Negative Breast Cancer Cell Lines Research Articles

Development of triphala churna extract mediated iron oxide nanoparticles as novel treatment strategy for triple negative breast cancer

The present work demonstrates the eco-friendly green synthesis of Iron oxide nanoparticles from the aqueous extract of Triphala churna (TIONPs) using ferric chloride hexahydrate (FeCl3.6H2O) precursor in a single-step process. The formulated TIONPs were characterized by various instrumental techniques such as UV–Visible spectrophotometry (UV–Vis), Atomic absorption spectroscopy (AAS), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), Scanning electron microscope (SEM), Dynamic light scattering- Zeta potential (DLS-Zeta sizer), and Superconducting quantum interference device (SQUID) to establish their physico-chemical characteristics. The average size range of obtained spherical TIONPs was found in between 29 and 74 nm. Further, the MTT assay and Nuclear staining assay were carried out on Triple negative breast cancer (TNBC) cell lines along with a breast cancer associated epidermoid skin carcinoma cell line. The MTT assay revealed prominent cytotoxic activity of TIONPs against the tested cancer cell lines. Additionally, the nuclear staining assay revealed that the cytotoxic activity of these nanoparticles occurred via apoptosis mode by exhibiting nuclear fragmentation, cytoplasmic shrinkage, and cell blebbing. These TIONPs were also found to possess superparamagnetic property which may be additionally beneficial for their rapid and organ targeted delivery. Hence, the green synthesized TIONPs can be explored in near future as a new promising option for TNBC management.

Design, synthesis, bioevaluation, DFT, docking, and molecular dynamic simulation for selected novel 1,3,4-Oxadiazole - indole derivatives hybrid against estrogen receptor alpha

Breast tumors overexpress ER alpha and it initiates, as well as the progression of breast cancer. Studies have proven that anti-estrogen therapy (tamoxifen) is used to treat breast cancer. For the first time, in vitro bioevaluation, molecular docking, and DFT simulations were used to investigate the essential interactions of the estrogen receptor (ER) alpha with selected novel 1,3,4-Oxadiazole - indole derivatives hybrid against Triple Negative Breast Cancer (TNBC) cell lines. The key step of this synthesis is the oxidative cyclization of N-acylhydrazones, 2(a-j), in 20 min using reagent BTI [bis(trilfuoroacetoxy)iodobenzene]. Synthesized compounds, 3(a-j), were screened against a series of cancer cell lines, and among these compounds, the compound, 3d ((5′-Bromo-1H-indol-1-yl) methyl)-5′’-(3-bromo-4-methylphenyl)-1,3,4-Oxadiazole), as a potent molecule which displayed IC50 values of 10.56 and 22.61 µM against MDA MB 468, and MDA MB 231 cell lines respectively. In vitro antibody specific for Human Estrogen Receptor alpha assay showed an IC50 value of 5.27 µM for compound 3d. The structures were optimized to their molecular geometry (3d) by the DFT method with B3LYP hybrid functional and 6–311++g(d,p) basis set. The electrostatic potential map was plotted to understand the energy distribution and chemical reactive regions of the molecule. The optimized structures were used to perform in silico studies. Molecular docking followed by molecular dynamics trajectories is essential to investigate the stable binding of 3d to estrogen receptor alpha. Overall, results show that 3d has the potential to be further explored for the treatment of metastatic breast cancer.

In vitro radiosensitization of breast cancer with hypoxia-activated prodrugs.

KP167 is a novel hypoxia‐activated prodrug (HAP), targeting cancer cells via DNA intercalating and alkylating properties. The single agent and radiosensitizing efficacy of KP167 and its parental comparator, AQ4N, were evaluated in 2D and 3D cultures of luminal and triple negative breast cancer (TNBC) cell lines and compared against DNA damage repair inhibitors. 2D normoxic treatment with the DNA repair inhibitors, Olaparib or KU‐55933 caused, as expected, substantial radiosensitization (sensitiser enhancement ratio, SER0.01 of 1.60–3.42). KP167 induced greater radiosensitization in TNBC (SER0.01 2.53 in MDAMB‐231, 2.28 in MDAMB‐468, 4.55 in MDAMB‐436) and luminal spheroids (SER0.01 1.46 in MCF‐7 and 1.76 in T47D cells) compared with AQ4N. Significant radiosensitization was also obtained using KP167 and AQ4N in 2D normoxia. Although hypoxia induced radioresistance, radiosensitization by KP167 was still greater under 2D hypoxia, yielding SER0.01 of 1.56–2.37 compared with AQ4N SER0.01 of 1.13–1.94. Such data show KP167 as a promising single agent and potent radiosensitiser of both normoxic and hypoxic breast cancer cells, with greater efficacy in TNBCs.

