Xenografts In Athymic Mice Research Articles

Abstract PO3-14-08: Metaplastic Breast Cancer: Whole Genome Sequencing Analysis and Emerging Therapeutic Targets

Abstract Background: Metaplastic breast cancer (MpBC) is a rare histologic subtype of triple negative breast cancer (TNBC) with an aggressive behavior and poor outcome. Previous studies by whole-exome and targeted sequencing showed that MpBCs display a dominant mutational signature 3, associated to homologous recombination (HR) deficiency (HRD). Tumors displaying HRD are associated with response to therapies causing double strand breaks. Nonetheless, MpBCs consistently display significantly lower rates of response to neoadjuvant chemotherapy than non-metaplastic TNBCs and effective therapeutic alternatives are an unmet clinical need. Here we sought to assess the HR features of MpBCs using whole-genome sequencing (WGS), and to evaluate the expression in MpBCs of antigens that are targets of novel antibody drug conjugates (ADCs) in common forms of breast cancer. In addition, we sought to assess the efficacy of ADCs and drugs targeting the DNA damage response (DDR) using preclinical MpBC models. Materials and Methods: We subjected 25 primary MpBC to WGS. HRDetect, the ‘gold standard’ tool for assessment of HRD, was utilized. Mutational signatures were inferred using Signal. 31 MpBCs were subjected to RNA-sequencing to assess the gene expression levels ADC targets compared to non-MpBCs from The Cancer Genome Atlas (TCGA). Efficiency of ADCs and DDR drugs was evaluated using the MpBC cell models HCC1806, BT549 and Hs578T in vitroand/or using HCC1806 xenografts in athymic mice in vivo. Results: Although the majority of MpBCs (13/25) displayed a dominant SBS mutational signature 3, only a small subset of cases (3/25) were classified as HRD by HRDetect. These three bona fide HRD cases were found to harbor bi-allelic inactivation of BRCA1 (n=2) or RAD51C (n=1), and displayed various genomic features of HRD, including deletions with microhomology, enrichment in tandem duplications, rearrangement signatures 3 and 5, and high large scale state transitions (LST) and telomeric allelic imbalance (NtAI) scores. Most MpBCs (22/25), however, were not classified as HRD by HRDetect displaying an incomplete HRD phenotype, with only partial genomic features of HRD. 52% of MpBCs (13/25) displayed APOBEC mutagenesis exposure, which was present in the three bona fide HRD MpBCs. RNA-sequencing analyses revealed detectable ERBB2 and TROP2 expression in all MpBCs interrogated, although at lower levels than in non-MpBCs from TCGA. The HCC1806, BT549 and Hs578T MpBC cell models were found to be sensitive to Sacituzumab Govitecan and Trastuzumab Deruxtecan (T-DXd) in cell viability and colony formation assays, and to T-DXd in vivo. These MpBC cell models were also found to be sensitive to DDR compounds, such as ATR, WEE1 and PKMYT1 inhibitors in vitro, alone and in combination with T-DXd or Sacituzumab Govitecan. Conclusions: Most MpBCs display an incomplete HRD genomic scar, except for those cases harboring bi-allelic inactivation of HRD genes. APOBEC mutagenesis is operative in MpBCs and co-exists with HRD. MpBCs express ERBB2 and TROP2, and MpBC cell models are sensitive to T-DXd and Sacituzumab govitecan, as well as DDR compounds, targeting the incomplete HRD phenotype of MpBCs, alone and in combination. Taken together, these findings warrant further studies in patients with MpBCs testing the therapeutic efficacy of agents targeting the incomplete HRD phenotype, as well as the expression of ADC targets in these aggressive rare forms of TNBCs. Citation Format: Fresia Pareja, Andrea Gazzo, Higinio Dopeso, David Brown, Pier Selenica, Yingjie Zhu, Juan Blanco-Heredia, Laxmi Gusain, Xin Pei, Giacomo Montagna, Nour Abuhadra, Hanna Y Wen, Edi Brogi, Nadeem Riaz, Sarat Chandarlapaty, Maurizio Scaltriti, Larry Norton, Simon Powell, Jorge Reis-Filho, Britta Weigelt. Metaplastic Breast Cancer: Whole Genome Sequencing Analysis and Emerging Therapeutic Targets [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-08.

Abstract 4558: A triterpenoid derivative has strong antitumor activities in gemcitabine-resistant advanced bladder cancer models and promotes immune cell infiltration in a high grade murine syngeneic model

Abstract Bladder cancer (BC) presents a formidable challenge in treatment and patient survival, requiring particular attention to advanced stages, such as muscle-invasive stage (MIBC), due to its potential for rapid progression and substantial impact on patient morbidity and mortality. The five-year survival rate for MIBC is a meager 30%, which drops further down to 8% once tumors metastasize. Advanced bladder cancers (High grade T1) are treated with intravesical therapy with Gemcitabine and Cisplatin (G+C) combination and Stage T2 are treated neoadjuvant with G+C and cystectomy, thereby decreasing the quality of life for patients with BC. Furthermore, tumors progress beyond responding to other chemotherapy regimens such as dose-dense treatment with Methotrexate +Vinblastine + Adriamycin and Cis-platin (MVAC), all of which will further decrease the quality of life with persistent adverse toxicity. Our laboratory has developed a novel compound derived from the pentacyclic triterpenoid, Ursolic acid, termed N-methyl piperazine conjugated-Ursolic acid, or UA4, and have demonstrated its antitumor activities against both Gem-Sensitive (Gem-S) and Gem-Resistant (Gem-R) human advanced BC cell lines, T24 and 5637. We tested the combination of Gem, UA4 and Gem + UA4 in a murine non-MIBC model (MB49) to investigate the role of immune cell infiltration during tumor development and treatment response. UA4 treatment was equally effective in chemo-sensitive and chemo-resistant cell lines at dosages between 4-5µM. Previous in vitro mechanistic studies on UA4 and UA4+Gem combination treatments revealed cell death occurred through apoptosis and autophagy, along with a highly selective form of mitochondrial-mediated autophagy (mitophagy). These findings were confirmed with transmission electron microscopy ultrastructural analysis, cell cycle phase analyses, excess, changes in mitochondrial membrane potential and reactive oxygen species measurements. Gem-S and Gem-R xenografts in athymic mice further supported UA4’s efficacy in vivo, demonstrating a significant reduction in both tumor weight and volume following oral gavage (100mg/kg), with mice exhibiting no signs of systemic toxicity. Combination treatment with UA4 and Gem synergistically increased antiproliferative activity. MB49 syngeneic mice model further supported UA4 as an effective therapy, and immunofluorescence assays with immune-related markers, suggested an anti-inflammatory effect in vivo. Overall, we demonstrate UA4 has a potential to be used as an efficacious, non-toxic compound for further adjuvant use in clinical settings. Citation Format: Juliette Rose Seremak, Kunj Bihari Gupta, Siva S. Panda, Bal L. Lokeshwar. A triterpenoid derivative has strong antitumor activities in gemcitabine-resistant advanced bladder cancer models and promotes immune cell infiltration in a high grade murine syngeneic model [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 4558.

