MCF-7 Xenografts Research Articles

XS-2, a novel potent dual PI3K/mTOR inhibitor, exhibits high in vitro and in vivo anti-breast cancer activity and low toxicity with the potential to inhibit the invasion and migration of triple-negative breast cancer

Breast cancer has become the most commonly diagnosed cancer, surpassing lung cancer, with 2.26 million new breast cancers worldwide in 2020. Hence, there is an urgent need to develop effective molecularly targeted therapeutic drugs to treat breast cancer. In this paper, we designed, synthesized and screened a novel thiophene-triazine derivative, XS-2, as a potent dual PI3K/mTOR inhibitor for the treatment of breast cancer. Also, XS-2 was found to be potentially effective against triple-negative breast cancer (TNBC) in vitro during the investigation. We evaluated the in vitro inhibitory effect of XS-2 on 10 cancer cell lines by MTT and 6 kinases to investigated its in vivo antitumor activity in MCF-7 xenograft tumor-bearing BALB/c nude mice. In addition, the in vitro/in vivo toxicity to mice was also assessed by hemolytic toxicity, H&E staining and blood biochemical analysis. In order to investigate the antitumor mechanism of XS-2, a series of experiments were carried out in vitro/in vivo animal model and molecular biological levels such as the cell cycle and the apoptosis assay, real-time PCR, western blot, docking and molecular simulations analysis, etc. What’s more, wound healing assay, Transwell and Western Blot were applied to explore the ability of XS-2 to inhibit the cell invasion and migration. The results showed that XS-2 exhibited strong antitumor activity both in vitro and in vivo. The inhibitory activities of XS-2 on ten cancer cell lines were ranging from 1.07 ± 0.11 to 0.002 ± 0.001 μM, which were 1565 times better than that of the lead compound GDC-0941, inhibitory activities against PI3Kα and mTOR kinases were 291.0 and 60.8 nM, respectively. Notably, XS-2 not only showed significant in vivo antitumor activity and low toxicity, with the tumor inhibition rate of 57.0 %, but also exhibited strong inhibitory in the expression of related proteins of PI3K pathway in tumor tissues. In addition, XS-2 significantly inhibited breast cancer MCF-7 and MDA-MB-231 cells in a concentration- and time-dependent manner, and inhibited the migration and invasion ability of MDA-MB-231 and MCF-7 cells. More than that, XS-2 could inhibit the increase of the expression levels of N-cadherin and vimentin upregulated by EGF and reversed the E-cadherin expression down regulated by EGF, resulting in inhibiting EMT in MCF-7 and MDA-MB-231 cells. The results showed that XS-2 was expected to be successfully developed as a high-efficiency and low-toxicity breast cancer therapeutic drug with the potential to inhibit the invasion and migration of TNBC. This provides a new research idea for the treatment of TNBC, which is of great significance.

Novel zinc(II)−curcumin molecular probes bearing berberine and jatrorrhizine derivatives as potential mitochondria-targeting anti-neoplastic drugs