Abstract 115: Combined treatment of letrozole or olaparib with aspirin inhibits cell proliferation and induces apoptosis through upregulation of wild p53 and modulation of aurora kinases in human breast cancer cell lines

Abstract Although, there are several breakthroughs in the treatment of breast cancer on the past few decades, high incidence of relapse and progression after conventional therapies is deeply concerning, indicating a great need for developing new therapeutics for breast cancer. In the present study, the anti-cancer effects of combinational therapy of Letrozole (Aromatase inhibitor) or Olaparib (PARP inhibitor) with Aspirin, non-steroidal anti-inflammatory drugs (NSAIDs), were examined on well differentiated ER-positive and poorly differentiated triple negative breast cancer cell lines, T-47D and MDA-MB-231, respectively. In addition, the effect of combined treatment was also examined by detecting cell survival, cell cycle analysis, and alterations in signaling pathway that play a role in breast cancer progression such as β -catenin, STAT3, cyclin D1, Bax, c-PARP, wild and Mutant p53. In addition the effect of combined treatments were examined on gene expression levels of Cytochromes; CYP1A1, CYP1B1, Aurora kinases; Aurora-A and Aurora-B were determined by RT-PCR.Our data showed that combined treatments of 50 nmol Letrozole or 250 nmol Olaparib with 5 mmol, 2.5mmol Aspirin for 24 and 48 h, respectively, promoted apoptosis induction through enhanced histone release and caspase-3 activation compared to control and cells treated with each drug alone. Furthermore, apoptotic induction was associated with upregulation of Bax and downregulation of Bcl-2, cyclin D1, β-catenin, and STAT3 signaling pathways in both tested cell lines compared to control, suggesting that Aspirin may enhance the sensitivity of tested cells to Letrozole or Olaparib in both (ER) positive and triple negative breast cancer cell lines. In addition, cell cycle analysis showed a cell arrest at G0/G1 phase in both cell lines. Interestingly, in T-47D cells, combined treatment of Letrozole with Aspirin significantly upregulated wild p53 at protein level and downregulated CYP1A1, CYP1B1, Aurora-A, and Aurora-B expression at gene level compared to control, suggesting that combined treatment may inhibit breast cancer cell proliferation via down-regulation of Aurora-A and Aurora-B. In MDA-MB-231 cells, combined treatment of Olaparib with Aspirin showed a significant downregulation of Mutant p53 at protein level and CYP1A1, CYP1B1, Aurora-B and upregulation of Aurora-A at gene level compared to control, suggesting that there is a crosstalk between Olaparib/Aspirin and P53 In conclusion, combined treatment of Letrozole or Olaparib with Aspirin might be a promising approach to inhibit both ER (+) and triple negative breast cancer cell lines survival and proliferation. Citation Format: Marwa Elsayed ElKamel, Salah Sheweita, Ahmed Sultan. Combined treatment of letrozole or olaparib with aspirin inhibits cell proliferation and induces apoptosis through upregulation of wild p53 and modulation of aurora kinases in human breast cancer cell lines [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 115.

Abstract 4060: Identification of anti-neoplastic compounds on a triple negative breast cancer cell line from T. violacea

Abstract Background: The incidence of Invasive triple negative breast cancer (TNBC) is higher than average in South Africa, with 90% of patients being Black South Africans. Current treatments for breast cancer are associated with severe side effects. Additionally, TNBC is resistant to hormone therapy commonly used to treat other forms of breast cancer. Tulbaghia violacea, a plant native to Southern Africa, is a medicinal herb used in traditional medicine. This study aims to establish the impact of water soluble extracts from T. violacea on TNBC cancer cell lines and identify any anti-cancer compounds present in this extract. Methods: Following the determination of the IC50 value for this extract. The anti-proliferative and anti-metastatic activities of the extract on the TNBC cell line were determined using scratch assays and cell invasion and migration assays, performed using real time cell analysis. The pro-apoptotic action of the extract on the TNBC cell line was determined using flow cytometry cell death assays. Once the anti-cancer activity of the extract was confirmed, the composition of the extract was determined in order to establish what known or possible anti-cancer compounds are present within it. This was performed using gas chromatography-mass spectroscopy (GC-MS). The structure of these identified compounds was then confirmed using Nuclear Magnetic Resonance (NMR). Results: The IC50 value for the T. violacea water soluble extract against MDA MB 231 cells was determined to be ~350 µg/mL. The extract was also shown to have anti migration and invasion as well as pro-apoptotic effects on these cells. Fourteen known anti-cancer compounds were identified in this water soluble extract. In addition to this multiple other compounds were shown to also be present in the extract. Discussion: The water soluble T. violacea leaf extract has cytotoxic effects and the ability to inhibit proliferation and metastasis in a TNBC cell line. The identification of fourteen compounds with known anti-cancer activity may explain the effects this extract has on these TNBC cells, however, the contribution made by any of the other compounds to this activity remains to be determined. Conclusion: These findings suggest that one or more of the compounds present in T. violacea may serve as lead compounds for the development of new treatments for TNBC. Citation Format: Zodwa Dlamini, Clement Penny, Rodney Hull, Mohammed Alouna. Identification of anti-neoplastic compounds on a triple negative breast cancer cell line from T. violacea [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 4060.