Novel Anti-Melanoma Compounds Are Efficacious in A375 Cell Line Xenograft Melanoma Model in Nude Mice.

Despite the successes of immunotherapy, melanoma remains one of the deadliest cancers, therefore, the need for innovation remains high. We previously reported anti-melanoma compounds that work by downregulating spliceosomal proteins hnRNPH1 and H2. In a separate study, we reported that these compounds were non-toxic to Balb/C mice at 50 mg/kg suggesting their utility in in vivo studies. In the present study, we aimed to assess the efficacy of these compounds by testing them in A375 cell-line xenograft in nude athymic mice. Animals were randomized into four groups (n = 12/group): 10 mg/kg vemurafenib, and 25 mg/kg 2155-14 and 2155-18 thrice a week for 15 days along with a control group. The results revealed that both 2155-14 and 2155-18 significantly decreased the growth of A375 tumors, which was comparable to vemurafenib. These results were confirmed by tumor volume, weight, and histopathological examination. In conclusion, these results demonstrate the therapeutic potential of targeting spliceosomal proteins hnRNPH1 and H2.

Sodium selenite inhibits proliferation and metastasis through ROS‐mediated NF‐κB signaling in renal cell carcinoma

BackgroundSodium selenite (SSE) has been reported to exert anti-tumor effects in several cancer cells. However, the underlying mechanisms in renal cancer are yet to be elucidated. The effects of SSE on the proliferation, metastasis, and apoptosis of renal cancer cells, as well as its mechanism, were investigated in this study.MethodsACHN and 786-O renal cancer cells were treated with different concentrations of SSE, MTT, and colony formation assays were used to detect the proliferation ability of cells. The migration of cells was detected using scratch-wound-healing and transwell-migration assays. The effect of SSE on apoptosis was assessed by AnnexinV-FITC/PI double staining. Besides, Western blotting was employed to detect the protein-expression level and elucidate the underlying pathways. We also made subcutaneous xenografts in athymic mice to verify the effect of SSE on tumor growth in vivo.ResultsOur results demonstrated that treatment with SSE resulted in significant inhibition of cell proliferation and migration. Flow cytometry and Western blot confirmed that SSE induced apoptosis via the endogenous apoptotic pathway. We also confirmed that SSE treatment causes an increase in intracellular reactive oxygen species (ROS) levels, resulting in the inhibition of nuclear transcription factor-κB (NF-κB) signaling. Modulation of the ROS level by the chemical inhibitor N-acetyl-cysteine reversed the effect of SSE on cells. Similarly, subcutaneous xenografts in athymic mice models showed that SSE inhibits tumor growth in vivo. ConclusionThese results indicate that SSE inhibits proliferation and migration and induces apoptosis via ROS mediated inhibition of NF-κB signaling in renal cancer cells.

The recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis shows potent efficacy against pancreatic cancer

KRAS mutation and NF-κB both play crucial role in pancreatic cancer; in addition, defensin, the peptide mediator in innate immunity, can inhibit NF-κB. Assuming a strategy that targets both NF-κB and concomitantly the mutated KRAS indirectly via intensive macropinocytosis, we designed and generated a recombinant protein DF2-HSA which consists of two molecules of human beta-defensin 2 (HBD2) and a moiety of human serum albumin (HSA). As shown, the recombinant protein DF2-HSA markedly down-regulated NF-κB in both KRAS mutant MIA PaCa-2 cells and wild type BxPC-3 cells. Determined by confocal microscopy, the uptake of DF2-HSA in MIA PaCa-2 cells was more intense than that in BxPC-3 cells. The uptake was blocked by the specific inhibitor EIPA, indicating that DF2-HSA internalized via macropinocytosis. DF2-HSA displayed more potent cytotoxicity to cancer cells than HBD2. DF2-HSA induced apoptosis in cancer cells. Notably, DF2-HSA inhibited tumor cell spheroid formation, an effect comparable to that of salinomycin. DF2-HSA inhibited tumor cell migration and invasion. As detected with scanning electron microscopy, DF2-HSA strongly depleted filopodia on cell surface; and salinomycin induced similar changes. By in vivo imaging, DF2-HSA displayed intense tumor-site accumulation and lasting retention for over 14 days; however, HBD2 showed much less tumor-site accumulation and a shorter retention time for only 24 h. DF2-HSA suppressed the growth of pancreatic cancer MIA PaCa-2 xenograft in athymic mice; and its combination with gemcitabine achieved higher antitumor efficacy. In summary, the recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis is highly effective against pancreatic cancer.