Berberine and jatrorrhizine are major bioactive components that are emerging as potential anti-cancer drugs. However, no zinc(II) – berberine/jatrorrhizine – curcumin compounds have been reported in the literature to date. Therefore, the molecular mechanisms associated with their cytotoxicity remain unexplored. To investigate the potential mitochondria-targeting ability, anti-neoplastic activity, and utility in cell imaging of berberine and jatrorrhizine derivates, four novel zinc(II) complexes, [Zn(Ber)(H2O)Cl2] (Zn(Ber)), [Zn(Ber)(Cur)Cl] (Zn(CurBer)), [Zn(Jat)(H2O)Cl2] (Zn(Jat)), and [Zn(Jat)(Cur)Cl] (Zn(CurJat)) bearing the berberine-derived ligand 2,2,2-trifluoroacetate 10-methoxy-9-((9-((2-(pyridin-2-yl)ethyl)amino)nonyl)oxy) −5,6-dihydro- [1,3]dioxolo[4,5-g]isoquinolino [(Torre et al., 2015; de Ruijter et al., 2020) 3,23,2-a]isoquinolin-7-ium (Ber), the jatrorrhizine-derived ligand 2,2,2-trifluoroacetate 2,9,10-trimethoxy-3-((9- ((2-(pyridin-2-yl)ethyl)amino)nonyl)oxy)-5,6-dihydroisoquinolino [(Torre et al., 2015; de Ruijter et al., 2020) 3,23,2-a]isoquinolin-7-ium (Jat), and/or curcumin (H-Cur) were first synthesised in this study. Zn(Ber), Zn(CurBer), Zn(Jat), and Zn(CurJat) showed higher cytotoxicity against human MCF-7 (breast adenocarcinoma) cells than did cisplatin, with IC50 values ranging from 0.21 to 4.45 μM. The anti-neoplastic activities of the zinc(II) – berberine/jatrorrhizine – curcumin complexes were in the following order: Zn(CurBer) > Zn(CurJat) > Zn(Ber) > Zn(Jat) > cisplatin > H-Cur > Ber > Jat > ZnCl2. Among these, Zn(CurBer) displayed the highest cytotoxicity (0.21 ± 0.06 μM). Furthermore, mechanistic investigations revealed that Zn(CurBer) and Zn(CurJat) could accumulate in the mitochondria, exhibit red fluorescence, and trigger mitophagy and apoptosis. In vivo anti-cancer evaluations also suggested that Zn(CurBer) inhibited MCF-7 xenograft tumour growth more effectively than cisplatin and Zn(CurJat). This is the first report describing the synthesis of zinc(II) – berberine/jatrorrhizine – curcumin complexes and their potential use as molecular probes and mitochondria-targeting anti-neoplastic drugs.

Effect of Bevacizumab on a Human Breast Cancer Model that Exhibited Palbociclib-resistance by RB Knockout.

Although CDK4/6 inhibitors have been increasingly used in combination with hormonal agents to treat hormone-receptor positive and human epithelial growth factor receptor 2-negative breast cancer, the mechanism of CDK4/6 inhibitor resistance and its impact on established therapy for post-resistance, especially bevacizumab combined with chemotherapy, are unclear. Sensitivity of RB knockout MCF7 clones to CDK4/6 inhibitors was evaluated in vitro. One RB knockout clone was subcutaneously implanted in nude mice and the effects of bevacizumab on volume and microvessel density (MVD) of tumors were investigated. Palbociclib did not exhibit antitumor efficacy against the RB knockout tumor, in contrast to the parental MCF7 xenograft model. Bevacizumab significantly exhibited antitumor efficacy and suppressed the MVD both in RB knockout and parental MCF7 xenograft models. Bevacizumab inhibited tumor growth by suppressing MVD in the CDK4/6 inhibitor-resistant tumor acquired due to RB loss, suggesting its efficacy also in patients after treatment with CDK4/6 inhibitors.

A novel form of docetaxel polymeric micelles demonstrates anti-tumor and ascites-inhibitory activities in animal models as monotherapy or in combination with anti-angiogenic agents.

Malignant ascites (MA) is caused by intraperitoneal spread of solid tumor cells and results in a poor quality of life. Chemotherapy is a common first-line treatment for patients with MA. Taxotere ® (DTX) is widely used in solid tumor therapies. However, the low water solubility and side effects caused by additives in the formulation restrict the clinical application of docetaxel. HT001 is a clinical stage docetaxel micelle developed to overcome the solubility issue with improved safety profiles. To support clinical development and expand clinical application of HT001, this study used in vitro and in vivo approaches to investigate the anti-tumor effects of HT001 when applied as monotherapy or in combination with anti-angiogenic agents. HT001 demonstrated comparable anti-proliferative activities as docetaxel in a broad range of cancer cell lines in vitro. Furthermore, HT001 suppressed tumor growth in a dose-dependent manner in A549, MCF-7, and SKOV-3 xenograft tumor mouse models in vivo. In a hepatocellular carcinoma H22 malignant ascites-bearing mouse model, HT001 presented a dose-dependent inhibition of ascites production, prolonged animal survival, and reduced VEGF levels. When dosed at 20 mg/kg, the HT001-treated group exhibited curative results, with no ascites formation in 80% of mice at the end of the study while all the mice in the vehicle control group succumbed. Similar results were obtained in HT001 treatment of mice bearing malignant ascites produced by human ovarian cancer ES-2 cells. Notably, the combination of HT001 with Endostar not only significantly reduced ascites production but also prolonged survival of H22 ascites-bearing mice. HT001 showed similar PK and tissue distribution profiles as DTX in non-rodent hosts. Collectively, these results demonstrate potent anti-tumor activity of HT001 in multiple solid tumor models or malignant ascites models, and reveal synergistic effects with anti-angiogenic agents, supporting the clinical development and clinical expansion plans for HT001.

