in lung NETs cells expressing high levels of wt-RET. Citation Format: Jaume Roca-Arias, Jordi Bertran-Alamillo, Ruth Román, Cristina Aguado, Laura López, Leticia Ferro-Leal, Beatriz Garcia-Pelaez, Rodrigo de Oliveira Cavagna, Silvia Teixeira, Rafael Rosell, Rui Manuel Reis, Miguel A. Molina-Vila, Ivana Sullivan. RET overexpression is frequent in lung neuroendocrine tumors (NET) and associates with response to RET tyrosine kinase inhibitors (RET TKIs) in NET cell lines. [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 4039.

Targeting of Glucose Transport and the NAD Pathway in Neuroendocrine Tumor (NET) Cells Reveals New Treatment Options

(1) Background: the potency of drugs that interfere with glucose metabolism, i.e., glucose transporters (GLUT) and nicotinamide phosphoribosyltransferase (NAMPT) was analyzed in neuroendocrine tumor (NET, BON-1, and QPG-1 cells) and small cell lung cancer (SCLC, GLC-2, and GLC-36 cells) tumor cell lines. (2) Methods: the proliferation and survival rate of tumor cells was significantly affected by the GLUT-inhibitors fasentin and WZB1127, as well as by the NAMPT inhibitors GMX1778 and STF-31. (3) Results: none of the NET cell lines that were treated with NAMPT inhibitors could be rescued with nicotinic acid (usage of the Preiss-Handler salvage pathway), although NAPRT expression could be detected in two NET cell lines. We finally analyzed the specificity of GMX1778 and STF-31 in NET cells in glucose uptake experiments. As previously shown for STF-31 in a panel NET-excluding tumor cell lines, both drugs specifically inhibited glucose uptake at higher (50 μM), but not at lower (5 μM) concentrations. (4) Conclusions: our data suggest that GLUT and especially NAMPT inhibitors are potential candidates for the treatment of NET tumors.

Wnt/β-catenin modulating drugs regulate somatostatin receptor expression and internalization of radiolabelled octreotide in neuroendocrine tumor cells.

Differentiated neuroendocrine tumors (NETs) express somatostatin receptors (SSTRs), targets for therapy with either unlabeled or radioactively labeled somatostatin analogs (SSA). Associated with worse prognosis, dedifferentiated NET loose SSTR expression, which may be linked to deregulation of Wnt/β-catenin signaling on an intracellular level. The aim of the present study was to investigate the effect of Wnt/β-catenin signaling pathway alterations on SSTR expression and its function in NET. The NET cell lines BON-1 and QGP-1 were incubated with the Wnt-inhibitors 5-aza-2'-deoxycytidine (5-aza-CdR), Quercetin, or Niclosamide, or the Wnt activator lithium chloride (LiCl). Expression of SSTR1, SSTR2, and SSTR5 was determined by quantitative RT-PCR (qRT-PCR), immunocytomicroscopy and western blot. Changes in the Wnt pathway were analyzed by qRT-PCR of selected target genes and the TaqMan Array Human WNT Pathway. Receptor-associated function was determined by measuring the cellular uptake of [125I-Tyr3] octreotide. The mRNAs of SSTRs 1-5 were expressed in both cell lines. Wnt inhibitors caused downregulation of Wnt target genes, while 5-aza-CdR had the highest inhibitory effect. LiCl lead to an upregulation of Wnt genes, which was more marked in QGP-1 cells. SSTR expression increased in both cell lines upon Wnt inhibition. All three Wnt inhibitors lead to a marked increase in the specific uptake of [125I-Tyr3]octreotide, with 5-aza-CdR showing the greatest effect (increase by more than 50% in BON-1 cells), while a decreased uptake of [125I-Tyr3]octreotide was seen upon activation of Wnt signaling by LiCl. We demonstrate here that Wnt signaling orchestrates SSTR expression and function in a preclinical NET model. Wnt inhibition increases [125I-Tyr3]octreotide uptake offering an opportunity to enhance the efficacy of SSTR-targeted theranostic approaches.