A recombinant scFv antibody-based fusion protein that targets EGFR associated with IMPDH2 downregulation and its drug conjugate show therapeutic efficacy against esophageal cancer

The present study aimed to evaluate the anti-tumor efficacy of the epidermal growth factor receptor (EGFR)-targeting recombinant fusion protein Fv-LDP-D3 and its antibody-drug conjugate Fv-LDP-D3-AE against esophageal cancer. Fv-LDP-D3, consisting of the fragment variable (Fv) of an anti-EGFR antibody, the apoprotein of lidamycin (LDP), and the third domain of human serum albumin (D3), exhibited a high binding affinity for EGFR-overexpressing esophageal cancer cells, inhibited EGFR phosphorylation and down-regulated inosine monophosphate dehydrogenase type II (IMPDH2) expression. Fv-LDP-D3 was taken up by cancer cells through intensive macropinocytosis; it inhibited the proliferation and induced the apoptosis of esophageal cancer cells. In vivo imaging revealed that Fv-LDP-D3 displayed specific tumor-site accumulation and a long-lasting retention over a 26-day period. Furthermore, Fv-LDP-D3-AE, a pertinent antibody-drug conjugate prepared by integrating the enediyne chromophore of lidamycin into the Fv-LDP-D3 molecule, displayed highly potent cytotoxicity, inhibited migration and invasion, induced apoptosis and DNA damage, arrested cells at G2/M phase, and caused mitochondrial damage in esophageal cancer cells. More importantly, both of Fv-LDP-D3 and Fv-LDP-D3-AE markedly inhibited the growth of esophageal cancer xenografts in athymic mice at well tolerated doses. The present results indicate that Fv-LDP-D3, and Fv-LDP-D3-AE exert prominent antitumor efficacy associated with targeting EGFR, suggesting their potential as promising candidates for targeted therapy against esophageal cancer.

Abstract P5-08-18: Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate transport and glutaminase

Abstract OBJECTIVES: Upregulated glutamine metabolism is an important metabolic signature for aggressive and treatment-resistant cancers, including many triple negative breast cancers (TNBCs). Glutamate (Glu) produced from glutamine (Gln) via mitochondrial glutaminase (GLS) feeds into the TCA cycle to provide energy and biosynthetic precursors for tumor growth. Recent studies from our lab and others have revealed that Glu also plays an important role in maintaining redox homeostasis which is essential for survival of cancer cells. Glu is a component amino acid of glutathione, the most abundant cellular antioxidant. Furthermore, via the xCT antiporter (SLC7A11), Glu is exported for import of cystine, a rate-limiting step for glutathione synthesis. We assessed the effect of metabolic inhibition of GLS and/or xCT pathways on cellular reactive oxygen species (ROS) level and induction of apoptosis. To track cytosolic Glu transport cross plasma membrane in vivo, we tested (4S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), a non-metabolized structural analog of Glu. We hypothesize that mitochondrial Glu supply is necessary for maintaining cellular redox homeostasis and [18F]FSPG PET can serve as a tool to track Glu transport in glutaminolytic TNBC and guide treatment targeting GLS and redox balance leading to unmitigated oxidative stress and cell death. METHODS: A human glutaminolytic TNBC cell line (HCC1806) and a non-glutaminolytic estrogen-receptor positive cell line (MCF-7) were used to generate xenografts in athymic mice. Cells were exposed to culture media containing GLS inhibitor CB839 (1 µM), xCT inhibitor Erastin (ERA, 3 µM) and/or chemotherapy doxorubicin (DOX, 0.2 µM) for 24 h. ROS were measured by DHE and CellROX Green assays; early apoptosis was estimated by flow cytometry analysis of cells double-stained with Annexin V-FITC and TOR-PR-3. In vivo dynamic [18F]FSPG PET imaging (1 h) was performed on HCC1806 and MCF-7 xenograft models. Lipid peroxidation induced by treatments was examined using C11-Bodipy, a fluorescent sensor. RESULTS: ROS level in HCC1806 cells increased robustly with CB839 (20% over untreated cells), ERA (30%), or ERA plus CB839 (50%), whereas ROS responses in MCF-7 cells were more blunted. Compared to untreated cells, DOX treatment increased ROS level in HCC1806 cells by 40% whereas DOX combined with CB839 plus ERA led to a remarkable increase of cellular ROS by more than 200%. Combined treatment also led to 31% of cells undergoing early apoptosis, compared to 3% in DOX only and 2% in untreated cells. Time-activity curves (TACs) from [18F]FSPG PET of these xenograft models were fit by a reversible one-compartment model in keeping with the known biology of the tracer. The tumor-to-blood ratio (T/B) averaged from the last 6 points (30 min) of the TACs reveal a large difference between HCC1806 vs. MCF-7 by a factor > 2. C11-Bodipy signal in HCC1806 cells was not changed after CB839 but increased substantially after ERA only (45%) or DOX only (68%) treatment and after combined treatment of CB839, ERA and DOX (111%). DISCUSSIONS & CONCLUSION: Our preliminary data support the efficacy of dual targeting of glutaminolysis and Glu transport for sensitizing glutaminolytic TNBC to cytotoxic chemotherapy. T/B of [18F]FSPG PET appears to be distinct for glutaminolytic versus non-glutaminolytic breast cancers. Future work will focus on developing [18F]FSPG PET, complementing our prior work with a Gln analog as a measure of Gln metabolism. The two PET tracers may guide treatment targeting both Gln and Glu usage to overcome chemoresistant TNBC. SUPPORT: R01CA211337 and Komen SAC130060. Reference: 1. Gross MI, et al. Mol Cancer Ther 2014;13(4):890-901. 2. Viswanath V, et al. J Nucl Med 2020. 3. Zhou R, et al. Cancer Res 2017;77(6):1476-84. Citation Format: Hoon Choi, Hsiaoju Lee, Austin Pantel, Christopher Hensley, David Mankoff, Rong Zhou. Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate transport and glutaminase [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-18.