The γ-Glutamylcyclotransferase Inhibitor Pro-GA Induces an Antiproliferative Effect Through the Generation of Mitochondrial Reactive Oxygen Species.

γ-Glutamylcyclotransferase (GGCT) is up-regulated in a broad range of cancers, including breast cancer, and GGCT inhibition has been shown to be a promising strategy for therapy. Herein, we evaluated the efficacy and mechanism of action of pro-GA, a GGCT enzymatic inhibitor, in MCF7 breast cancer cells. Proliferation was evaluated by WST-8 and trypan blue dye exclusion assays. Western blot analysis was conducted to examine the expression of cyclin-dependent kinase inhibitors (CDKI), including p21, p27, and p16. Induction of senescence was assessed by senescence-associated β-galactosidase staining. Generation of mitochondrial superoxide reactive oxygen species (ROS) was assessed using flow cytometry. The effect of N-acetylcysteine (NAC) on pro-GA dependent inhibition of proliferation, ROS generation, and senescence was also studied. The efficacy of systemic administration of pro-GA was evaluated in a MCF7 xenograft mouse model. Treatment with pro-GA inhibited proliferation of MCF7 cells, increased CDKI expression and mitochondrial ROS, and induced cellular senescence. We found that cotreatment with NAC restored proliferation in pro-GA treated cells. NAC similarly suppressed CDKI expression, mitochondrial ROS generation, and senescence induced by pro-GA. Furthermore, the systemic administration of pro-GA in an MCF7 xenograft model had significant antitumor effects without toxicity. Pro-GA may be a promising therapeutic agent for the treatment of breast cancer.

Identification of phenolics from miracle berry (Synsepalum dulcificum) leaf extract and its antiangiogenesis and anticancer activities.

Miracle berry is well-known for its ability to convert sour foods to sweet. In this study, the secondary metabolites of miracle berry leaves (MBL) were identified by UPLC-DAD-MS, and its antiangiogenesis and anticancer activities were evaluated by using a zebrafish model and the MCF-7 xenograft mouse model, respectively. The result showed that 18 phenolic compounds were identified in MBL extract, and dominated by the derivatives of quercetin and myricetin. The MBL extract showed low toxicity and high antiangiogenesis activity, it significantly inhibited the subintestinal vein vessels development in zebrafish at very low concentration. Furthermore, the MBL extract could promote the apoptosis of tumor cells and significantly inhibit the growth of MCF-7 xenograft tumor. In addition, the analysis of metabolites revealed that the MBL extract inhibited tumor growth by activating the metabolic pathways of unsaturated fatty acids and purines. Overall, this study suggests that MBL extract can be used as a natural anticancer adjuvant in the fields of functional foods.

Abstract 5642: Discovery of SCR-8079, a novel and selective CDK2/4/6 inhibitor overcomes the resistance to CDK4/6 inhibition and demonstrates broad anti-tumor activities in breast cancers