Cancer-Associated Fibroblasts Induce Proliferation and Therapeutic Resistance to Everolimus in Neuroendocrine Tumors through STAT3 Activation

Introduction: Cancer-associated fibroblasts (CAF) have been identified as relevant contributors to cancer progression and drug resistance in many tumors. Although neuroendocrine tumors (NET) are often associated with a strong stromal reaction, no study has addressed whether CAF are involved in progression and therapeutic resistance in NET. The aim of this study was to characterize the role of CAF in NET. Methods: We established primary CAF cultures derived from NET liver metastases to study the effect on NET cell lines NT-3 and BON. Immunohistochemistry was performed on tissue sections of primary and metastatic NET tissue. Results: Immunohistochemistry identified CAF dispersed in between tumor cells and within fibrotic bands separating tumor cell clusters in NET. Stimulating NET cells with CAF decreased expression of SSTR2 and chromogranin A and induced expression of CXCR4. CAF induced a 2.3-fold increase in proliferation and completely reversed the response to everolimus in NT-3 cells. We identified STAT3 as the main signaling pathway induced by CAF. STAT3 targeting by small interfering RNA knockdown and inhibitors prevented CAF-induced proliferation and restored everolimus responsiveness. STAT3 activation in NET tissue was associated with decreased chromogranin A expression, increased Ki-67 index, and decreased 5-year overall and progression-free survival. CAF directly influence proliferation and therapeutic response in NET cells. Conclusion: Identifying STAT3 as the contributing pathway of this so far neglected tumor-stroma interaction has the potential to become a new therapeutic target to halt tumor growth and to restore therapeutic responsiveness in NET.

Novel preclinical gastroenteropancreatic neuroendocrine neoplasia models demonstrate the feasibility of mutation-based targeted therapy

PurposeGastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) form a rare and remarkably heterogeneous group of tumors. Therefore, establishing personalized therapies is eminently challenging. To achieve progress in preclinical drug development, there is an urgent need for relevant tumor models.MethodsWe successfully established three gastroenteropancreatic neuroendocrine tumor (GEP-NET) cell lines (NT-18P, NT-18LM, NT-36) and two gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) cell lines (NT-32 and NT-38). We performed a comprehensive characterization of morphology, NET differentiation, proliferation and intracellular signaling pathways of these five cell lines and, in addition, of the NT-3 GEP-NET cell line. Additionally, we conducted panel sequencing to identify genomic alterations suitable for mutation-based targeted therapy.ResultsWe found that the GEP-NEN cell lines exhibit a stable neuroendocrine phenotype. Functional kinome profiling revealed a higher activity of serine/threonine kinases (STK) as well as protein tyrosine kinases (PTK) in the GEP-NET cell lines NT-3 and NT-18LM compared to the GEP-NEC cell lines NT-32 and NT-38. Panel sequencing revealed a mutation in Death Domain Associated Protein (DAXX), sensitizing NT-18LM to the Ataxia telangiectasia and Rad3 related (ATR) inhibitor Berzosertib, and a mutation in AT-Rich Interaction Domain 1A (ARID1A), sensitizing NT-38 to the Aurora kinase A inhibitor Alisertib. Small interfering RNA-mediated knock down of DAXX in the DAXX wild type cell line NT-3 sensitized these cells to Berzosertib.ConclusionsThe newly established GEP-NET and GEP-NEC cell lines represent comprehensive preclinical in vitro models suitable to decipher GEP-NEN biology and pathogenesis. Additionally, we present the first results of a GEP-NEN-specific mutation-based targeted therapy. These findings open up new potentialities for personalized therapies in GEP-NEN.

Baicalein activates 5’ adenosine monophosphate-activated protein kinase, inhibits the mammalian target of rapamycin, and exhibits antiproliferative effects in pancreatic neuroendocrine tumors in vitro and in vivo

BackgroundThe mammalian target of rapamycin inhibition has been shown to prolong progression-free survival in patients with pancreatic neuroendocrine tumors. The natural compound baicalein indirectly inhibits the mammalian target of rapamycin, but it is unknown if baicalein exhibits such effects at physiologically achievable concentrations or exhibits synergy. MethodsPancreatic neuroendocrine tumor cell lines were cultured with baicalein, everolimus, and/or a synthetic 5’ adenosine monophosphate-activated protein kinase activating agent alone and in combination. Cell viability assays and immunoblotting were performed. Female severe combined immunodeficient-beige mice were injected with BON-1 cells and treated with baicalein and COH-SR4 solutions via oral gavage. Tumor volumes were compared at 30 days. ResultsImmunoblotting revealed that treatment of baicalein induced 5’ adenosine monophosphate-activated protein kinase activation and the mammalian target of rapamycin inhibition. Treatment with baicalein alone led to a significant decrease in the ratio of viable cells compared with controls at 72 hours at concentrations ≥5 μM (P = .021). The addition of COH-SR4 led to significantly greater effect on cell viability than with baicalein alone (P < .001, P < .001). The combination of baicalein with everolimus resulted in significantly lower cell viability than with everolimus alone (P = .005, P < .001). Tumor volume in vivo was significantly decreased with the combination of baicalein and COH-SR4 compared with controls (P = .003). ConclusionBaicalein exhibits antiproliferative effects against pancreatic neuroendocrine tumor cell lines at doses ≥5 μM and demonstrates synergy.