IMB5036, a novel pyridazinone compound, inhibits hepatocellular carcinoma growth and metastasis.

Background Hepatocellular carcinoma (HCC) is one of the most common cancers with a high mortality rate due to metastasis and relapse. Purpose Here, we reported a small-molecule pyridazinone compound, designated as IMB5036. Its antitumor activity against HCC and underlying mechanism were studied. Methods In vitro cytotoxicity, apoptosis, DNA breaks, and cell motility assays were performed. Protein expression was analyzed by Western blot and microarray analysis. A xenograft tumor model in athymic mice was used to evaluate the antitumor activity. Results IMB5036 displayed potent cytotoxicity against various HCC cell lines. It caused double DNA breakages and induced cell death via apoptosis. It also significantly inhibited the motility of HCC cells. Western blot showed that IMB5036 induced the up-regulation of E-cadherin, while down-regulation of N-cadherin. The gene expression profile analysis and Western blot assay revealed that IMB5036 down-regulated the expression of Tau protein. Analysis of the TCGA dataset revealed that high expression of Tau decreased the survival rate of HCC patients. In vivo experiments proved that IMB5036 significantly inhibited the growth of HCC xenografts in athymic mice. Conclusions These results collectively demonstrate IMB5036 can be a promising therapeutic candidate for patients with HCC.

Triptolide Downregulates the Expression of NRF2 Target Genes by Increasing Cytoplasmic Localization of NRF2 in A549 Cells.

We have identified triptolide as a novel NRF2 inhibitor, which significantly attenuates ARE-luciferase activity at nanomolar concentrations. Triptolide did not affect the level of NRF2, but significantly inhibited the expression of NRF2 target genes in A549 cells. We found that NRF2 possesses a previously unrecognized NES in the Neh2 domain, and that triptolide promotes an interaction between NRF2 and CRM1. Triptolide also decreased nuclear accumulation of NRF2, suggesting that it promotes nuclear export of NRF2. In addition, we show that triptolide decreased the expression of NRF2 target genes and increased intracellular oxidative stress, suppressing invasion and promoting cisplatin-induced apoptosis in A549 cells. Finally, oral administration of triptolide suppressed the growth of A549 xenografts in athymic mice by decreasing the expression of NRF2 target genes and promoting oxidative damages via the nuclear export of NRF2 and CRM1 in vivo. To the best of our knowledge, triptolide is the first type of compound to inhibit NRF2 by increasing cytoplasmic localization of NRF2.

Nano-Targeting of Thyrointegrin αvβ3 Receptor in Solid Tumors and Impact on Radiosensitization.

Tetraiodothyroacetic acid is a ligand of thyrointegrin αvβ3, a protein that is highly expressed in various solid tumors and surrounding neovascular regions. Its nano derivative, Nano-diamino-tetrac (NDAT), has anticancer properties in preclinical models, enhances radiosensitivity, and inhibits cancer cell growth in vitro after X-ray irradiation. Using a novel experimental system developed to deliver accurate radiation dose to tumors under sterile conditions, this study establishes NDAT's radiosensitizing effect in SUIT-2 pancreatic cancer and H1299 non-small cell lung carcinoma xenografts in athymic mice for tumor-targeted radiation. In this work, low-melting-point Lipowitz alloy was used to shield normal organs and allow accurate tumor-targeted irradiation. Over a three-week period, mice with SUIT-2 xenografts received daily NDAT treatment at different doses (0, 1, 3, or 10 mg/kg body weight) and tumor-targeted irradiation (1 or 5 Gy). Validation was performed with a test dose of 30 Gy to mice bearing SUIT-2 xenografts and resulted in more than 80% reduction in tumor weight, compared to nonirradiated tumor weight. The results of this work showed that NDAT had a radiosensitizing effect in a dose-dependent manner in decreasing tumor growth and viability. An enhanced anticancer effect of NDAT (1 mg/kg body weight) was observed in mice with H1299 xenografts receiving 5 Gy tumor-targeted irradiation, indicated by decreased tumor weight and increased necrosis, compared to nonirradiated tumors. This technique demonstrated accurate tumor-targeted irradiation with new shielding methodology, and combined with thyrointegrin antagonist NDAT treatment, showed anticancer efficacy in pancreatic cancer and non-small cell lung carcinoma.

Development of a novel multi-functional integrated bioconjugate effectively targeting K-Ras mutant pancreatic cancer

Folate receptor (FR) overexpression occurs in a variety of cancers, including pancreatic cancer. In addition, enhanced macropinocytosis exists in K-Ras mutant pancreatic cancer. Furthermore, the occurrence of intensive desmoplasia causes a hypoxic microenvironment in pancreatic cancer. In this study, a novel FR-directed, macropinocytosis-enhanced, and highly cytotoxic bioconjugate folate (F)-human serum albumin (HSA)-apoprotein of lidamycin (LDP)-active enediyne (AE) derived from lidamycin was designed and prepared. F-HSA-LDP-AE consisted of four moieties: F, HSA, LDP, and AE. F-HSA-LDP presented high binding efficiency with the FR and pancreatic cancer cells. Its uptake in wild-type cells was more extensive than in K-Ras mutant-type cells. By in vivo optical imaging, F-HSA-LDP displayed prominent tumor-specific biodistribution in pancreatic cancer xenograft-bearing mice, showing clear and lasting tumor localization for 360 h. In the MTT assay, F-HSA-LDP-AE demonstrated potent cytotoxicity in three types of pancreatic cancer cell lines. It also induced apoptosis and caused G2/M cell cycle arrest. F-HSA-LDP-AE markedly suppressed the tumor growth of AsPc-1 pancreatic cancer xenografts in athymic mice. At well-tolerated doses of 0.5 and 1 mg/kg, (i.v., twice), the inhibition rates were 91.2% and 94.8%, respectively (P<0.01). The results of this study indicate that the F-HSA-LDP multi-functional bioconjugate might be effective for treating K-Ras mutant pancreatic cancer.

