Growth Of Cell Lines Research Articles

Abstract A040: The SRG OncoRat supports growth of numerous RAS mutant cell lines, expanding pre-clinical RAS-inhibitor testing

Abstract RAS family proteins are important drug targets in multiple cancers due to their central role in hypertrophic and pro-mitotic signaling. KRAS is a small GTPase that is activated in response to growth factors including HGF, TGFα, and EGF. KRAS then activates the MAP kinase (MAPK; RAF/MEK/ERK) signaling pathway, driving ERK translocation to the nucleus, and resulting in transcription of pro-mitotic and pro-growth genes. Additionally, KRAS contributes to PI3K/AKT/mTOR signaling, leading to transcriptional activation as well as hypertrophic protein synthesis via mTOR. We sought to validate several RAS mutant cell-derived tumor xenograft models in the SRG OncoRat. Cells were inoculated subcutaneously in the flank of SRG rats. To date, we have validated tumor growth using the HT-1080 fibrosarcoma NRAS mutant (Gln61Lys (c.181C>A)) cell line. We have also validated a suite of KRAS mutant lines in the SRG OncoRat, including: A-549 lung adenocarcinoma (Gly12Ser (c.34G>A)); NCI-H2122 NSCLC adenocarcinoma (Gly12Cys (c.34G>T)); MIA PaCa-2 pancreatic ductal adenocarcinoma and H-358 NSCLC adenocarcinoma (Gly12Cys (c.34G>T)); Capan-2 pancreatic ductal adenocarcinoma (Gly12Val (c.35G>T)); NCI-H441 NSCLC papillary adenocarcinoma (Gly12Val (c.35G>T)); NCI-H1734 NSCLC adenocarcinoma (Gly13Cys (c.37G>T)); and MDA-MB-231 breast adenocarcinoma (Gly13Asp (c.38G>A)). All cell lines showed robust and consistent tumor growth when injected subcutaneously into the SRG OncoRat. These models provide valuable tools for pre-clinical testing of RAS inhibition. Citation Format: R. Grace Walton, Diane Begemann, Cynthia Dunn, Valeria Steffey, Abigail Ross, Razoanul Haque, Fallon K. Noto. The SRG OncoRat supports growth of numerous RAS mutant cell lines, expanding pre-clinical RAS-inhibitor testing [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A040.

Abstract B006: Novel direct RAS inhibitors for pancreatic cancer

Abstract RAS oncogenes are frequently activated by mutations in pancreatic and lung cancer, where they appear to act as driving mutations. This study examines the activity of a series of novel direct RAS inhibitors with a predicted unique interaction region. We have used in silico library screening followed by Medicinal Chemistry optimization to develop the compounds. We have validated the agents using Microscale Thermophoresis to quantify binding to recombinant RAS protein as well as NMR analysis of the drug/RAS complex. We used 3D growth inhibition assays in mutant RAS cell lines and protein-based RAS signaling assays to quantify and characterize the action of the family of inhibitors. We have identified compounds that bind wild type K-RAS and H-RAS but exhibit preferential binding to the K-RAS-G12D and KRAS-G12C mutants. We can suppress the association of mutant RAS protein with its effector RAF-1 in treated cells. We also observe suppression of mutant RAS signaling and inhibition of 3D cell growth of mutant RAS cell lines with the agents. As the compound family are predicted to bind to a different site than either AMG-510 (K-RAS G12C specific) or MRTX-1133 (K-RAS G12D specific), agents we have also tested co-operative activity with these drugs. Our compounds enhanced the effects of MRTX-1133 against K-RAS G12D cells and had a similar effect on AMG-510 in K-RAS G12C cell lines. These agents may serve as novel anti-RAS therapeutics and may have potential to enhance the activity or suppress resistance to AMG-510 and MRTX-1133. Citation Format: Geoff Clarke, Tariq Arshad, Howard Donninger, Becca von Baby, Rachel Ferrill, Mike Sabo, Joe Burlison, John Trent. Novel direct RAS inhibitors for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B006.

Berberine Inhibited Growth and Migration of Human Colon Cancer Cell Lines by Increasing Phosphatase and Tensin and Inhibiting Aquaporins 1, 3 and 5 Expressions.