Abstract 2308: Inhibition of fatty acid synthesis and concurrent lipid deprivation decreases neuroendocrine cancer cell proliferation and colony formation

Abstract Introduction. Gastroenteropancreatic neuroendocrine tumors (GEP-NETs), the second most common digestive cancer in terms of prevalence, can be particularly challenging to treat due to resistance to current clinical regimens. Lipid metabolism provides an important energy source for cancer cells; cells increase lipid supply by endogenous synthesis or exogenous uptake. Both processes can become dysregulated in cancer, but little is known about the role of either pathway in neuroendocrine tumors (NETs). The goal of this project was to determine whether inhibition of lipid synthesis with concurrent lipid deprivation affects NET cell proliferation and survival. Methods. (i) Expression and localization of fatty acid synthase (FASN) in NET patient samples (n=39) and human cell lines (BON, QGP-1, and NT-3) were examined with immunohistochemistry and immunoblotting, respectively. (ii) NET neutral lipid stores were determined with immunofluorescent analysis of Bodipy 493/503 staining. (iii) To study the role of lipid synthesis and lipid uptake on colony formation, NET cells were treated with TVB-3664 with or without FA depletion for 10d. Colonies were visualized with sulforhodamine B (SRB) dye staining and quantified with photospectrometry. (iv) To study the effect of lipid synthesis and uptake on proliferation, NET cells were treated with TVB-3664 with or without FA depletion for 72h, 96h, and 120h. Cellular growth was determined with SRB assays. (v) The effect of TVB-3664 on cyclin D1 and β-catenin expression in NET cells was determined with immunoblotting. Results. (i) FASN was detected in the cytoplasm of all 39 NET primary and metastatic tissue samples; scores of 5 or 6 indicated robust expression in 38/39 samples. Among cell lines, FASN expression was highest in BON cells followed by similar levels in QGP-1 and NT-3 cells. (ii) TVB-3664 treatment or lipid deprivation for 5d reduced neutral lipid storage in BON cells; combined TVB-3664 treatment with FA depletion for 5d fully depleted BON lipid stores. (iii) TVB-3664 treatment, but not FA deprivation, reduced NET colony formation by ~25% in BON cells and ~50% in QGP-1 cells. TVB-3664 treatment with FA deprivation reduced colony formation by ~65% in BON cells and ~75% in QGP-1 cells. (iv) TVB-3664 treatment or FA deprivation reduced NET cell proliferation at 72h, 96h, and 120h. (v) TVB-3664 treatment reduced cyclin D1 expression in BON and QGP-1 cells and β-catenin levels in BON cells. Conclusions. Our findings indicate that FASN is robustly expressed in NET patient samples and cell lines. Inhibition of endogenous lipid synthesis with concurrent lipid deprivation reduces colony formation and proliferation of NET cells. Importantly, our findings show that lipid metabolism promotes NET cell growth and that targeting de novo lipogenesis may be a potential treatment strategy for advanced GEP-NETs. Citation Format: Jeremy Johnson, Yekaterina Y. Zaytseva, Jennifer Castle, B. Mark Evers, Piotr Rychahou. Inhibition of fatty acid synthesis and concurrent lipid deprivation decreases neuroendocrine cancer cell proliferation and colony formation [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 2308.

Phase 1b study of tarlatamab, a half-life extended bispecific T-cell engager (HLE BiTEimmune therapy) targeting DLL3, in de novo or treatment emergent neuroendocrine prostate cancer (NEPC).