Abstract 738: Targeting metabolic vulnerabilities of endocrine resistant breast cancers using XPO1 and NAMPT inhibition

Abstract Introduction: Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing the potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis with a 5-year relative overall survival rate of just 24%. Previously, we described metabolic changes in metastatic breast cancer cells that are driven by single therapies and the metastatic organ microenvironment. Combining selinexor (SEL), a small molecule inhibitor of exportin 1 (XPO1) approved by the FDA for treatment of some hematologic malignancies, with the current therapies used for advanced breast cancers (e.g. tamoxifen) decreased metabolic adaptations in response to individual drug treatments and prevented colony formation in hydrogels and tumor growth in xenograft models. Since we observed a reliance on NAD+ metabolism in cancer cells that are treated with single therapies, in the current study we investigated co-targeting metabolic alterations using SEL and the NAMPT inhibitor KPT-9274. Methods: We established 3D cell culture systems in novel hydrogels derived from decellularized bone, lung, and liver tissues - the major sites of metastasis of ER+ breast cancers, and compared with standard Matrigel culture. Endocrine responsive (MCF7 and T47D) and resistant (MCF7 and T47D cells with ESR1 Y537S or D538G mutations) breast cancer cell lines were treated with SEL or KPT-9274. Tumorigenicity was assessed using in vitro cell viability and colony formation assays and in vivo breast cancer cell line xenografts in athymic mice. Metabolic changes were assessed using whole metabolite profiling by GC-MS and a Seahorse XF analyzer. Results: Combining an FDA-approved XPO1 small molecule inhibitor, Selinexor (SEL) with the NAMPT inhibitors decreased metabolic adaptations in response to individual drug treatments, and prevented colony formation in hydrogels and tumor growth in xenograft models. Conclusions: Targeting drug and metastatic-site induced adaptations to regenerate new vulnerabilities in endocrine-resistant breast tumors are novel. Given the need for better strategies for improving therapy response of metastatic ER+ tumors, our findings show uncovering the role XPO1-NAMPT crosstalk plays in metastatic breast cancer could lead to new combined therapies that reduce mortality. Citation Format: Qianying Zuo, Ayca Mogol, Ozan Imir, Yosef Landesman, Zeynep Madak Erdogan. Targeting metabolic vulnerabilities of endocrine resistant breast cancers using XPO1 and NAMPT inhibition [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 738.

NR4A1 Ligands as Potent Inhibitors of Breast Cancer Cell and Tumor Growth.

Simple SummaryBis-indole derived (CDIMs) bind the orphan nuclear receptor 4A1 (NR4A1) and inhibit NR4A1-regulated cancer cell and tumor growth. In this study a subset of 3,5-disubstituted phenyl CDIM compounds that bound NR4A1 were investigated in a breast cancer model. All of these analogs were potent inhibitors of breast tumor growth in a xenograft model using MDA-MB-231 cells at doses ≤ 1 mg/kg/d.Nuclear receptor 4A1 (NR4A1, Nur77, TR3) is more highly expressed in breast and solid tumors compared to non-tumor tissues and is a pro-oncogenic factor in solid tumor-derived cancers. NR4A1 regulates cancer cell growth, survival, migration, and invasion, and bis-indole-derived compounds (CDIMs) that bind NR4A1 act as antagonists and inhibit tumor growth. Preliminary structure-binding studies identified 1,1-bis(3′-indolyl)-1-(3,5-disubstitutedphenyl)methane analogs as NR4A1 ligands with low KD values; we further investigated the anticancer activity of the four most active analogs (KD’s ≤ 3.1 µM) in breast cancer cells and in athymic mouse xenograft models. The treatment of MDA-MB-231 and SKBR3 breast cancer cells with the 3-bromo-5-methoxy, 3-chloro-5-trifluoromethoxy, 3-chloro-5-trifluoromethyl, and 3-bromo-5-trifluoromethoxy phenyl-substituted analogs decreased cell growth and the expression of epidermal of growth factor receptor (EGFR), hepatocyte growth factor receptor (cMET), and PD-L1 as well as inhibited mTOR phosphorylation. In addition, all four compounds inhibited tumor growth in athymic nude mice bearing MDA-MB-231 cells (orthotopic) at a dose of 1 mg/kg/d, which was not accompanied by changes in body weight. These 3,5-disubstituted analogs were the most potent CDIM/NR4A1 ligands reported and are being further developed for clinical applications.

Abstract 5675: Metastatic organ-specific changes in ERα and XPO1 signaling and metabolic adaptations dictate the therapy responses in endocrine resistant breast cancers