Abstract The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival and overall survival in HR positive and HER2-negative breast cancer in combination either with letrozole or fulvestrant. However, resistance to the CDK4/6 inhibition inevitably develops. Preclinical models and clinical transcriptome specimen analysis disclosed that abnormal activation of Cyclin E/CDK2 due to CCNE gene amplification was one of the major reasons to CDK4/6 inhibition resistance. And inhibition of CDK2 either by gene knockdown of CCNE or by pharmaceutic approach rescued the sensitivity of resistant tumor cells to CDK4/6 inhibition. To overcome the resistance, a novel and selective CDK2/4/6 inhibitor, SCR-8079 was developed. In biochemical assay, SCR-8079 was demonstrated high potency on inhibiting CDK2/4/6 with IC50 of 3 nM, 1 nM and 2 nM, respectively. And SCR-8079 showed good selectivity over CDK1/9 isoforms (>60 selectivity folds). SCR-8079 demonstrated closed inhibitory activities both on CDK4/6 sensitive and resistant lines. In MCF-7, a breast cancer cell line sensitive to PAL, SCR-8079 potently inhibited Rb phosphorylation (IC50, 53 nM) and cell proliferation (IC50, 28 nM). Meanwhile, SCR-8079 showed high potency on inhibiting on the growth of OVCAR3, an ovarian cancer cell line with elevated CDK2 activation and resistance to PAL. SCR-8079 inhibited Rb phosphorylation (IC50, 41 nM) and cell proliferation (IC50, 21 nM). In order to further mimic CDK4/6 resistance, a PAL resistant line (MCF-7R) was generated via induction of MCF-7 by gradient increased PAL. SCR-8079 potently inhibited MCF-7R cells growth (IC50, 3 nM) but PAL didn’t (IC50 >1 µM). Moreover, SCR-8079 showed high potency on growth inhibition in a broad of breast cancer cell lines, including HR-positive, Her2-positive and triple negative lines. SCR-8079 displayed good oral bioavailability in multiple pre-clinical species and robust anti-tumor activities both in OVCAR3 and MCF7 xenograft models. In summary, SCR-8079 is a selective CDK2/4/6 inhibitor, which overcomes the CCNE-mediated resistance to CDK4/6 inhibition. Broad anti-tumor activity suggests SCR-8079 central role in breast cancer treatment. Citation Format: Feng Zhou, Guimei Yang, Feng Tang, Yan Zhang, Wenqing Yang, Liting Xue, Ping Chen, Renhong Tang. Discovery of SCR-8079, a novel and selective CDK2/4/6 inhibitor overcomes the resistance to CDK4/6 inhibition and demonstrates broad anti-tumor activities in breast cancers [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 5642.

Abstract 2697: Inhibition of estrogen receptor signaling as a strategy for radiosensitization of ER+ breast cancers

Abstract Purpose: The estrogen receptor (ER) is expressed in over 80% of breast tumors and has been shown to be a significant driver of breast cancer (BC) pathogenesis and therefore a target of effective first-line therapies. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of ER+ BC, the effect of ET on tumor radiosensitization remains unclear, with concerns it may be radioprotective based on G1 cell arrest with ET treatment. Here we assessed the efficacy and mechanism of ER-mediated radiosensitization using various pharmacologic approaches in ER+ BC. Methods: Radiosensitization with ER inhibitors (tamoxifen [TAM], fulvestrant [FULV], AZD9496) was assessed using clonogenic survival assays. DNA damage was assessed by the neutral comet assay. Efficiency of homologous recombination (HR) or non-homologous end joining (NHEJ) as well as changes in cell cycle, apoptosis, and senescence were assessed. The efficacy of TAM with RT in vivo was assessed with an MCF-7 xenograft model. Results: The selective estrogen receptor modulator TAM radiosensitized ER+ MCF-7 (enhancement ratio [enhR]: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells but not ER-negative SUM-159 cells (enhR: 0.99-1.02). The selective estrogen receptor degrader (SERD) FULV had similar radiosensitization effects in MCF-7 (enhR: 1.33-1.76) and T47D cells (enhR: 0.97-2.81) with no radiosensitization observed in SUM-159 cells (enhR: 1.01-1.03). The novel oral SERD AZD9496 radiosensitized MCF-7 cells (enhR: 1.36-1.56). MCF-7 cells treated with TAM and RT had an increase in dsDNA breaks compared to RT alone as measured by the comet assay (p<0.05) and a decrease in NHEJ-mediated repair with TAM (p<0.05). No changes were observed in HR-mediated repair by Rad51 foci or a reporter (p=NS). RT alone and in combination with TAM or FULV induced similar levels of cell cycle arrest, suggesting that radiosensitization with the combination therapy is cell-cycle independent. There were no significant changes in apoptosis with TAM, FULV, RT, or the combination (p=NS). Although TAM or FULV did induce senescence, ET with RT increased senescence induction (p<0.05). In vivo, combination RT and TAM led to a significant delay in days to tumor doubling (control: 17, TAM: 40, RT: 32, TAM+RT: undefined; p<0.0001), and a significant difference in tumor growth between mice treated with TAM or RT alone compared combination treatment, with no increased toxicities or skin lesions from the combination treatment. Conclusion: Our data suggest that TAM, FULV, or AZD9496 can radiosensitize ER+ breast tumors, and these agents with RT may be more effective for radiosensitization. This work also supports further clinical investigation of the timing of RT for patients receiving ET, including using ET during RT, especially as initiating ET prior to RT has been increasingly utilized as a bridging therapy followed by concurrent ET+RT during the COVID-19 pandemic. Citation Format: Anna R. Michmerhuizen, Lynn M. Lerner, Andrea M. Pesch, Connor Ward, Rachel Schwartz, Kari Wilder-Romans, Meilan Liu, Charles Nino, Kassidy Jungles, Ruth Azaria, Alexa Jelley, Nicole Zambrana Garcia, Alexis Harold, Amanda Zhang, Bryan Wharram, Daniel F. Hayes, James M. Rae, Lori J. Pierce, Corey W. Speers. Inhibition of estrogen receptor signaling as a strategy for radiosensitization of ER+ breast cancers [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 2697.