Abstract 2637: Inhibition of novel kinase MAP3K19 reverses EMT and kills triple negative breast cancer cell lines

Abstract The Triple-Negative Breast Cancer (TNBC) subtype is a breast cancer subpopulation whose phenotype lacks estrogen and progesterone growth receptors, and lacks HER2 amplification. This contrasts with the Luminal and HER2+ subpopulations for which clinical precision therapies exist to target cancer proliferation and improve prognoses. Consequently, the discovery of new targets is necessitated by the TNBC subtype, which has the poorest patient outcomes. Our studies have identified MAP3K19 as one such potential target. MAP3K19 an understudied protein kinase with poorly characterized physiological functions outside of its roles in the Mitogen-Activated Protein Kinase (MAPK) signaling cascade. Broadly, the MAPK signaling has been associated with cell proliferation and differentiation in pathways both intracellular and extracellular, which in the context of cancer cell biology warrants investigation into cancer cell behavior as well as the associated tumor microenvironment (TME). The Published Kinase Inhibitor Set (PKIS) is an open-access drug panel designed by GlaxoSmithKline to identify novel kinases. Preliminary PKIS cell-viability screening identified GSK1440913, GW494601, and GSK907232A as compounds with the most adverse effects on cell phenotype and growth in TNBC lines MDA-MB-231 and PDX-derived TU-BcX-49S. Analysis of common targets revealed MAP3K19 a common target, suggesting its importance in cancer cell growth and proliferation. AT-9283, a more specific MAP3K19 inhibitor, was used to support the initial results. Treatment with AT-9283 resulted in a dose-dependent killing effect. Furthermore, MAP3K19 inhibition resulted in decreased EMT gene signatures by qPCR. To summarize, MAP3K19 was identified by the PKIS screen as a major kinase in TNBC. Treatment with AT-9283, a more specific MAP3K19 inhibitor, resulted in cellular death and reversal in EMT gene signature by qPCR. Future directions for this project involve genetic knock-down and overexpression of MAP3K19 to evaluate its roles in biology and its signaling mechanisms. Citation Format: Khoa Nguyen, Thomas Cheng, Madlin Alzoubi, Steven Elliott, Sean Lee, David Drewry, Bridgette Collins-Burow, John Brognard, Matthew Burow. Inhibition of novel kinase MAP3K19 reverses EMT and kills triple negative breast cancer cell lines [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 2637.

Abstract 5827: Increased miR150 expression in tumors enhances lymphocytes infiltration causing immunosuppression through inflammatory chemokines

Abstract Dysregulated miRNAs play key roles in multiple biological processes and cancer. miR-150-5p, a cancer-related miRNA, has been reported to be aberrantly expressed in various cancers. We found that triple-negative and HER2+ breast cancer patients have elevated expression of miR150 in the TCGA and METABRIC patient cohort. Both cohorts showed significantly increased expression of miR150 in grade IV patients. Further studies explained that miR150 is strongly correlated with CD8+ T cells and B cells in accord with increased expression of immune-checkpoint markers PD-1, PDL-1, and CTLA4. We, therefore, hypothesized that higher expression of miR150 in tumors attracted more immune cells in the tumor microenvironment. We use two triple negative breast cancer cell lines MDA-231 and BT-549 cells and overexpressed miR150 followed by co-culture them with Jurkart and B cells. Our data revealed that both jurkart and B cells had increased migration towards cancer cells overexpressed miR150 mimic compared to miR150 mimic negative control. Interestingly, overexpressed miR150 mimic in cancer cells inhibited migration in both triple-negative breast cancer cells. Overexpression of miR150 mimic in cancer cells did not alter cell proliferation. To understand further the role of miR150 in breast cancer, we overexpressed miR150 mimic and its corresponding negative control in MDA-231 and did RNA seq analysis. RNA seq data explained that approximately 4927 genes are upregulated with miR150 mimic overexpression. Interestingly several chemokines and mediators of metastasis (CXCL2, CXCL1 CCL20, IGBP3, and ANGPTL4) are upregulated among the top 50 upregulated genes. In addition, gene enrichment analysis explained that TNF-α signaling pathways, Hypoxia, epithelial-mesenchymal pathways, E2F signaling, and Inflammatory pathways are a few upregulated pathways with miR150 mimic overexpression compared to the negative control. We also found miR150 high tumor enriched correlated with interferon-alpha and gamma signaling, inflammatory response pathways similar with cohort analysis data. In conclusion, miR150 in cancer plays a key role in interferon and inflammatory signaling pathways attracting more immune cells and suppressing the tumor microenvironment. Citation Format: Aparna Maiti, Masanori Oshi, Rongrong Wu, Nitai Hait, Kazuaki Takabe. Increased miR150 expression in tumors enhances lymphocytes infiltration causing immunosuppression through inflammatory chemokines [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 5827.

Abstract 1640: Enhanced development of functional human innate immune cells in a novel FLT3nullNSG mouse strain expressing human FLT3L