TPS197 Background: NEPC is an aggressive cancer with no standard treatment approach and poor prognosis. It is usually treatment-emergent, occurring in 15%–20% of patients (pts) with metastatic castration-resistant prostate cancer following treatment with androgen signaling inhibitors (ASI) and is characterized by histological transformation from adenocarcinoma to a high-grade neuroendocrine tumor.1 The tumor associated antigen delta-like ligand 3 (DLL3) has been identified as a promising target in both NEPC and small cell lung cancer (SCLC), as it is highly expressed in these tumors and minimally expressed on normal tissue. Tarlatamab is a DLL3-targeting HLE BiTE® immune therapy designed to bind DLL3 on cancer cells and CD3 on T cells, resulting in T cell activation and expansion and T cell-dependent killing of tumor cells. In preclinical studies, tarlatamab induced T-cell dependent lysis of DLL3-expressing neuroendocrine tumor cell lines, including NEPC cells.2 Interim results of an ongoing first-in-human study in pts with SCLC (NCT03319940) show evidence for tarlatamab efficacy with an acceptable safety profile.3 Together, these findings support a clinical study of tarlatamab in NEPC. Methods: NCT04702737 is an open-label, phase 1b study evaluating tarlatamab infusion in pts with metastatic de novo or treatment-emergent NEPC, consisting of dose exploration and then dose expansion. Key eligibility criteria include adults (≥18 y) with metastatic NEPC whose disease progressed/recurred after ≥1 prior line of systemic therapy (platinum-based regimen for de novo NEPC or an ASI if treatment-emergent), measurable disease per RECIST 1.1 with Prostate Cancer Working Group 3 modifications, and ECOG performance status ≤2. Primary objectives are to evaluate safety and tolerability and determine the maximum tolerated dose or recommended phase 2 dose of tarlatamab. Secondary objectives are to evaluate antitumor activity (as assessed by objective response, duration of response, progression-free survival, overall survival, and disease control rate) and characterize pharmacokinetics. Four US and international study sites have been activated with two enrolled pts.

High-Throughput Analysis to Identify Activators of Notch Signaling.

The Notch pathway regulates many cellular functions in a context-dependent manner. Depending on the cell type, either the activation or inhibition of Notch signaling can influence many processes such as cellular proliferation, specification, differentiation, and survival. The activation of Notch signaling has been shown to have therapeutic advantages in some cancers, thus having a method to identify Notch-activating compounds is needed. In this chapter we outline a method for high-throughput analysis of potential Notch pathway activators in a pancreatic neuroendocrine tumor cell line as an example. We also include the steps for subsequent validation of results and preclinical testing.

Ex Vivo Modeling of Human Neuroendocrine Tumors in Tissue Surrogates.

Few models exist for studying neuroendocrine tumors (NETs), and there are mounting concerns that the currently available array of cell lines is not representative of NET biology. The lack of stable patient-derived NET xenograft models further limits the scientific community’s ability to make conclusions about NETs and their response to therapy in patients. To address these limitations, we propose the use of an ex vivo 3D flow-perfusion bioreactor system for culturing and studying patient-derived NET surrogates. Herein, we demonstrate the utility of the bioreactor system for culturing NET surrogates and provide methods for evaluating the efficacy of therapeutic agents on human NET cell line xenograft constructs and patient-derived NET surrogates. We also demonstrate that patient-derived NET tissues can be propagated using the bioreactor system and investigate the near-infrared (NIR) dye IR-783 for its use in monitoring their status within the bioreactor. The results indicate that the bioreactor system and similar 3D culture models may be valuable tools for culturing patient-derived NETs and monitoring their response to therapy ex vivo.

Abstract P024: Antitumoral effect of telotristat ethyl as a single agent in neuroendocrine tumor cell lines and potential synergies