Abstract 1865: Tumor microenvironment ameliorating effect of E7130 sensitized ER+ breast cancer against fulvestrant

Abstract Introduction and objectives: E7130 is a novel anti-cancer agent created from a total synthetic study of the natural compound norhalichondrin B, and is evaluated in a clinical study currently (NCT03444701). Previous pre-clinical study reported that E7130 had both cancer cell proliferation inhibitory activity and tumor microenvironment ameliorating activities featuring vascular remodeling effect and anti-CAF (cancer-associated fibroblasts) effect. Intratumoral hypoxia induces tumor malignancy and causes resistance against anti-cancer drugs including anti-estrogen agents (eg fulvestrant). It is hypothesized that vascular remodeling effect of E7130 mitigates hypoxia-induced resistant mechanism of ER (estrogen receptor) + breast cancer against anti-estrogen agents, resulting in improving anti-tumor activity of fulvestrant in combination with E7130. Experimental procedure: ERα expression levels in human ER+ breast cancer cell lines under hypoxic or normoxic culture condition were examined by FCM (flow cytometry) analysis. The histological changes after treatment with E7130 were examined by immunohistochemical analyses in the MCF-7 subcutaneous xenograft model. Anti-tumor activity of E7130 in combination with fulvestrant were examined in concurrent and in sequential treatment in the MCF-7 model. Summary: In vitro hypoxic culture condition down-regulated ERα expression in MCF-7 and T-47D compared with normoxic condition. E7130 had no effect on the ERα expression level in the cancer cells in vitro. On the contrary, E7130 increased ERα expression level in the cancer cells concomitantly with an increase of CD31-positive endothelial cell number (ie vascular remodeling effect) in MCF-7 xenograft tumor. These results suggested that E7130 up-regulated ERα expression in cancer cells not by cell-autonomous manner but by mitigating intratumoral hypoxia via its vascular remodeling effect. Concurrent combination therapy of E7130 at 90 μg/kg with fulvestrant at 250 mg/kg significantly inhibited the tumor growth compared with each monotherapy. In sequential combination therapy, pretreatment of E7130 followed by fulvestrant showed superior anti-tumor activity to posttreatment of E7130 following fulvestrant. Severe body weight loss was not observed in any treatment groups. Conclusions: The vascular remodeling effect of E7130 increased ERα expression in cancer cells in vivo, and sensitized ER+ breast cancer against fulvestrant to achieve promising combinational anti-tumor activity. Our data provides compelling evidence that E7130 attenuates tumor malignancy by its tumor microenvironment ameliorating effects and improves current anti-cancer therapy in combination with other drugs. Citation Format: Takanori Abe, Satoshi Kawano, Ken Ito, Taro Semba, Kimiyo Tabata, Hiroki Jozawa, Osamu Asano, Yoshito Kishi. Tumor microenvironment ameliorating effect of E7130 sensitized ER+ breast cancer against fulvestrant [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 1865.