Abstract Introduction: Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing a potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis with a 5-year relative overall survival rate of just 24%. The tumor microenvironment (TME) plays a key role in the behavior of cancer cells and their response to endocrine therapies. However, there is a critical need for relevant 3D models of different metastatic sites to use for studying the molecular mechanisms driving resistance in these tumor phenotypes. Methods: We established 3D cell culture systems in novel hydrogels derived from decellularized bone, lung and liver tissues - the major sites of metastasis of ER+ tumors, and compared with standard Matrigel culture. Endocrine responsive (MCF7 and T47D) and resistant (MCF7 and T47D cells with ESR1 Y537S or D538G mutations) breast cancer cell lines were treated with fulvestrant (FUL), tamoxifen (TAM) and palbociclib (PAL). Tumorigenicity was assessed using in vitro cell viability and colony formation assays and in vivo breast cancer cell line xenografts in athymic mice. ERα cistromes and transcriptomes were assessed using ChIP-Seq and RNA-Seq analysis. Metabolic changes were assessed using whole metabolite profiling by GC-MS, a Seahorse XF analyzer, and flux analysis with labeled glucose and glutamine. Results: We observed metastatic site-specific responses to each drug. For example, TAM blocked colony formation in Matrigel and lung hydrogel but increased colony formation in bone and liver hydrogels. FUL acted as an agonist and increased colony number and size in all three-tissue matrices, but not in Matrigel. PAL increased colony size and number in Matrigel and liver hydrogel but not in bone or lung hydrogel. Genome-wide ERα recruitment to chromatin was reduced in liver hydrogels, consistent with decreased clinical response to endocrine agents in patients with liver metastasis. We found that treatment with individual drugs activated novel metabolic pathways to increase survival in different metastatic sites. Using RNA-Seq analysis, we identified increased expression of nuclear export pathway components (XPO1, KPNA2, etc.) in various metastatic sites. Combining an FDA-approved XPO1 small molecule inhibitor, Selinexor (SEL) with the current therapies used for advanced breast cancers (FUL, TAM or PAL) decreased metabolic adaptations in response to individual drug treatments and prevented colony formation in hydrogels and tumor growth in xenograft models. Conclusions: Further developing and characterizing 3D metastatic site models will enable us to elucidate the underlying mechanisms of metastasis and resistance, and target these mechanisms with novel drug combinations in already metastasized patients. Targeting metastatic-site specific adaptations to regenerate new vulnerabilities in endocrine-resistant breast tumors is novel. Given the need for better strategies for improving therapy response of metastatic ER+ tumors, our findings show uncovering the role ERα-XPO1 crosstalk plays in metastatic breast cancer could lead to new combined therapies that reduce mortality. Citation Format: Qianying Zuo, Eylem Kulkoyluoglu Cotul, Ashlie Santaliz-Casiano, Ozan Berk Imir, Elif Tunc, Chengjian Mao, David J. Shapiro, Ben Ho Park, Yosef Landesman, Zeynep Madak-Erdogan. Metastatic organ-specific changes in ERα and XPO1 signaling and metabolic adaptations dictate the therapy responses in endocrine resistant breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5675.

Abstract 4172: Oral administration of novel compound targeting triple negative breast cancer (TNBC) through the inhibition of SERCA2

Abstract Background: Cancer survival is dependent on the increase of protein synthesis for cell migration, proliferation and metastasis. Cancer adaptive mechanisms include upregulation of endoplasmic reticulum (ER) activities to accommodate increases in misfolded proteins. Sarcoendoplasmic reticulum calcium ATPase (SERCA) are important regulators of calcium found in the endoplasmic reticulum, where inhibition can lead to protein misfolding. Since breast cancer cells express higher level of SERCA2, its overexpression maybe a target for drug development. Method: Using an in vitro screening method to guide our development, we have developed a pharmacophore and made structural changes to enhance anti-cancer activity while reducing of COX-2 inhibition. In this effort, we have identified C1, a coxib analogue with improved antitumor activities with minimal COX-2 inhibitory properties. We have further tested C1 in nu/nu athymic mice xenograft with human breast cancer cell line (MDA MB 231). Tumor volume and animal weights were measured three times a week for 28 days. At the end of the study, tumor volume was correlated with molecular markers using RT-PCR and IHC staining. Results: Our screening identified C1 as a promising anti-cancer compound in vitro and affirmed in xenograft triple negative breast cancer (TNBC) animal model. C1 has EC50 of &amp;lt;20 µM which was more potent than Di-methyl Celecoxib (DMC) and Celecoxib (CBX) with EC50 of ~30 µM and ~40 µM, respectively. Molecular dissection using western blot showed that C1 inhibited SERCA2 in both a time- and exposure-dependent manner. SERCA2 inhibition correlated with blocking the maturation of Notch 1. In vivo studies using oral administration of C1 showed optimal antitumor activity between 25-50 mg/kg/day, which resulted in &amp;gt;85% tumor reduction when compared to vehicle after 28 days of treatment. The safety of C1 was assessed for 48 days at 100 mg/kg/day, where no detectable biochemical, hematologic or histological toxicities were detected. RT-PCR analysis of residual tumor correlated with cell death indicated through the activation of CHOP and DR5 marker. Finally, C1 treatment also decreased angiogenesis markers VEGF-α and IL-8, corresponding to reduction in microvessel formation. Conclusion: C1 is a bioavailable coxib analogue with minimal COX-2 inhibition while possessing broad spectrum antitumor activity, including TNBC. Treatment with C1 leads to dosage dependent antitumor properties as a consequence of specific SERCA2 inhibition. In dosage escalation studies up to 100 mg/kg/day, no detectable biochemical and histological toxicities were detected. With these properties, C1 may be an effective component in the treatment of TNBCs, where effective therapy(ies) is much needed. Citation Format: Tracey J. Lin1, Julie Yoo2, Priyal Dave, Andrew Mead, Kabir Ahluwalia, Tiange Dong, Eugene Zhou, Hua Pei, Isaac Asante, Nicos Petasis, Stan Louie Citation Format: Tracey Lin, Julie Yoo, Priyal Dave, Andrew Mead, Kabir Ahluwalia, Tiange Dong, Eugene Zhou, Hua Pei, Isaac Asante, Nicos Petasis, Stan Louie. Oral administration of novel compound targeting triple negative breast cancer (TNBC) through the inhibition of SERCA2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4172.