Abstract Humanized mice are being applied widely to study human immune system homeostasis, function, and as a testing platform for cancer immunotherapies. A major limitation for many humanized mouse models is the lack of functional and mature human innate immune cells, which are critical for effective human immune system-tumor interactions. Previous studies have demonstrated that delivery of human FLT3L into immunodeficient mice that lack mouse FLT3, promotes the development of human innate immune cell subsets following engraftment with human hematopoietic stem cells (HSC). Here we describe a NOD-scid IL2rgnull (NSG) mouse that lacks the expression of mouse FLT3 and expresses human FLT3L transgenically (NSG-mFLT3nullTg (HuFLT3L) or NSG-FLT3L. In these studies, NSG-FLT3L and NSG mice were engrafted with human umbilical cord blood (UCB) CD34+ HSC and compared for human immune system development and function. HSC-engrafted NSG-FLT3L and NSG mice show similar levels of total human CD45+ cells in blood over the course of 18 weeks post-engraftment. However, HSC-engrafted NSG-FLT3L mice show significantly higher levels of human CD141+ and CD1c+ DC subsets, CD123+ pDC, CD14+ monocytes, CD56+ NK cells and CD3+ T cells in the blood as compared to NSG mice. CD34-FLT3L mice also show increased levels of human immune cell infiltration into the gut mucosa. CD56+ NK cells and CD3+ T cells from HSC-engrafted NSG-FLT3L mice express granzyme A and granzyme B, indicating cytotoxic activity. Following treatment of HSC-engrafted NSG-FLT3L mice with LPS, heightened levels of human cytokines were detected in serum samples, confirming innate immune system function. The growth kinetics of tumor cells from a triple negative breast cancer cell line MDA-MB-231 and a lung PDX model LG1306 in HSC-engrafted NSG-FLT3L mice are work in progress and will be compared with HSG-engrafted NSG mice. Overall these results demonstrate that the NSG-FLT3L model supports enhanced development of functional, human innate immune cells and is a novel tool to study human immuno-oncology. Citation Format: Li-Chin Yao, Shantashri Vaidya, Pali Kaur, James G. Keck, Leonard D. Shultz, Dale L. Greiner, MIchael A. Brehm. Enhanced development of functional human innate immune cells in a novel FLT3nullNSG mouse strain expressing human FLT3L [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 1640.

Abstract 1338: Humanized mouse model for in vivo evaluation of bispecific antibody efficacy

Abstract Bispecific antibodies have shown several promising advantages over monoclonal antibodies and are emerging as one of growing cancer immunotherapies. Preclinical in vivo models to assess efficacy associated with immune cell engagement are needed. Here we evaluated an EGFR bispecific T cell engager (EGFRXCD3 BiTE) using xenograft models in humanized NSG™ (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice that have been engrafted with human CD34+ HSC and have human T cells in circulation. Three EGFR+ tumor models were used in this report including LG1202 lung PDX and two triple negative breast cancer cell lines MDA-MB-231 with a high EGFR expression level and MDA-MB-453 with a low EGFR expression. Interestingly we found that all three models were resistant to EGFR monoclonal antibody drug cetuximab despite the expression of EGFR but responded to EGFRXCD3 BiTE. BiTE-induced T cell activation was evident through the analysis of tumor infiltrated lymphocytes by flow cytometry at the end of the study. BiTE treatment for 3 weeks effectively reduced the LG1202 tumor growth whereas the effect of cetuximab on the tumor growth was minimal. In the BiTE-treated group, CD3+ T cell percentage increased as compared to the vehicle group, and both CD4+ and CD8+ T cell subsets showed an increased frequency of CD69+ cells. We also observed elevated levels of human cytokines IFN-γ, IL-6, IL-2, and IL-10 in circulation 4 h after animals received the first BiTE dose. We evaluated the dose response of BiTE treatments on MDA-MB-231 model with a high EGFR expression. Low dose BiTE reduced tumor growth whereas high dose BiTE effectively prevented or even regressed some tumor growth. Flow analysis showed that tumor CD3+ T cell frequency remained similar to the vehicle group but CD8+ T frequency increased after high dose BiTE. High CD69+CD8+ T cell frequency was found in the vehicle group and the percentage further increased after high dose BiTE. For the low EGFR-expressing MDA-MB-453 model, BiTE treatment for 4 weeks also effectively reduced the tumor growth. In comparison, while cetuximab treatment decreased EGFR expression on tumor cells, it did not reduce the tumor growth. Flow analysis showed that BiTE-treated group had increased T cell infiltration into the tumors when compared to the PBS and cetuximab groups. The frequency and cell number of CD25+ and CD69+ CD4+ T and CD8+ T cells in the tumor increased after BiTE treatment. Together, these data support our humanized mouse model to be used to validate T-cell dependent bispecific antibody efficacy. Citation Format: Li-Chin Yao, James G. Keck, Danying cai. Humanized mouse model for in vivo evaluation of bispecific antibody efficacy [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 1338.

Abstract 3326: Uncovering the molecular mechanisms which predict sensitivity to a novel src kinase inhibitor NXP900 to inform personalized healthcare strategies

Abstract We have previously described the discovery of a highly potent, selective and orally bioavailable Src kinase inhibitor NXP900 with a unique binding mode relative to other Src inhibitors under clinical development or approved (Temps et al 2021). This unique binding mode locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions. Also, in contrast to other Src inhibitors in the clinic, NXP900 exhibits a unique target selectivity profile with 1000 fold selectivity for Src over Abl kinase. The aim of this current study was to perform broad cancer cell line panel screening to inform disease positioning and identify predictive biomarkers of sensitivity for this novel class of Src kinase inhibitor. We describe the results of extensive cancer cell line panel profiling across a suite of 2-dimensional(D) and 3-D in vitro cell viability and high content phenotypic profiling assays that has revealed clear patterns of cell line sensitivity and insensitivity. Among the most sensitive cell line subtypes are, squamous cell carcinoma, Arid1A mutant ovarian clear cell carcinoma and subtypes of breast luminal A and triple negative breast cancer cell lines. HER2+ breast cancer lines appear insensitive to NXP900 indicating HER2 as a potential driver of resistance to NXP900. Further in-depth bioinformatic analysis of genetic and transcriptomic profiles of sensitive cell lines has identified putative biomarkers to support patient selection and personalized healthcare strategies. These studies demonstrate the identification of cancer cell lines which exhibit high sensitivity to NXP900 and which can be grouped into discrete molecular subtypes based on gene mutation status, transcriptomics and histotype. Our result provide an indication of patient subgroups which may exhibit optimal therapeutic response to NXP900 and provide data to guide further preclinical, biomarker and clinical development. Temps C et al., Cancer Res. 2021 Nov 1;81(21):5438-5450. doi: 10.1158/0008-5472.CAN-21-0613. Citation Format: Neil Carragher, Rebecca Hughes, Valerie Brunton, Asier Unciti-broceta, Iñigo Lanzagorta-Calvillo, Carolin Temps, Enrique Poradosu. Uncovering the molecular mechanisms which predict sensitivity to a novel src kinase inhibitor NXP900 to inform personalized healthcare strategies [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 3326.