Discovery of Novel Bicyclic Phenylselenyl-Containing Hybrids: An Orally Bioavailable, Potential, and Multiacting Class of Estrogen Receptor Modulators against Endocrine-Resistant Breast Cancer.

Breast cancer (BC) is a multifactorial disease and is prone to drug resistance during treatment. In this study, we described a new class of multifunctional estrogen receptor (ER) modulators ground on a prerogative indirect antagonism skeleton (OBHS, oxabicycloheptene sulfonate) of ER containing a phenylselenyl group. Compound 34b showed significant antiproliferative activities against tamoxifen-sensitive (MCF-7) and -resistant (LCC2) cells. Moreover, hexokinase 1 (HK1) was identified as a direct target of 34b. Further mechanism investigations proved that 34b induced apoptosis, which was associated with mitochondrial dysfunction caused by the synergistic effects of downregulating mitochondrial-bound HK1 protein and promoting reactive oxygen species generation. In vivo, 34b had a favorable pharmacokinetic profile with a bioavailability of 23.20% and exhibited more potent tumor suppression than tamoxifen both in MCF-7 and LCC2 tumor xenograft models. Collectively, our studies showed that 34b is a promising new multifunctional candidate compound for ERα+ BC treatment, particularly for tamoxifen-resistant BC.

New Cluster Analysis Method for Quantitative Dynamic Contrast-Enhanced MRI Assessing Tumor Heterogeneity Induced by a Tumor-Microenvironmental Ameliorator (E7130) Treatment to a Breast Cancer Mouse Model.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can provide insight into tumor perfusion. However, a method that can quantitatively measure the intra-tumor distribution of tumor voxel clusters with a distinct range of Ktrans and ve values remains insufficiently explored. Two-dimensional cluster analysis may quantify the distribution of a tumor voxel subregion with a distinct range of Ktrans and ve values in human breast cancer xenografts. Prospective longitudinal study. Twenty-two female athymic nude mice with MCF-7 xenograft, treated with E7130, a tumor-microenvironmental ameliorator, or saline. 9.4 Tesla, turbo rapid acquisition with relaxation enhancement, and spoiled gradient-echo sequences. We performed two-dimensional k-means clustering to identify tumor voxel clusters with a distinct range of Ktrans and ve values on Days 0, 2, and 5 after treatment, calculated the ratio of the number of tumor voxels in each cluster to the total number of tumor voxels, and measured the normalized distances defined as the ratio of the distance between each tumor voxel and the nearest tumor margin to a tumor radius. Unpaired t-tests, Dunnett's multiple comparison tests, and Chi-squared test were used. The largest and second largest clusters constituted 44.4% and 27.5% of all tumor voxels with cluster centroid values of Ktrans at 0.040 min-1 and 0.116 min-1 , and ve at 0.131 and 0.201, respectively. At baseline (Day 0), the average normalized distances for the largest and second largest clusters were 0.33 and 0.24, respectively. E7130-treated group showed the normalized distance of the initial largest cluster decreasing to 0.25, while that of the second largest cluster increasing to 0.31. Saline-treated group showed no change. A two-dimensional cluster analysis might quantify the spatial distribution of a tumor subregion with a distinct range of Ktrans and ve values. 1 TECHNICAL EFFICACY: Stage 1.

Gene Expression Changes by Diallyl Trisulfide Administration in Chemically-induced Mammary Tumors in Rats.