Introduction: Berberine is a natural isoquinoline alkaloid with anti-cancer properties. Nevertheless, the underlying mechanism of its action in human colorectal cancer (CRC) has not been thoroughly elucidated. We investigated the anti-cancer effect of berberine on HT-29, SW-480 and HCT-116 human CRC cell lines. Methods: Cell proliferation, migration and invasion were studied by MTT assay, wound healing, transwell chambers and flow cytometry. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunostaining were used to evaluate the expression of aquaporins (AQPs) 1, 3 and 5 in colon cancer cell lines before and after treatment with berberine (10, 30 and 100 µM). RT-qPCR and Western blotting were used to further explore the PI3K/AKT signaling pathway and the molecular mechanisms underlying berberine-induced inhibition of cell proliferation. Results: We demonstrated that treatment of these CRC cell lines with berberine inhibited cell proliferation, migration and invasion through induction of apoptosis and necrosis. HT-29, SW-480 and HCT-116 stained positively for AQP 1, 3 and 5, and berberine treatment down-regulated the expression of all three types of AQPs. Berberine also modulated PI3K/AKT pathway activity through up-regulating PTEN and down-regulating PI3K, AKT and p-AKT expression as well as suppressing its downstream targets, mTOR and p-mTOR at the protein level. Discussion/Conclusions: These findings indicate that berberine inhibited growth, migration and invasion of these colon cancer cell lines via down-regulation of AQP 1, 3 and 5 expressions, up-regulating PTEN which inhibited the PI3K/AKT pathway at the gene and protein levels, and that AQP 1, 3 and 5 expression level can be used as prognostic biomarkers for colon cancer metastasis.

Celecoxib, a Non-Steroidal Anti-Inflammatory Drug, Exerts a Toxic Effect on Human Melanoma Cells Grown as 2D and 3D Cell Cultures.

Cutaneous melanoma (CM) remains one of the leading causes of tumor mortality due to its high metastatic spread. CM growth is influenced by inflammation regulated by prostaglandins (PGs) whose synthesis is catalyzed by cyclooxygenases (COXs). COX inhibitors, including non-steroidal anti-inflammatory drugs (NSAIDs), can inhibit tumor development and growth. In particular, in vitro experiments have shown that celecoxib, a NSAID, inhibits the growth of some tumor cell lines. However, two-dimensional (2D) cell cultures, used in traditional in vitro anticancer assays, often show poor efficacy due to a lack of an in vivo like cellular environment. Three-dimensional (3D) cell cultures, such as spheroids, are better models because they can mimic the common features displayed by human solid tumors. Hence, in this study, we evaluated the anti-neoplastic potential of celecoxib, in both 2D and 3D cell cultures of A2058 and SAN melanoma cell lines. In particular, celecoxib reduced the cell viability and migratory capability and triggered the apoptosis of melanoma cells grown as 2D cultures. When celecoxib was tested on 3D melanoma cell cultures, the drug exerted an inhibitory effect on cell outgrowth from spheroids and reduced the invasiveness of melanoma cell spheroids into the hydrogel matrix. This work suggests that celecoxib could represent a new potential therapeutic approach in melanoma therapy.

The Potential Anti-Cancerous Activity of Prunus amygdalus var. amara Extract

The extract of P. amygdalus var. amara is known for its health benefits, which include lowering diabetes and heart disease risks. In eight human tumor cell lines, P. amygdalus var. amara demonstrated potent anti-cancer activity, including NB4, Huh-7, A-549, SKOV-3, PC-3, T-24, U937, and Hep-2. There was a notable change in the morphology of nearly all cancer cell lines, and cancer cells continued to exist. Incubation for 12 h, 24 h, 48 h, or 72 h resulted in the lowest viable cell concentration at 48 h, which was 34.65% lower than that of the non-treated cells. During exposure to the extract, the majority of cells lost their typical morphology and shrank in size. According to the cell viability data, the P. amygdalus var. amara treatment significantly decreased the cancer cells’ growth in most cancer cell lines when doses and time were taken into consideration.

231 Eribulin inhibits cell growth in cutaneous squamous cell carcinoma cell lines and a novel patient-derived xenograft harboring TP53 and ARID2 mutations

231 Eribulin inhibits cell growth in cutaneous squamous cell carcinoma cell lines and a novel patient-derived xenograft harboring TP53 and ARID2 mutations

Gallium maltolate shows synergism with cisplatin and activates nucleolar stress and ferroptosis in human breast carcinoma cells.