Abstract 1781: Up284, a novel inhibitor to treat quadruple negative breast cancer

Abstract Quadruple negative breast cancer (QNBC), lacking the expression of ER (estrogen receptor), PR (progesterone receptor), HER2 (human epidermal growth factor receptor 2) and AR (androgen receptor), is the breast cancer subtype with the worst prognosis, and QNBC disproportionately afflicts African Americans. It has no standard-of-care treatment targets and thus efficacious and safe treatments are urgently sought for this unmet medical need, and to address the disparity in breast cancer outcomes. The current proposal is motivated by data showing elevated expression of proteasome subunit RPN13 is associated with both African American race and lower survival in QNBC patients, that RPN13 selectively targeted by a small molecule Up284 developed by Up Therapeutics. Both triple negative breast cancer and QNBC cell lines show evidence of greater vulnerability to proteasome inhibitors. However, licensed 20S proteasome inhibitors, e.g. bortezomib, have proven ineffective against solid tumors, with emergence of resistance, and dose limiting toxicities including thrombocytopenia and neutropenia. Up284 has a target and structure designed to overcome the limitations of the licensed drugs with respect to drug resistance. Up284 blocks substrate recognition and deubiquitination rather than just one of the three 20S catalytic activities), poor activity against solid tumors (Up284 has a novel spiro structure with evidence of improved drug access to tumor as compared to peptide-based 20S inhibitors), key toxicities of thrombocytopenia and neutropenia (unlike 20S inhibitors, Up284 does not target the immunoproteasome expressed by hematopoietic cells and does not show these toxicities). Up284 shows broad anticancer activity in vitro, including against QNBC lines with a robust therapeutic index, a promising safety profile and pharmacodynamics, and the ability to control xenograft tumor. By inhibiting proteasome ubiquitin receptor RPN13 function and its associated deubiquitinase activity, Up284 triggers more rapid accumulation and increased molecular weight polyubiquinated protein aggregates than is induced by 20S inhibitors. These toxic misfolded protein aggregates produce an unresolved ER stress, activate the canonical Unfolded Protein Response (UPR) signaling cascade and more rapidly triggers apoptosis than 20S inhibitor. The safety parameters and promising efficacy of Up284 against breast cancer lines encourages us to validate Up284 efficacy in more QNBC lines and animal models. We discuss Up284 efficacy and pharmacological analysis in our presentation. Citation Format: Balasubramanyam Karanam, Clayton Yates, Ravi Anchoori. Up284, a novel inhibitor to treat quadruple negative breast cancer [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 1781.

Abstract 3913: Quantitative phosphoproteomic analysis of AXL signaling network in breast cancer

Abstract Background: As a key receptor tyrosine kinase (RTK) regulating tumorigenesis, AXL is overexpressed in multiple different types of cancers. AXL can be activated by homodimerization after binding with its ligand, growth arrest specific 6 protein (Gas6) or by heterodimerization with other RTKs. AXL overexpression and/or activation have been reported to promote cancer cell proliferation, migration/invasion and to induce resistance to targeted therapy. However, the AXL downstream signaling network has not yet been fully elucidated. Methods: We employed a CRISPR-CAS9-based knockout technology to create AXL inducible knockout cells in a human triple negative breast cancer (TNBC) cell line, HCC1395. In order to explore the downstream signaling events driven by AXL, we induced AXL knockout for 72 hours and used a 6-plex TMT-labeling-based quantitative proteomic approach to characterize the protein phosphorylation alterations induced by AXL knockout. IMAC and anti-phosphotyrosine antibody (pY1000)-based phosphopeptide enrichment methods were used to comprehensively quantify the phosphoproteome changes induced by AXL knockout. Results: In this study, we identified 38,525 phospho serine/threonine sites and 1,427 phosphotyrosine sites. In response to AXL knockout, 1,581 phospho serine/threonine sites and 40 phosphotyrosine sites showed significant differences between AXL-KO and AXL-WT groups with >1.5-fold change and p<0.05 as the cutoff. Among them, 982 phosphoserine/threonine sites and 30 phosphotyrosine sites were decreased, while 599 phospho serine/threonine sites and 10 phosphotyrosine sites were increased in AXL-KO cells. Signaling pathway enrichment analysis revealed that suppressing AXL expression could inhibit multiple important cancer related signaling pathways, including cell cycle, focal adhesion, MAPK signaling pathway, p53 signaling pathway and mTOR signaling pathway. Conclusion: Quantitative analysis of the phosphoproteome driven by AXL demonstrated that AXL plays a pivotal role in regulating canonical oncogenic signaling but also signaling pathways involved in RNA processing and DNA repair. Our data also suggest that targeting AXL could suppress these oncogenic signaling pathways and have therapeutic potential to improve clinical outcomes for patients with AXL overexpression. Citation Format: Li Wang, Nicole Pearson, Yuning Xiong, Santosh Renuse, Ran Cheng, Jodi M. Carter, Akhilesh Pandey, Xinyan Wu. Quantitative phosphoproteomic analysis of AXL signaling network in breast cancer [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 3913.