Diallyl trisulfide (DATS) was shown to be a potent inhibitor of luminal-type MCF-7 xenograft growth in vivo. The present study was conducted to determine the preventive effect of DATS administration using an N-methyl-N-nitrosourea (MNU)-induced rat mammary tumor model, which shares molecular resemblance to luminal-type human breast cancers. The DATS administration (50 mg/kg body weight, 5 times/week) was safe, but did not reduce mammary tumor latency, incidence, burden or multiplicity. Therefore, we conducted RNA-seq analysis using mammary tumors from control and DATS-treated rats (n = 3 for each group) to gain insights into lack of mammary tumor prevention by this phytochemical. The gene ontology and the Kyoto encyclopedia of genes and genomes pathway analyses of the RNA-seq data revealed upregulation of genes associated with ribosomes, translation, peptide biosynthetic/metabolic process, and oxidative phosphorylation but downregulation of genes associated with mitogen-activated protein kinases. A total of 33 genes associated with ribosomes were significantly upregulated by DATS treatment, including RPL11 and RPS14. Western blotting confirmed upregulation of RPL11 and neurofascin protein expression in mammary tumors from DATS-treated rats when compared to controls. A statistically significant increase in protein level of c-Jun N-terminal kinase 2 was also observed in tumors from DATS-treated rats when compared to controls. On the other hand, expression of complex I subunits NDUFV1 or NDUFS1 was not affected by DATS treatment. These results offer potential explanations for ineffectiveness of DATS in the chemically-induced rat mammary tumor model. Inhibitors of the proteins upregulated by DATS may be needed to improve chemopreventive efficacy of this phytochemical.

Site-Specific Integration of TRAIL in iPSC-Derived Mesenchymal Stem Cells for Targeted Cancer Therapy.

Mesenchymal stem cells (MSCs) are a promising cellular vehicle for transferring anti-cancer factors to malignant tumors. Currently, a variety of anti-cancer agents, including the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been loaded into MSCs derived from a range of sources through different engineering methods. These engineered MSCs exhibit enormous therapeutic potential for various cancers. To avoid the intrinsic defects of MSCs derived from tissues and the potential risk of viral vectors, TRAIL was site-specifically integrated into the ribosomal DNA (rDNA) locus of human-induced pluripotent stem cells (iPSCs) using a non-viral rDNA-targeting vector and transcription activator-like effector nickases (TALENickases). These genetically modified human iPSCs were differentiated into an unlimited number of homogeneous induced MSCs (TRAIL-iMSCs) that overexpressed TRAIL in both culture supernatants and cell lysates while maintaining MSC-like characteristics over continuous passages. We found that TRAIL-iMSCs significantly induced apoptosis in A375, A549, HepG2, and MCF-7 cells in vitro. After intravenous infusion, TRAIL-iMSCs had a prominent tissue tropism for A549 or MCF-7 xenografts and significantly inhibited tumor growth through the activation of apoptotic signaling pathways without obvious side effects in tumor-bearing mice models. Altogether, our results showed that TRAIL-iMSCs have strong anti-tumor effects in vitro and in vivo on a range of cancers. This study allows for the development of an unlimited number of therapeutic gene-targeted MSCs with stable quality and high homogeneity for cancer therapy, thus highlighting a universal and safe strategy for stem cell-based gene therapy with high potential for clinical applications.

Estrogen receptor inhibition mediates radiosensitization of ER-positive breast cancer models

Endocrine therapy (ET) is an effective first-line therapy for women with estrogen receptor-positive (ER + ) breast cancers. While both ionizing radiation (RT) and ET are used for the treatment of women with ER+ breast cancer, the most effective sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we sought to understand the effects of inhibiting estrogen receptor (ER) signaling in combination with RT in multiple preclinical ER+ breast cancer models. Clonogenic survival assays were performed using variable pre- and post-treatment conditions to assess radiosensitization with estradiol, estrogen deprivation, tamoxifen, fulvestrant, or AZD9496 in ER+ breast cancer cell lines. Estrogen stimulation was radioprotective (radiation enhancement ratios [rER]: 0.51–0.82). Conversely, when given one hour prior to RT, ER inhibition or estrogen depletion radiosensitized ER+ MCF-7 and T47D cells (tamoxifen rER: 1.50–1.60, fulvestrant rER: 1.76–2.81, AZD9496 rER: 1.33–1.48, estrogen depletion rER: 1.47–1.51). Combination treatment resulted in an increase in double-strand DNA (dsDNA) breaks as a result of inhibition of non-homologous end joining-mediated dsDNA break repair with no effect on homologous recombination. Treatment with tamoxifen or fulvestrant in combination with RT also increased the number of senescent cells but did not affect apoptosis or cell cycle distribution. Using an MCF-7 xenograft model, concurrent treatment with tamoxifen and RT was synergistic and resulted in a significant decrease in tumor volume and a delay in time to tumor doubling without significant toxicity. These findings provide preclinical evidence that concurrent treatment with ET and RT may be an effective radiosensitization strategy.