Breast cancer is the most common cancer in women. Triple-negative breast cancer (TNBC) is an aggressive disease with poor outcomes. TNBC lacks effective targeted treatments, and the development of drug resistance limits the effectiveness of chemotherapy. It is crucial to identify new drugs that can enhance the efficacy of traditional chemotherapy to reduce drug resistance and side effects. TNBC cell lines, MDA-MB-231 and Hs 578T, and a normal cell line, MCF-10A, were included in this study. The cells were treated with gallium maltolate (GaM), and their transcriptome was analyzed. Ferroptosis and nucleolar stress markers were detected by qPCR, western blotting, fluorescence microscopy, and flow cytometry. The impairment of ribosome synthesis was evaluated by northern blotting and sucrose gradients. GaM triggered cell death via apoptosis and ferroptosis. In addition, GaM impaired translation and activated nucleolar stress. Cisplatin (DDP) is a chemotherapeutic agent for advanced breast cancer. While single treatment with GaM or DDP at low concentrations did not impact cell growth, co-administration enhanced cell death in TNBC but not in normal breast cells. The enhancement of ferroptosis and nucleolar stress could be observed in TNBC cell lines after co-treatment. These results suggest that GaM synergizes with cisplatin via activation of nucleolar stress and ferroptosis in human breast carcinoma cells. GaM is marginally toxic to normal cells but impairs the growth of TNBC cell lines. Thus, GaM has the potential to be used as a therapeutic agent against TNBC.

Identification, Phytochemistry and Biological Activities of Paris polyphylla on Hepatocellular Carcinoma.

<b>Background and Objective:</b> Liver cancer is the common cause of cancer death. <i>Paris polyphylla</i> is used as a traditional folk medicine in Vietnam to treat pneumonia, mastitis, bruises and fractures but no study was available regarding its ability to treat liver cancer or slow its growth. In this study, <i>Paris polyphylla</i> samples were identified and evaluated cytotoxic activity against the liver cancer cells. <b>Materials and Methods:</b> <i>Paris polyphylla</i> species were collected from various areas in Yen Bai, Vietnam, which were identified by comparative morphological method and DNA barcoding for the <i>18S, matK</i> genes and <i>ITS</i> region. <i>Paris polyphylla</i> samples were dried until constant weight, ground into a fine powder and extracted in various solvents. The bioactivity of these extracts were done by the MTT assay. <b>Results:</b> The sequences of <i>18S, matK</i> genes and <i>ITS</i> region were high similarity to sequences of <i>P. polyphylla</i> in the National Center for Biotechnology Information. The N-hexane and ethyl acetate fractions were produced from the methanol extract of <i>P. polyphylla</i>. The TLC results showed that there was a significant difference in the component of n-hexane and ethyl acetate fraction. The N-hexane fraction contains mainly low-polarity and non-polarity substances. While ethyl acetate fraction consists mainly of polar substances. In addition, ethyl acetate fraction was shown the strongest cytotoxic activity on the cancer cell lines HepG2 and Huh7 with the evaluation of IC<sub>50</sub> = 115.11±2.77 μg mL<sup>1</sup> and IC<sub>50</sub> = 148.11±1.78 μg mL<sup>1</sup>. <b>Conclusion:</b> The extract of <i>Paris polyphylla</i> demonstrated strong potential to inhibit the growth of the liver cancer cell line. The ethyl acetate fraction has the highest ability for cytotoxicity on the liver and cell line at a concentration of 200 μg mL<sup>1</sup> through MTT.

Abstract LB163: A microfluidic platform for developing circulating tumor cells (CTCs) organoids for precision medicine in pancreatic cancer