Abstract 5450: New se-nsaid hybrid molecules as potent and selective cytotoxic agents against breast cancer cells

Abstract Breast cancer is currently the most frequently diagnosed cancer and one of the leading causes of mortality among women worldwide. The introduction of a selenium atom (Se) in the structure of organic molecules has been demonstrated to be a valid approach in the design of novel chemotherapeutic agents with several biological effects, including antitumoral activity. In addition, several epidemiological, preclinical, and clinical studies support the chemopreventive and chemotherapeutic potential use of non-steroidal anti-inflammatory drugs (NSAIDs).On the basis of these findings, and taking into account the results of previous studies by our research group, we have developed a new series of twenty-five NSAID analogs bearing Se in the form of selenoesters modulated with different functional groups. The new compounds were tested in four cancer cell lines (HTB-54, DU-145, HT-29 and MCF-7) and two non-malignant cells (184B5 and BEAS-2B). The cytotoxicity of the five most potent and selective molecules was further studied in other cancer cell lines as well. Compounds 4a and 4d stood out as the most selective compounds with a selectivity index at least 5-fold higher in breast cancer cells. Compound 4d showed IC50 values below 10 µM and was found to be more potent than 4a in almost every cell line tested. Additionally, 4d effectively induced apoptosis in triple negative (MDA-MB-231) and estrogen receptor-positive (MCF-7) breast cancer cell lines. Taken together, this study supports the design of new derivatives containing Se as potential anticancer agents. Citation Format: Sandra Ramos-Inza, Ignacio Encío, Asif Raza, Arun Sharma, Carmen Sanmartín, Daniel Plano. New se-nsaid hybrid molecules as potent and selective cytotoxic agents against breast cancer cells [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 5450.

Combinational silencing of components involved in JAK/STAT signaling pathway

Combinatorial silencing of more than one protein via small interfering RNA (siRNA) is a new strategy that can enhance the effect of RNA interference on cell function. To explore this strategy, we selected JAK/STAT axis as a major signaling pathway that contributes to several mechanisms involved in cancer cell proliferation and survival. We focused on four proteins involved in this pathway to explore the possibility of identifying a combinatorial targeting strategy (as the proof of concept) with enhanced efficiency: gp 130 (a co-receptor for IL6 cytokines), JAK2, STAT3, and importin α3 (the nuclear transporter reportedly involved in translocation of activated STAT3 to nucleus). Selected proteins were targeted by siRNA in two selected Triple Negative Breast Cancer cell lines (MDA-MB-231 and MDA-MB-468). The effect of individual and selected combinations of siRNAs on selected downstream antiapoptotic proteins, pro-apoptosis proteins, and cell-cycle regulating proteins was explored. Combinatorial silencing of JAK2/gp 130 enhanced the effect of RNA interference on downstream proteins significantly, and demonstrated enhanced effect in reducing cell viability, cell migration, and the level of activation of STAT3. In conclusion, the promising results of simultaneous targeting of JAK2 and gp 130 might be an example for potential combinatorial silencing strategies in cancer treatment.

Phase Ib study of selinexor and eribulin combination in advanced solid tumors and triple-negative breast cancer.

3108 Background: Selinexor (KPT-330) is potent inhibitor of Exportin-1. In vitro, Selinexor was found to be synergistic with eribulin in triple negative breast cancer (TNBC) cell lines and enhanced antitumor activity of eribulin in TNBC patient-derived xenografts (PMID 28810913). Methods: We conducted a phase Ib trial in combination of selinexor and eribulin using 3 + 3 design in dose escalation for patients with advanced solid tumors and in TNBC in dose expansion cohort. Eribulin could be discontinued after combination for 6 cycles at physician discretion. Primary objectives: Safety, Recommended Phase 2 Dose (RP2D). Secondary Objectives: Objective Response Rate (ORR), Duration of Response (DOR), Disease Control Rate (DCR), Overall Survival (OS) and Progression Free Survival (PFS). Results: 31 patients, TNBC (n = 19), sarcoma (n = 8), others (n = 4) enrolled in dose escalation (n = 10) and dose expansion phases (n = 21). Median prior therapies:4 (1–6). Study initiated selinexor at 60mg twice weekly and eribulin 1.4mg/m2 on Day1, Day8 every 3 weeks which led to 1 Dose Limiting Toxicity (DLT) and hence, selinexor 80mg once weekly and eribulin 1mg/m2 was elected as RP2D due to efficacy and tolerability. As of 01/15/2022, of 29 patients (94%) who have discontinued treatment, 24 (77%) were due to progressive disease, 3 (10%) withdrew consent and 2 (6%) due to toxicities (G1 pneumonitis; G3 neutropenia) while 2 (6%) remain on trial. All 31 patients had at least one treatment emergent adverse event (TEAE) while most prevalent TEAEs (all grades) were leukopenia (77%), nausea (71%), anemia and neutropenia (68%) and fatigue (48%). The most common G3/4 TEAE were leukopenia (26%) and neutropenia (29%). 2 DLTs occurred; 1 in first dose level (DL); 1 in second DL dosed at selinexor 80 mg once weekly due to G3 neutropenia. ORR for all was 10% while DCR (SD+PR+CR) > 6 months seen in 3 (15%) TNBC and 2 (20%) sarcoma patients. The median OS and PFS for all were 12.3 (7.3, 27.3) months and 2.3 (1.6, 4.1) months. In dose escalation cohort, ORR was 10% where one patient (3%) with vaginal SCC had confirmed PR (-44%) for 2.1 months. Five patients (62.5%) with sarcoma had stable disease (SD). One patient with high grade sarcoma has SD for 68 months and remains on selinexor after 4 months of eribulin and selinexor. In TNBC dose expansion (n = 19), ORR was 10.5% with 2 confirmed PRs and median duration of response (DOR) of 10.8 months. One patient who has remained on treatment for 18 months, and after receiving 8 months of eribulin and selinexor, remains on selinexor with 100% target regression and an indeterminate brain lesion. Conclusions: Selinexor with eribulin is safe with manageable toxicity profile and modest overall clinical efficacy. Durable responses and disease control were observed with metastatic TNBC. Further study is needed to examine the determinants of response to this combination. Clinical trial information: NCT02419495.