Abstract Neuroendocrine tumors (NET) comprise a rare and heterogeneous family of tumors arising from cells throughout the diffuse endocrine system. Carcinoid syndrome, a common gastrointestinal and lung NET complication, is mainly caused by serotonin release into the bloodstream, promoting diarrhea, flushing and carcinoid heat disease. By inhibiting the tryptophan hydroxylase, telotristat ethyl diminishes the synthesis of serotonin and improves carcinoid-related bowel movements. However, to our knowledge, there is no data showing the potential activity of telotristat ethyl as anti-tumoral agent and not only as a symptom-reliever . The purpose of this study is to analyze possible anti-tumoral effects of telotristat ethyl in NETs. For this, we used the pancreatic NET cell lines BON-1 and QGP1, and the gastrointestinal cell line HROC57. We treated them with telotristat ethyl as a single agent or in combination with common NET therapies, such as somatostatin analogues, mTOR inhibitors, standard chemotherapy and tyrosine kinase inhibitors. We analyzed the effects of these treatments on cellular viability, apoptosis markers, cell cycle and signaling pathways. Telotristal ethyl as single agent induced apoptosis measured by MTT in all NET cell lines after 72 hours of treatment, with IC50 in the µM range. Telotristat ethyl maintained this apoptotic effect even after serotonin addition. Surprisingly, neither the somatostatin analog octreotide nor the standard NET chemotherapy capecitabin/temozolomide induced apoptosis in BON-1, QGP-1 nor HROC57, suggesting that these cell lines are somatostatin analog- and chemotherapy-resistant. mTOR inhibition by everolimus treatment in NET cell lines induced apoptosis as monotherapy with IC50 in the µM range. Telotristat ethyl combination with everolimus produced a strong synergetic effect, dramatically decreasing cell viability in all NET cell lines. Mechanistically, combination of telotristat ethyl with everolimus promoted increasing levels of the apoptosis marker Anexin V with no major effects on cell cycle. mTOR inhibition by everolimus, alone and in combination with telotristat ethyl, decreased phosphorylated levels of the mTOR pathway readout protein S6 and elevated phosphorylated p42/44 MAPK levels, with no clear effects of telotristat ethyl. Our results show that telotristat ethyl has the capacity to inhibit pancreatic and GI NET cells tumor growth as a single agent and seems synergistic when combined with mTOR inhibitors but not with antiangiogenics or chemotherapy. Citation Format: Arantzazu Sierra Ramirez, Marinela Méndez, Adrián Plaza, Andrés Pastor, Javier Molina Cerrillo, Pablo José Fernández Marcos, Teresa Alonso Gordoa, Enrique Grande Pulido. Antitumoral effect of telotristat ethyl as a single agent in neuroendocrine tumor cell lines and potential synergies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P024.

Comparing the Effect of Multiple Histone Deacetylase Inhibitors on SSTR2 Expression and [111In]In-DOTATATE Uptake in NET Cells.

Simple SummaryPatients diagnosed with neuroendocrine tumors (NETs) are often treated with peptide receptor radionuclide therapy (PRRT). This therapy targets the somatostatin type-2 receptors (SSTR2) frequently overexpressed on these types of tumors. Although this therapy has proven to be effective, complete responses are rare and therapy improvement is desirable. We aimed to increase SSTR2 expression on NET cells, potentially increasing the number of patients eligible for SSTR2-targeted PRRT and improving clinical outcomes. We used histone deacetylase inhibitors (HDACis) to manipulate the epigenetic machinery and hereby aimed to increase SSTR2 gene transcription. Our results showed that the HDACis increased SSTR2 expression in several NET cell lines. Moreover, the uptake of radiolabeled DOTATATE, the tracer used for PRRT, was enhanced. The observed reversibility profile after HDACi withdrawal of the induced effects suggests that proper timing of HDACi treatment is likely essential.The aim of this study was to increase somatostatin type-2 receptor (SSTR2) expression on neuroendocrine tumor (NET) cells using histone deacetylase inhibitors (HDACis), potentially increasing the uptake of SSTR2-targeted radiopharmaceuticals and subsequently improving treatment efficacy of peptide receptor radionuclide therapy (PRRT). Human NET cell lines BON-1, NCI-H727, and GOT1 were treated with HDACis (i.e., CI-994, entinostat, LMK-235, mocetinostat, panobinostat, or valproic acid (VPA); entinostat and VPA were the HDACis tested in GOT1 cells) to examine SSTR2 mRNA expression levels and uptake of SSTR2-targeting radiotracer [111In]In-DOTATATE. Reversibility of the induced effects was examined after drug-withdrawal. Finally, the effect of VPA on radiosensitivity was investigated. A strong stimulatory effect in BON-1, NCI-H727, and GOT1 cells was observed after HDACi treatment, both on SSTR2 mRNA expression levels and [111In]In-DOTATATE uptake. The effects of the HDACis were largely reversible over a period of seven days, demonstrating largest reductions within the first day. The reversibility profile of the induced effects suggests that proper timing of HDACi treatment is most likely essential for a beneficial outcome. In addition to increasing SSTR2 expression levels, VPA enhanced the radiosensitivity of all cell lines. In conclusion, HDACi treatment increased SSTR2 expression, and radiosensitivity was also enhanced upon VPA treatment.