Abstract Introduction Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second leading cause of cancer death in the US by 2030. Besides the late detection and lack of effective treatments, the greatest treatment hurdle to PDAC is the ineffectiveness of patient-derived tumor models. Precision medicine-based treatment decisions require the generation of consistent and trackable complex organoid models that is not achievable by tissue biopsies. Liquid biopsies such as blood, instead, contain circulating biomarkers released by the tumor, among which especially interested in for this study are circulating tumor cells (CTCs). This work develops microfluidic 3D organoids from CTCs to be used as a precision medicine tool to predict and guide the drug response of early stage I-IIB (PDAC) patients. Experimental Methods CTCs are isolated from PDAC patient blood samples using microfluidic Labyrinth following removal of red blood cells with 6% dextran. Collected cells post-isolation are immunofluorescence (IF) stained with DAPI (nuclei), CD45 (leukocyte), pancytokeratin (type I/II cytokeratin 1-8, 10, 14- 16 and 19), Vimentin, and Epithelial cell adhesion molecule (EpCAM) to identify CTCs and characterize their phenotypes. The mold of the microfluidic platform for CTC organoids is fabricated with the standard SU-8 photolithography. The PDMS-based device is tested with a low seeding number of a pancreatic cancer CTC cell line labeled fluorescently. Decellularization of cancer associated fibroblasts (CAFs) generates the in vitro matrix that supplies amino-acid precursors for PDAC metabolism, promoting CTC organoid formation. Serial images of cell growth are obtained throughout the course using a Ti-20 microscope. After each 7-day culture, organoids are dissociated from the device and IF stained using a similar panel as above (with Ki67 marker instead of CD45 for cell proliferation). Preliminary gemcitabine dosage-performance analysis is performed on-chip. Results CTC count from the tested patient panel (n=16) averages 17.0±8.4/mL of blood. CAFs are successfully decellularized on-chip and the embedded pancreatic CTC cell line growth curve is generated. IF staining of the dissociated cells from the organoids confirms the proliferation of the loaded pseudo-CTCs. Conclusion The work above demonstrates the successful isolation of CTCs from PDAC patient blood samples and an engineered microfluidic platform to expand low abundance of cells into organoids. It has a potential to be tested and employed to expand CTCs from PDAC into organoids for drug screening. Citation Format: Yu Zhang, Scott Smith, Minal Nenwani, Valerie Gunchick, Vaibhav Sahai, Sunitha Nagrath, Deepak Nagrath. A microfluidic platform for developing circulating tumor cells (CTCs) organoids for precision medicine in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB163.

Hyaluronan-estradiol nanogels as potential drug carriers to target ER+ breast cancer cell line

An innovative hyaluronan-based nano-delivery system is proposed for the active targeting towards ER+ breast cancer. Hyaluronic acid (HA), an endogenous and bioactive anionic polysaccharide, is functionalized with estradiol (ES), a sexual hormone involved in the development of some hormone-dependent tumors, to give an amphiphilic derivative (HA-ES) able to spontaneously self-assemble in water to form soft nanoparticles or nanogels (NHs). The synthetic strategy used to obtain the polymer derivatives and the physico-chemical properties of the obtained nanogels (ES-NHs) are reported. ES-NHs ability to entrap hydrophobic molecules has also been investigated, by loading curcumin (CUR) and docetaxel (DTX), both able to inhibit the growth of ER+ breast cancer. The formulations are studied for their capability to inhibit the growth of the MCF-7 cell line, thus evaluating their efficacy and potential as a selective drug delivery systems. Our results demonstrate that ES-NHs have not toxic effects on the cell line, and that both ES-NHs/CUR and ES-NHs/DTX treatments inhibit MCF-7 cell growth, with ES-NHs/DTX effect higher than that of free DTX. Our findings support the use of ES-NHs to deliver drugs to ER+ breast cancer cells, assuming a receptor-dependent targeting.

Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic.

Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA-approved medication for asthma, is a pan-thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC50 in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor-dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock-treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker-only (CA-125) relapsed ovarian cancer, where the rate of rise of CA-125 was significantly reduced following treatment with zafirlukast, while no severe adverse events were reported. Thiol isomerase inhibition with zafirlukast represents a novel, well-tolerated therapeutic in the treatment of ovarian cancer.

Potent antitumor effects of the conditioned medium of bone marrow-derived mesenchymal stem cells via IGFBP-4.

Cell transfer therapy using mesenchymal stem cells (MSCs) has pronounced therapeutic potential, but concerns remain about immune rejection, emboli formation, and promotion of tumor progression. Because the mode of action of MSCs highly relies on their paracrine effects through secretion of bioactive molecules, cell-free therapy using the conditioned medium (CM) of MSCs is an attractive option. However, the effects of MSC-CM on tumor progression have not been fully elucidated. Herein, we addressed this issue and investigated the possible underlying molecular mechanisms. The CM of MSCs derived from human bone marrow greatly inhibited the in vitro growth of several human tumor cell lines and the in vivo growth of the SCCVII murine squamous cell carcinoma cell line with reduced neovascularization. Exosomes in the MSC-CM were only partially involved in the inhibitory effects. The CM contained a variety of cytokines including insulin-like growth factor binding proteins (IGFBPs). Among them, IGFBP-4 greatly inhibited the in vitro growth of these tumors and angiogenesis, and immunodepletion of IGFBP-4 from the CM significantly reversed these effects. Of note, the CM greatly reduced the phosphorylation of AKT, ERK, IGF-1 receptor beta, and p38 MAPK in a partly IGFBP4-dependent manner, possibly through its binding to IGF-1/2 and blocking the signaling. The CM depleted of IGFBP-4 also reversed the inhibitory effects on in vivo tumor growth and neovascularization. Thus, MSC-CM has potent inhibitory effects on tumor growth and neovascularization in an IGFBP4-dependent manner, suggesting that cell-free therapy using MSC-CM could be a safer promising alternative for even cancer patients.