Hyaluronic acid targeted metal organic framework based on iron (III) for delivery of platinum curcumin cytotoxic agent to triple negative breast cancer cell line

Platinum‐based drugs are an essential class of chemotherapy drugs in breast cancer. However, suffer from side effects and drug resistance; structural changes and applying drug carriers can lead to new drug candidates in this class. The metal‐organic frameworks (MOFs) as porous carriers have recently attracted much attention in drug delivery. In this study, for the first time, hyaluronic acid (HA)‐targeted MOF (NH2‐MIL‐101 (Fe)) nanoparticles (Pt‐CUR@MIL@HA NPs) were evaluated in delivery of platinum‐curcumin (Pt‐CUR) prodrug to the MDA‐MB‐231 triple‐negative breast cancer cell line; in this regard, the cytotoxicity, ROS production, and cellular uptake of designed NPs have been studied. Different analysis methods confirmed chemical structures, the prepared NPs had a uniform morphology, and the hydrodynamic size of the optimized non‐targeted loaded particles increased to about 252 nm with a zeta potential of +26.9 mV, after targeting with HA, the size increased to 310 nm, and the zeta potential changed to −28 mV. Based on TGA and atomic absorption (ICP‐MS) results, the drug loading percent was determined to be about 30%–35%. Drug release from the HA targeted Pt‐CUR@MIL@HA system in the neutral condition was slow and sustained, and after 36 h, a maximum of 60% of the drug was released, but in acidic conditions, the release was increased, and by 18 h, the release was about 80%. The cytotoxicity of MOF NPs containing Pt‐CUR was more significant than that of the free drug, and HA targeted has resulted in more cellular uptake than the non‐targeted NPs. In conclusion, these new MOF‐ based HA‐coated NPs of PT‐CUR can be introduced to pre‐clinical researches after completing in vitro and in vivo studies.

Hormone receptor binding, selectivity and cytotoxicity of steroid D-homo lactone loaded chitosan nanoparticles for the treatment of breast and prostate cancer cells

Chemically modified steroids have a long history as anti-neoplastic drugs. Incorporation of a lactone moiety in the steroid nucleus, as in previously obtained 3β-acetoxy-17-oxa-17a-homoandrost-5-en-16-one (A) and 3β-hidroxy-17-oxa-17a-homoandrost-5-en-16-one (B), often results in enhanced anticancer properties. In this work, chitosan-based (Ch) nanoparticles were created and loaded with potent anticancer steroidal compounds, A and B. Changes to hormone receptor binding and cytotoxicity were then measured. In agreement with our previous results for A and B, A- and B-loaded Ch displayed cytotoxic properties against cancer cell lines. Both A-Ch and B-Ch showed activity toward estrogen negative breast cancer (MDA-MB-231) and androgen negative prostate cancer cell lines (PC-3). Greater selectivity toward cancer cells versus healthy lung fibroblast (MRC-5) was observed for B-Ch particles. Cell viability and cytotoxicity measurements after a recovery period indicate more robust recovery of healthy cells versus malignant cells. Compounds A and B or their Ch-encapsulated forms were shown to have negligible affinity for the ligand binding domain of estrogen receptor β or the androgen receptor in a fluorescent yeast screen, suggesting a lack of estrogenicity and androgenicity. Steroid-loaded chitosan nanoparticles display strong cytotoxicity towards MDA-MB-231 and PC-3 with a lack of hormone activity, indicating their safety and efficacy.