Abstract 2450: Methyl CpG Binding Protein 2 suppresses Myc targeting miRNAs to promote context dependent tumor proliferation

Abstract Background: Epigenetic reader proteins maintain an imbalance between differentiation and self-renewal in cancer. Genetic alterations in the DNA methylation machinery (e.g., TET, DNMTs) and chromatin remodelers (e.g., KDMs, EZH1/2) are a hallmark of cancer. Methyl CpG binding domain protein 2 (MBD2) is an epigenetic reader protein which modulates regional gene transcription by recruiting co-repressor complexes to sites of CpG methylation. Our goal is to develop a novel class of anti-neoplastic epigenetic therapies targeting MBD2 to selectively reprogram tumor cells towards a terminally differentiated state and sensitize them to chemotherapy, radiation, and immunotherapy. Methods: To explore conserved mediators of MBD2 function in cancer, we used MBD2 targeting shRNA lentivirus to stably knockdown MBD2 in prostate cancer (PC3, DU145, LnCap), leukemia (SigM5, TET2WTK562 or TET2KOK562) and triple negative breast cancer (MDA-MB231-nanog-GFP) cell lines. Inhibition of MBD2 expression was confirmed by qRT-PCR and Western Blot. Proliferative potential was determined by cell counting and clonogenic potential was determined by methylcellulose-based colony forming assays. RNAseq analysis was performed on prostate cancer and leukemia cell lines. miRNA expression was analyzed using miScript miRNA PCR (Qiagen). In vivo tumor initiation capacity was analyzed using orthotropic and heterotopic xenografts in athymic NSG mice. Results: Our results show that MBD2 is required for the proliferation of triple negative breast cancer (TNBC), prostate cancer (PCa), and TET mutant leukemias (TML), in vitro. The functional mediators of MBD2's growth promoting effects were tissue/tumor context dependent. In leukemias, MBD2's growth promoting and tumor initiating effects were most pronounced in TET2 null cells (which accumulate 5mC). In TNBC, nanog-GFP reporter positive cells were more sensitive to MBD2 knockdown than reporter negative cells. In PCa and TNBC, RNAseq analysis revealed that knockdown of MBD2 led to downregulation of Myc pathway genes and increased the expression of Myc targeting microRNAs, miR33-5, miR34a, miR148a and miR363. Western blot analysis confirmed that MBD2 knockdown coordinately downregulated cMyc expression and activated p27 expression. We further demonstrated that inhibition of MBD2 diminished the tumor initiating capacity of TNBC and PCa in xenograft models and the in vivo engraftment rate of patient derived TET-/- AML. Conclusions: MBD2 knockdown diminished the proliferative capacity of PCa, TNBC and primary TET2 mutant leukemia cells in a genetic (TET2) and phenotypic (nanog+ stem/progenitor) context dependent manner. Delayed peak effect and altered differentiation markers after MBD2 inhibition suggest epigenetic reprogramming as the mechanism of growth suppression. MBD2 targets miRNA's upstream of cMyc in PCa and TNBC. MBD2 murine knockout models are developmentally normal, suggesting an acquired function in cancer with a favorable therapeutic window for targeting. Citation Format: Aysegul Balyimez, Yihong Guan, Emily Esakov, Shinjini Ganguly, Ofer Reizes, Daniel J. Lindner, Jaroslaw Maciejewski, Babal Jha, Omar Y. Mian. Methyl CpG Binding Protein 2 suppresses Myc targeting miRNAs to promote context dependent tumor proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2450.

Ginsenoside 20(S)-protopanaxadiol induces cell death in human endometrial cancer cells via apoptosis

Background20(S)-protopanaxadiol (20(S)-PPD), one of the aglycone derivatives of major ginsenosides, has been shown to have an anticancer activity toward a variety of cancers. This study was initiated with an attempt to evaluate its anti-cancer activity toward human endometrial cancer by cell and xenograft mouse models. MethodsHuman endometrial cancer (HEC)-1A cells were incubated with different 20(S)-PPD concentrations. 20(S)-PPD cytotoxicity was evaluated using MTT assay. Apoptosis was detected using the annexin V binding assay and cell cycle analysis. Cleaved poly (ADP-ribose) polymerase (PARP) and activated caspase-9 were assessed using western blotting. HEC-1A cell tumor xenografts in athymic mice were generated by inoculating HEC-1A cells into the flank of BALB/c female mice and explored to validate 20(S)-PPD anti-endometrial cancer toxicity. Results20(S)-PPD inhibited HEC-1A cell proliferation in a dose-dependent manner with an IC50 value of 3.5 μM at 24 h. HEC-1A cells morphologically changed after 20(S)-PPD treatment, bearing resemblance to Taxol-treated cells. Annexin V-positive cell percentages were 0%, 10.8%, and 58.1% in HEC-1A cells when treated with 0, 2.5, and 5 μM of 20(S)-PPD, respectively, for 24 h. 20(S)-PPD subcutaneously injected into the HEC-1A cell xenograft-bearing mice three times a week for 17 days manifested tumor growth inhibition by as much as 18% at a dose of 80 mg/kg, which sharply contrasted to controls that showed an approximately 2.4-fold tumor volume increase. These events paralleled caspase-9 activation and PARP cleavage. Conclusion20(S)-PPD inhibits endometrial cancer cell proliferation by inducing cell death via a caspase-mediated apoptosis pathway. Therefore, the 20(S)-PPD-like ginsenosides are endowed with ample structural information that could be utilized to develop other ginsenoside-based anticancer agents.