Acetyl-Click Screening Platform Identifies Small-Molecule Inhibitors of Histone Acetyltransferase 1 (HAT1).

HAT1 is a central regulator of chromatin synthesis that acetylates nascent histone H4. To ascertain whether targeting HAT1 is a viable anticancer treatment strategy, we sought to identify small-molecule inhibitors of HAT1 by developing a high-throughput HAT1 acetyl-click assay. Screening of small-molecule libraries led to the discovery of multiple riboflavin analogs that inhibited HAT1 enzymatic activity. Compounds were refined by synthesis and testing of over 70 analogs, which yielded structure-activity relationships. The isoalloxazine core was required for enzymatic inhibition, whereas modifications of the ribityl side chain improved enzymatic potency and cellular growth suppression. One compound (JG-2016 [24a]) showed relative specificity toward HAT1 compared to other acetyltransferases, suppressed the growth of human cancer cell lines, impaired enzymatic activity in cellulo, and interfered with tumor growth. This is the first report of a small-molecule inhibitor of the HAT1 enzyme complex and represents a step toward targeting this pathway for cancer therapy.

A novel tumour enhancer function of Insulin-like growth factor II mRNA-binding protein 3 in colorectal cancer

CRC cells evolve a variety of strategies to limit or circumvent apoptosis cell death. RNA binding proteins (RBPs) regulate many of the molecular mechanisms that underlie the development of cancer. The insulin-like growth factor II mRNA-binding proteins (IMP) family are oncofoetal RBPs, consisting of IMP1, IMP2 and IMP3, which have an important role in RNA metabolism. IMP3 is highly expressed in colorectal cancer (CRC) tissue, where its expression often correlates with poor prognosis. However, the role of IMP3 in CRC is not fully understood. IMP3 expression was analysed using a public database and by Western blotting and immunohistochemistry in human colon samples derived from patients with sporadic CRC and healthy subjects. To address whether IMP3 controls cancer cell survival, we analysed cell death pathways in in vitro and in vivo experiments after IMP3 downregulation by siRNA or an antisense oligonucleotide. IMP3 was highly expressed in CRC samples compared to normal control tissues. The knockdown of IMP3 enhanced a caspase-independent cell death in CRC cell lines. Furthermore, the treatment of CRC cells with IMP3 siRNA did not alter the expression of GSDMD, GPX-4 and the activated form of RIP3, three key molecules that govern pyroptosis, ferroptosis and necroptosis, respectively. Abrogation of IMP3 in CRC significantly reduced Bcl-2 and Bcl-xL mRNA and was associated with an altered mitochondrial membrane potential that allowed the nuclear migration of the apoptosis-inducing factor (AIF). Moreover, specific immunoprecipitation experiments on CRC human cell lines indicated that IMP3 binds Bcl-2 and Bcl-xL mRNA, suggesting that IMP3 acts as a regulator of the intrinsic apoptotic pathway through the surveillance of anti-apoptotic Bcl mRNA metabolism. Finally, we showed that IMP3 block inhibited the growth of CRC cell lines in vivo after transplantation into immunodeficient mice. Altogether, these data support a novel role for IMP3 in controlling the intrinsic caspase-independent apoptotic pathway in CRC.

Abstract 5458: TONSL is an immortalizing oncogene of the chromosome 8q24.3 amplicon and new therapeutic target in breast cancer