Hollow mesoporous SiO2–ZnO nanocapsules and effective in vitro delivery of anticancer drugs against different cancers with low doses of drugs

Spherical hollow and mesoporous SiO2–ZnO nanocapsules were synthesized through sol-gel synthesis approach. Metabolic activity and biocompatibility of these nanocapules were tested and confirmed that SiO2–ZnO nanocapsules are not toxic. Small biomolecules and drug can be loaded into the nanoparticles since they are mesoporous with hollow core. Further these nanocapsules are capable of loading a huge amount of anticancer drugs such as imatinib and paclitaxel. The sustained release of the drugs demonstrated that these functionalized nanocapsules are useful for various therapeutic applications with precise release of drugs such as for the cancer therapy with low doses. To check the therapeutic efficiency of these nanocapsules, HeLa cells (cervical cancer cell line), breast cancer cell lines such as MCF-7 (primary breast cancer) and MDA-MB-231 (triple negative breast cancer cell lines) were used for the study. For all cells the therapeutic efficiencies obtained are better than the treatment efficiencies obtained with the administration of the free drugs and it was found that the low doses are required for the treatment once the drug molecules are formulated with these nanocapsules. Results directed us that in future these nanocapsules may have a huge clinical potential to treat various diseases.

Abstract PR007: Homotypic cell-cell fusion in TNBC cells downregulates cancer testis antigen MAGEC2 and increases therapeutic sensitivity to paclitaxel and doxorubicin

Abstract Cell-cell fusion between cancer cells and genetically mismatched populations has been shown to generate genotypic diversity and more pathological phenotypes which may alter tumor evolution and treatment response, but little is known on how the process of cell-cell fusion itself affects progeny from genetically matched cells. To investigate the hypothesis that the process of cell-cell fusion alone leaves durable effects in daughter cell populations derived from homotypic cell-cell fusion, GFP and mCherry labeled cells from the triple negative breast cancer (TNBC) cell line HCC1806 were co-cultured and subclones were generated through single-cell FACS isolation of spontaneously generated fusion progeny (double positive cells; n=9) and control cells (single positive cells; n=9) from the same population. Subclonal populations were expanded for >19 generations (1 to ~106 cells) before experimental measurements and perturbations. The morphological features, growth rate, chemotactic ability, motility, karyotype, gene expression profile, and chemotherapeutic response of each fusion and control subclone were characterized. We find no durable differences between fusion and control subclones in terms of growth rate, chemotactic ability, and response to 5-fluorouracil. However, subclonal populations generated from homotypic cell-cell fusion show remarkably consistent and statistically significant increased sensitivity to paclitaxel and doxorubicin compared to control subclones. Further, differential expression analysis of RNA sequencing data revealed loss of expression of cancer-testis antigen (CTA) MAGEC2 in fusion cells compared to controls. The MAGE family of CTAs have shown promise as an immunotherapeutic target, but this study suggests that loss or downregulation of MAGEC2 after homotypic cell-cell fusion could allow immune escape. While fusion progeny may be more protected from immune threats, fusion progeny are simultaneously sensitized to paclitaxel and doxorubicin which highlights the potential need for standard chemotherapeutics after CTA immunotherapy. Where current literature posits cell-cell fusion as a pathologically disruptive event which may increase therapeutic resistance, this is the first study to show that fusion progeny are counterintuitively more sensitive to standard chemotherapeutics and to indicate homotypic cell-cell fusion as a potential mechanism of immunotherapeutic escape. Citation Format: Andrea Gardner, Lan Zheng, Kennedy Howland, Chisom Iloegbunam, Patrik Barrera, Carolina De Santiago, Amy Brock. Homotypic cell-cell fusion in TNBC cells downregulates cancer testis antigen MAGEC2 and increases therapeutic sensitivity to paclitaxel and doxorubicin [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR007.

Disentangling ERBB Signaling in Breast Cancer Subtypes-A Model-Based Analysis.

Simple SummaryBreast cancer subtypes are characterized by the expression and activity of estrogen-, progesterone- and HER2-receptors and differ by the treatment as well as patient prognosis. Tumors of the HER2-subtype overexpress this receptor and are successfully targeted with anti-HER2 therapies. We wanted to know if the HER2-receptor and the downstream signaling network act similarly also in the other subtypes and if this network could potentially be a therapeutic target beyond the HER2-positive subtype. To this end, we quantitatively assessed the wiring of signaling events in the individual subtypes to unravel the characteristics of HER-signaling. Our data along with a model-based analysis suggest that major parts of the intracellular signal transduction network are unchanged between the different breast cancer subtypes and that the clinical differences mostly come from the different levels at which these receptors are present in tumor cells as well as from the particular mutations that are present in individual tumors.Targeted therapies have shown striking success in the treatment of cancer over the last years. However, their specific effects on an individual tumor appear to be varying and difficult to predict. Using an integrative modeling approach that combines mechanistic and regression modeling, we gained insights into the response mechanisms of breast cancer cells due to different ligand–drug combinations. The multi-pathway model, capturing ERBB receptor signaling as well as downstream MAPK and PI3K pathways was calibrated on time-resolved data of the luminal breast cancer cell lines MCF7 and T47D across an array of four ligands and five drugs. The same model was then successfully applied to triple negative and HER2-positive breast cancer cell lines, requiring adjustments mostly for the respective receptor compositions within these cell lines. The additional relevance of cell-line-specific mutations in the MAPK and PI3K pathway components was identified via L1 regularization, where the impact of these mutations on pathway activation was uncovered. Finally, we predicted and experimentally validated the proliferation response of cells to drug co-treatments. We developed a unified mathematical model that can describe the ERBB receptor and downstream signaling in response to therapeutic drugs targeting this clinically relevant signaling network in cell line that represent three major subtypes of breast cancer. Our data and model suggest that alterations in this network could render anti-HER therapies relevant beyond the HER2-positive subtype.