DBDx-based drug combinations show highly potent therapeutic efficacy against human pancreatic cancer xenografts in athymic mice

ABSTRACT Previous studies have shown that DBDx, a combination consisting of dipyridamole, bestatin and dexamethasone is highly effective against several cancer xenografts in athymic mice. Here the therapeutic effects of DBDx and its combination with gemcitabine or capcitabine against human pancreatic cancer xenografts and the mechanism were studied. In vivo experiments performed in athymic mice showed that the antitumor efficacy of DBDx was much stronger than that of gemcitabine or capecitabine alone. Notably, the combination of DBDx and gemcitabine or capcitabine further enhanced the efficacy. In the case of DBDx (242 mg/kg) plus gemcitabine (100 mg/kg), tumor weight decreased about 97.7%, and tumor sizes were shrinking during the treatment. In the case of DBDx (242 mg/kg) plus capecitabine (718.7 mg/kg), tumor weight decreased about 94.9%. Moreover, DBDx and its combinations obviously prolonged theoverall survival of mice compared with gemcitabine or capcitabine alone. DBDx-based drug combination therapy showed no obvious systematic toxicity. The gene expression profile analysis showed that the genes changed by DBDx were related to immune system and tumor vasculature. The result of protein array showed that the changed proteins in the serum of treated mice were related to immune and inflammation system. These results show that DBDx-based drug combinations, a new strategy which integrates the use of low-cytotoxic drugs and cytotoxic chemotherapeutics, are highly effective regimens against human pancreatic cancer in athymic mice at well tolerated doses. DBDx-based drug combination therapy might provide new options for the treatment of pancreatic cancer.

Role of PLA2R1 and sPLA2 on Drug Release and Uptake of Liposome Nanoparticles in Prostate Cancer

Exploiting differences in tumor pathophysiology can enhance the delivery and antitumor activity of chemotherapeutics encapsulated in drug carriers. Long‐circulating nanoparticulate drug carriers, such as pegylated‐sterically‐stabilized liposomes (SSL), can stably entrap drug, alter drug disposition, improve antitumor activity and minimize toxicity. However, control of their drug‐release kinetics has limited their clinical potential. Secretory phospholipases A2 (sPLA2) are over expressed and excreted in a variety of tumors, e.g., up to 22‐fold in prostate. These enzymes degrade phospholipids preferentially and have been hypothesized as targets to control drug release from lipid‐based nanoparticles, such as liposomes. While they have shown activity in preclinical models, the clinical performance of these formulations has been limited. The goal of these studies was to gain insights into how sPLA2‐targeted liposome formulation (SPRL) alter drug release and uptake, specifically examining the role of sPLA2 and its membrane receptor (PLA2R1). Studies were performed using a metastatic‐derived human prostate adenocarcinoma cells (PC‐3) and a PLA2R1 knock‐down variant (PC‐3‐PLA2R‐KD) while varying supplementation of sPLA2 enzyme isoforms (IIA, X, V). Liposomes (SSL &amp; SPRL) were made as described previously and included DiR, a non‐exchangeable near‐infrared fluorescent dye. In vitro uptake and deposition of various formulations was determined using flow cytometry and fluorescence microscopy by measuring DiR and doxorubicin (fluorescent anticancer agent) entrapped within some of the formulations. Biodistribution and circulation times were determined non‐invasively on an IVIS Lumina XRMS and iThera Multispectral Optoacoustic Tomography (MSOT) systems in Athymic mouse xenograft models of prostate cancer. Liposomes formulations of SPRL and SSL were prepared at size of 120±1 and 118±2 nm. Circulation half‐life and tumor deposition was similar for both formulations, suggesting the enhanced pharmacological activity is related to release/uptake after drug extravasation. In‐vitro studies showed that PC‐3‐PLA2R‐KD cells had greater DiR fluorescence of both formulations than wild PC‐3 after 48 hr. Moreover, SPRL had greater uptake than SSL formulation (p&lt;0.05) beyond 24 hr. In‐vivo studies showed greater uptake of SSL and SPRL in the PC‐3‐PLA2R‐KD tumors. Overall these data suggest that PLA2R1 plays an important role in the performance of SSL and SPRL formulations. Further studies are needed to gain insights into the clinical significance and to determine if presence of PLA2R1 can be used to identify specific patient populations that would best respond to treatment with SPRL.Support or Funding InformationNIH/NIBIB 1R01EB016100‐01(RDA/BSC), 1R21EB008153 (RDA/BSC), and AU Research Initiative in Cancer “AURIC”.

Extracellular Vesicle-Mediated In Vitro Transcribed mRNA Delivery for Treatment of HER2+ Breast Cancer Xenografts in Mice by Prodrug CB1954 without General Toxicity.

Prodrugs are harmless until activated by a bacterial or viral gene product; they constitute the basis of gene-delivered prodrug therapies called GDEPT, which can kill tumors without major side effects. Previously, we utilized the prodrug CNOB (C16H7CIN2O4; not clinically tested) and enzyme HChrR6 in GDEPT to generate the drug MCHB (C16H9CIN2O2) in tumors. Extracellular vesicles (EVs) were used for directed gene delivery and HChrR6 mRNA as gene. Here, the clinical transfer of this approach is enhanced by: (i) use of CB1954 (tretazicar) for which safe human dose is established; HChrR6 can activate this prodrug. (ii) EVs delivered in vitro transcribed (IVT) HChrR6 mRNA, eliminating the potentially harmful plasmid transfection of EV producer cells we utilized previously; this has not been done before. IVT mRNA loading of EVs required several steps. Naked mRNA being unstable, we ensured its prodrug activating functionality at each step. This was not possible using tretazicar itself; we relied instead on HChrR6's ability to convert CNOB into MCHB, whose fluorescence is easily visualizable. HChrR6 mRNA-translated product's ability to generate fluorescence from CNOB vicariously indicated its competence for tretazicar activation. (iii) Systemic IVT mRNA-loaded EVs displaying an anti-HER2 single-chain variable fragment ("IVT EXO-DEPTs") and tretazicar caused growth arrest of human HER2+ breast cancer xenografts in athymic mice. As this occurred without injury to other tissues, absence of off-target mRNA delivery is strongly indicated. Many cancer sites are not amenable for direct gene injection, but current GDEPTs require this. In circumventing this need, a major advance in GDEPT applicability has been accomplished.