Abstract Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to lack of an appropriate isogenic model system. To address this technical challenge, we utilized primary breast luminal epithelial cells propagated from healthy donors of different genetic ancestry and their hTERT-immortalized counterparts to identify functional gene expression changes associated with immortalization. We identified elevated expression of TONSL (Tonsoku Like, DNA Repair Protein) as one of the earliest events during immortalization. TONSL is located on chromosome 8q24.3 and amplified in ~20% of breast cancers with significantly higher amplification in metastatic tumors. TONSL forms a complex with FACT and MMS22L1 to modulate multiple cellular pathways including DNA replication, repair through homologous recombination (HR) and functions as a post replicative histone/chromatin reader. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity. While TONSL overexpression alone was insufficient for neoplastic transformation, TONSL-immortalized primary cells modified to overexpress defined oncogenes generated estrogen receptor-positive adenocarcinomas in NSG mice. Analysis of breast tumor microarray with ~500 tumors revealed poor overall and progression free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors including NF-κB and limited access to the tumor suppressor p53. Most importantly, TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in HR and Fanconi Anemia pathways. Consistent with the effects of TONSL on HR-associated genes, TONSL overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was an essential gene for growth of TONSL-amplified breast cancer cell lines in vivo. Breast cancer cell lines with TONSL/chr8q24.3 amplification were sensitive to TONSL-FACT complex inhibitor CBL0137, both in vitro and in vivo. To our knowledge, TONSL is the only gene other than telomerase with immortalizing function and represents a new therapeutic target for breast cancer with chr8q24.3 amplification. Citation Format: Aditi S. Khatpe, Rebecca Dirks, Poornima Bhat-Nakshatri, Henry Mang, Katie Batic, Sarah Swiezy, Jacob Olson, Xi Rao, Yue Wang, Hiromi Tanaka, Sheng Liu, Jun Wan, Duojiao Chen, Yunlong Liu, Fang Fang, Sandra Althouse, Emily Hilsey, Maggie M. Granatir, Rebekah Addison, Constance J. Temm, George Sandusky, Audrey Lee-Gosselin, Kenneth Nephew Nephew, Kathy D. Miller, Harikrishna Nakshatri. TONSL is an immortalizing oncogene of the chromosome 8q24.3 amplicon and new therapeutic target in breast 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 5458.

Abstract 655: Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells

Abstract Programmed cell death (PCD) was originally limited to apoptosis, but now includes pyroptosis, ferroptosis, necroptosis, and paraptosis. Necroptosis has attracted particular interest as an approach for immunotherapy as activation of the RIPK1/RIPK3 necrosome phosphorylates MLKL to form membrane pores, leading to loss of integrity and enhanced ability to induce an antigen-specific immune response in models of vaccination. Here we sought to determine if necroptosis within tumor cells was an important regulatory of anti-tumor immunity. Deficiency in Ripk3 or Mlkl did not impact tumor incidence or growth in genetic models of mammary carcinoma or lung adenocarcinoma. Similarly, the growth of syngeneic cell lines was not altered by loss of Ripk3 or Mlkl in vitro or in vivo. We next examined the induction of apoptosis or necroptosis in syngeneic tumor models using genetic constructs with doxycycline-controlled expression. In line with published data, subcutaneous vaccination demonstrated that RIPK3-induced necroptosis promoted an antigen-specific CD8+ T cell response greater than that observed with caspase 8-induced apoptosis. Surprisingly however, when necroptosis was induced within established PyMT-B6 orthotopic mammary tumors, we observed significantly reduced survival, which correlated with higher neutrophil and macrophage recruitment, along with lower CD8+ T cell infiltration and IFN-γ expression. In vitro, necroptosis induced with either RIPK3 or MLKL greatly enhanced expression of Cxcl1 and Cxcl2, a property that could be recapitulated by transferring dead cell supernatant to live cells, suggesting the presence of an alarmin or damage-associated molecular pattern (DAMP). We therefore knocked out MyD88, which serves as a critical adapter protein to a number of surface receptors, including the TLR family, the IL1 receptor, and RAGE. The loss of MyD88 completely eliminated the ability of tumor cells to express Cxcl1 in response to supernatant from necrotic cells. Expression was not driven by release of HMGB1 or signaling through RAGE or TLRs. Instead, we found that blocking the interleukin (IL)1 receptor or IL1α, but not IL1β, prevented Cxcl1 expression. IL1α is a constitutively expressed by some tumors, suggesting a potential role for IL1α in regulating immune responses to therapies that induce necrotic-like forms of cell death. In support of this, Il1a expression was strongly predictive of progression free survival in triple-negative breast cancer. These findings suggest that IL1α has an unexplored role in regulating the tumor microenvironment. By understanding how IL1α regulates the immune response during cytotoxic therapy, our findings may explain why certain therapies are immunogenic (or not) in different cancers. Citation Format: Kay Hänggi, Aysenur Keske, Alycia Gardner, Jie Li, Olabisi Osunmakinde, Daiana Celias, Amer Beg, Brian Ruffell. Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells [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 655.