Targeted inhibition of the WEE1 kinase as a novel therapeutic strategy in neuroendocrine neoplasms

Neuroendocrine neoplasms (NENs) represent a rare and heterogeneous group of malignancies, sharing features of both neural and endocrine cells. NENs G3 appear as a highly aggressive subset with a poor prognosis and limited therapeutic options. The small-molecule inhibitor of the WEE1 tyrosine kinase, adavosertib (AZD1775), has previously demonstrated potent anti-tumor effects on various types of cancer in preclinical and clinical studies. However, the role of adavosertib in NENs G3 had remained elusive. We evaluated the effects of adavosertib on pancreatic (BON-1, QGP-1) and bronchopulmonary (NCI-H720) neuroendocrine tumor cell lines applying 2D and 3D spheroid models. We newly demonstrated that adavosertib is sufficient to reduce cell viability and proliferation in neuroendocrine cell lines with features of high-grade NENs. As underlying mechanisms, we identified adavosertib-mediated DNA double-strand breaks and a G2/M cell cycle checkpoint abrogation leading into mitotic catastrophe and cancer cell apoptosis. Silencing of WEE1 via siRNA transfection resulted in a phenotype similar to adavosertib treatment. Together, inhibition of the WEE1 tyrosine kinase applying adavosertib on NENs G3 outlines a promising novel therapeutic strategy.

Abstract 984: Targeting BCL-xL addiction with APG-1252 (pelcitoclax) to overcome apoptotic blockade in neuroendocrine neoplasm (NEN)

Abstract Current targeted therapies such as VEGFR inhibitor sunitinib and mTOR inhibitor everolimus extend progression-free survival (PFS) in patients with Grade 1 (G1) and G2 neuroendocrine tumors (NET), but the objective response rate remains low. The median PFS in patients with advanced neuroendocrine carcinoma (NEC) receiving platinum-based therapy is only 3 to 4 months. Hence, more effective therapy is needed to improve clinical outcomes. This study explored if BCL-2 family antideath proteins play a role in NEN tumorigenesis and whether clinical-stage dual BCL-2/BCL-xL inhibitor APG-1252 might overcome intrinsic apoptotic blockade in NEN. A panel of six NEN cell lines were exposed to APG-1252-M1, which is an active metabolite of APG-1252 typically used for in vitro studies. After 72 hours, the IC50 of APG-1252-M1 ranged from 0.43 to 10 μM. Among the panel, pancreatic NET (pNET) cell lines BON-1 and β-TC3 exhibited the highest sensitivity, with IC50 values of 0.43 and 0.55 µM, respectively, whereas NEC cell line NCI-H460 was insensitive to APG-1252-M1. Interestingly, Western blot analysis revealed higher expression of BCL-xL compared to BCL-2 and MCL-1 proteins in NET cell lines (BON-1, QGP-1, NCI-H727). Higher baseline levels of BCL-xL:BIM and BCL-xL:PUMA complexes in NEN cells indicated APG-1252-M1 sensitivity. Exposure to APG-1252-M1 disrupted these complexes, liberating BIM/PUMA to trigger the apoptotic cascade, as indicated by an increase in BAX:BAK complexes. Conversely, lower BCL-xL complex levels and higher MCL-1 levels correlated with insensitivity of NCI-H460 to APG-1252-M1. Immunohistochemistry staining of BCL-xL, BCL-2, and MCL-1 proteins was performed in formalin-fixed paraffin-embedded tissue sections of 106 NEN patient tumors (including stomach/intestine/pancreatic NEN), of which approximately 70% were NET and 30% NEC. Similar to data in NEN cell lines, BCL-xL expression was higher than BCL-2 expression in NEN patient samples, in which BCL-xL expression was indicated by more than 80% of samples with IHC scoring ≥6 on a scale of 0 to 10, and BCL-2 expression by 30% IHC scoring &amp;lt; 6 or undetectable (60% IHC scoring 0). Unexpectedly, however, MCL-1 expression was also high, with more than 80% of samples IHC scoring ≥6. In summary, the results suggest that BCL-xL plays an important role in NEN. Cellular sensitivity to BCL-2/BCL-xL inhibitor APG-1252-M1 correlates with baseline BCL-xL complex levels. In NEN patient samples, MCL-1 was also highly expressed, implicating its potential negative regulatory effect on sensitivity to APG-1252. Concurrent expression of BCL-xL and MCL-1 proteins suggests that a combination treatment targeting both proteins might be more effective in NEN. Our findings inform development of a BCL-2/BCL-xL inhibitor for NEN therapy. Citation Format: Yu Guo, Jing Deng, Lin Zhang, Xiaojing Huang, Kaixiang Zhang, Feng Zhou, Yuanbao Li, Fan Luo, Douglas D. Fang, Dajun Yang, Jie Chen, Yifan Zhai. Targeting BCL-xL addiction with APG-1252 (pelcitoclax) to overcome apoptotic blockade in neuroendocrine neoplasm (NEN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 984.