Abstract 6264: NUV-868, a novel BD2-selective BET inhibitor, in combination with enzalutamide or olaparib, inhibits growth of solid tumor xenografts

Abstract The bromodomain and extraterminal domain (BET) family of proteins play a vital role in gene transcription, making it an attractive therapeutic target for cancer. BET inhibitors can also combine with many anticancer agents to enhance activity. The bromodomains of the BET protein, BD1 and BD2, have unique functions and inhibiting either domain can result in differential responses. To date, non-selective BET inhibitors have failed during early clinical development due to significant on-target toxicity and limited benefit; however, selectively targeting specific bromodomains may result in a more favorable benefit/risk profile. NUV-868 is a novel and highly selective BD2 inhibitor of the BET protein family with ~1500-fold selectivity for BRD4-BD2 relative to BRD4-BD1. Herein, we describe NUV-868 and its activity in multiple in vitro and in vivo solid tumor models. Target engagement, selectivity of bromodomain inhibition, and regulation of BET-mediated gene expression were examined. Additionally, the antitumor activity of NUV-868 in combination with enzalutamide or olaparib was studied in tumor xenograft models of prostate, breast, and pancreatic cancer. NUV-868 demonstrated high selectivity for BD2 and regulated expression of several BET target genes. NUV-868 in combination with enzalutamide inhibited growth of several prostate cancer cell line- and patient-derived xenografts. NUV-868 in combination with olaparib inhibited tumor growth in models of breast, ovarian and pancreatic cancer. Our preclinical data demonstrate that NUV-868, a BD2-selective BET inhibitor, inhibits growth of tumor xenografts in combination with enzalutamide or olaparib and provides rationale for examination of these combinations in the clinic. An ongoing, phase 1 clinical study (NCT05252390) is evaluating NUV-868 as a monotherapy and in combination with olaparib or enzalutamide in patients with advanced solid tumors. Citation Format: Hitisha Patel, Jennifer Hertzog, Laura Heller, Spandana Vootukuri, Yan Zhang, Chris Miller, Gary Hattersley. NUV-868, a novel BD2-selective BET inhibitor, in combination with enzalutamide or olaparib, inhibits growth of solid tumor xenografts [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 6264.

Abstract 42: In vivo subcutaneous and orthotopic cancer xenograft modeling in the SRG immunodeficient rat

Abstract Human cancer xenografts are a vital tool for understanding tumor biology, growth kinetics, and therapeutic efficacy using animal models. Historically, immunodeficient mice have been the standard rodent species for cancer xenograft modeling. However, an immunodeficient rat that supports a wide variety of human cancer cell types would provide a larger rodent strain for easier surgical manipulation, serial blood sampling, and provide a single model in which efficacy, pharmacokinetics, and toxicology can be performed. We have created a Sprague Dawley Rag2 -/-, Il2rg -/- rat (SRGTM OncoRat®) that provides a highly supportive environment for growing tumors of human origin. The SRG rat lacks B, T, and NK cells and readily supports the growth of multiple human cancer cell lines. The SRG rat is more immunodeficient than the Nude rat, suggesting it may be permissive to a wider variety of human cancer types. Here we demonstrate the utility of the SRG rat for both subcutaneous and orthotopic xenograft modeling. The SRG rat supports the growth of both lung and liver orthotopic cancers. In addition, the SRG rat supports the growth of orthoptic human glioblastoma multiforme in the brain. We use in vivo imaging to visualize tumor establishment and growth in subcutaneous, orthotopic, and metastatic models. Furthermore, our data show the ability of the SRG rat to support the growth of multiple different human cancer cell types subcutaneously in two different matrices, Matrigel® and VitroGel®. These data confirm that the SRG rat is an excellent host for studying human cancer. Our data demonstrate that the SRG rat has a high utility for studies using in vivo imaging, orthotopic tumor implantation, and standard subcutaneous tumor modeling. As the most immunodeficient rat commercially available, the SRG rat supports the growth of multiple human cancer types in a larger rodent strain relative to commercially available mouse models. Citation Format: Diane Begemann, Aida Javidan, Cynthia Dunn, Nicolas Johnston, R. Grace Walton, Valeriya Steffey, Ian Corbin, Niveen Fulcher, Cleusa De Oliveira, Hu Xu, Mila Uzelac, Andrew Deweyert, John A. Ronald, Susanne Schmid, Matthew O. Hebb, Fallon K. Noto. In vivo subcutaneous and orthotopic cancer xenograft modeling in the SRG immunodeficient rat [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 42.

Abstract 1752: Darolutamide reverses osimertinib resistance in non-small cell lung cancer

Abstract Targeted therapies, such as EGFR inhibitors, have improved survival for patients with non-small cell lung cancer (NSCLC). Overall cure rates remain low due to eventual development of resistance. Osimertinib is a third-generation EGFR inhibitor that overcomes T790M-mediated resistance to earlier generation EGFRi. However, resistance to osimertinib has also emerged through mechanisms that are EGFR-dependent and EGFR-independent. To uncover novel strategies to overcome osimertinib resistance, we explored androgen receptor (AR) antagonists in combination with osimertinib as recent studies have demonstrated AR inhibition enhanced TKI-targeted therapy. For these experiments, we utilized lab-derived osimertinib-resistant PC9-AR and HCC827-AR NSCLC cell lines and matching parental lines in both in vitro and in vivo model systems. Clonogenic assays revealed that darolutamide in combination with osimertinib synergistically suppressed clonal growth in resistant cell lines. Similar results were observed with the combination of enzalutamide and osimertinib. Given this result, we next wanted to establish whether the promising in vitro activity of this combination translates into antitumor efficacy in vivo. Mice bearing either HCC827AR or PC9AR xenografts were treated orally daily with both drugs. At the doses administered, neither drug alone had antitumor activity. However, the combination significantly delayed tumor growth by more than 60%. We confirmed this ability to reverse resistance as a class effect using a different AR antagonist, enzalutamide in PC9AR-bearing mice. Significant tumor growth inhibition was observed with the combination of enzalutamide and osimertinib. To investigate the mechanism by which this combination may reverse osimertinib-resistance, RNA-seq studies were performed following drug exposure in vitro using parental and resistant cell lines under various experimental conditions. Comparison of gene expression profiles between parental and resistant cell lines under different therapeutic pressure and pathway enrichment analysis revealed important differentially expressed genes pointing plausible activated pathways that underline osimertinib resistance. AR antagonists have the potential to reverse EGFR inhibitor resistance in treatment of EGFR-mutant NSCLC. Citation Format: John C. Schmitz, Guojing Zhang, Zhentao Liu, Yufei Huang, Gabriel Sica, Shi-Yong Sun, Taofeek K. Owonikoko. Darolutamide reverses osimertinib resistance in non-small cell lung 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 1752.

Abstract 4847: Investigating the transcriptional regulation by estrogen-related receptor alpha (ERRα) under different metabolic conditions

Abstract Estrogen-related receptors (ERRs) are orphan nuclear receptors identified based on their high sequence similarity to estrogen receptors (ERs), but ERRs do not have a known endogenous ligand. ERRs play a primary role in regulating the transcription of genes involved in mitochondrial and lipid metabolism and are abundantly expressed across tissues. Because of ERR’s role in metabolism, it is suggested that they may also play a role in tumor metabolism, where dysfunction in lipid metabolism promotes tumor cell growth. The ERR subfamily is comprised of three isoforms: ERRα, ERRβ, and ERRγ. Studies targeting the ERR isoforms found that the absence of ERRα presents obesity and insulin resistance with an increase in bone mass, deletion of ERRβ causes placental abnormalities and embryonic lethality, and deletion of ERRγ leads to mitochondrial dysfunction. Together, these studies strongly suggest that ERRs, particularly ERR α and γ, function primarily as metabolic regulators, with ERRα being the predominant isoform expressed in the liver. In addition, the mechanisms leading to lipid accumulation vary under different feeding conditions. Previous studies from our lab showed that PTEN and PI3K/AKT signaling regulates ERRa expression and its function. Furthermore, we demonstrated that inhibiting ERRα blocks liver steatosis and steatohepatitis developed in a mouse model where loss of tumor suppressor PTEN drives both steatosis and cancer development. In addition, we found that ERR-PA, a small molecule inhibitor for ERR, attenuated cancer cell growth and proliferation in both mouse hepatocytes and human cancer cell lines. Here, we report transcriptome network regulated by ERRα under different metabolic conditions and further explored its regulation by the PI3K/AKT pathway. A better understanding of the role ERRα plays in physiology will allow further development of ERR-PA as a potential therapy for liver steatohepatitis, which progresses to end stage cirrhosis and ultimately liver cancer. Citation Format: Brittney A. Hua, Chien-yu Chen, Yang Li, Lina He, Bangyan Stiles, Ielyzaveta Slarve. Investigating the transcriptional regulation by estrogen-related receptor alpha (ERRα) under different metabolic conditions. [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 4847.