Abstract P5-05-04: Inhibition of HER2+ tumor growth with SDX-7320, a novel MetAP2 inhibitor, alone and in combination with capivasertib/AZD-5363: Reduced expression of hypoxia-inducible and innate-immune system genes

Abstract MetAP2 inhibitors have shown clinical anti-tumor activity, but CNS side effects and poor drug-like properties have generally limited their development. SDX-7320 is a polymer-drug conjugate of a novel fumagillin-derived MetAP2 inhibitor (SDX-7539) attached via a cleavable linker to a hydroxypropylmethacrylamide (HPMA) polymer backbone. This conjugation approach is intended to improve safety (i.e., limit CNS penetration) while also improving biodistribution and pharmacokinetics relative to small molecule MetAP2 inhibitors. SDX-7320 recently completed a phase I safety trial in late-stage cancer patients (NCT02743637) and was well-tolerated with no apparent CNS side effects. In prior models of breast cancer, SDX-7320 significantly inhibited the growth of syngeneic EO771 triple-negative breast cancers (TNBC) accelerated by obesity/metabolic dysfunction (i.e., “metabo-oncology”) and also synergized with the PI3Kα inhibitor alpelisib/Piqray® in ER+/Her2- MCF-7 xenografts. The objectives of these experiments were to determine the effect of SDX-7320 on the growth of Her2+ xenografts alone or in combination with the Akt/mTOR inhibitor capivasertib/AZD-5363 and to determine if SDX-7320 could attenuate hyperglycemia induced by capivasertib/AZD-5363. Female athymic, nude mice (9 weeks of age) were surgically implanted with slow-release estrogen pellets (90-day, 0.72 mg, inter-scapular) and three days later 2 x 107 BT474 cells (in Matrigel/RPMI-1640, 50/50, v/v) were injected into the fourth mammary gland. When the group mean tumor volume exceeded 100 mm3, treatment with test agents commenced. SDX-7320 (sc/q4d, 6, 12 mg/kg) exerted significant, dose-dependent inhibition of BT-474 tumor growth (TGI of 61 and 88% respectively), whereas capivasertib/AZD-5363 (po, qd, 100, 200 mg/kg) exhibited significant anti-tumor activity only at 200 mg/kg (54% TGI). The combination of SDX-7320 (12 mg/kg) plus capivasertib/AZD-5363 (200 mg/kg) produced additive effects in that a greater number of mice had tumor regression relative to mice treated with either SDX-7320 or capivasertib/AZD-5363 alone. Tumor tissue from a subset of treatment groups (Vehicle, SDX-7320-12 mg/kg, SDX-7320-12 mg/kg + AZD-5363-200 mg/kg, and AZD-5363-200 mg/kg) was snap frozen and stored at -80oC. PolyA+ RNA was isolated, cDNA libraries were constructed and RNASeq was conducted (range of 20-30 reads per million (RPM) base pairs). Analysis of RNASeq data showed that SDX-7320 alone significantly attenuated the expression of hypoxia-induced genes as well as genes related to the innate immune system. Based on these results, we conclude that SDX-7320 affected tumor growth by limiting the ability of tumors to adapt to a hypoxic microenvironment and by altering the innate tumor-immune microenvironment. In normal male, C57Bl/6 mice, capivasertib/AZD-5363 (200 mg/kg, po) elevated blood glucose after a single dose. Pretreatment with either one dose (24 hours before the Akt/mTOR inhibitor) or multiple doses (Q4D beginning 14 days prior to Akt/mTOR inhibitor) of SDX-7320 (8 mg/kg) significantly inhibited hyperglycemia induced by capivasertib/AZD-5363. In summary SDX-7320 significantly inhibited the growth of BT-474 xenografts, alone and in combination with capivasertib/AZD-5363, which was associated with suppression of hypoxia-induced genes, as well as genes of the innate immune system. In addition, SDX-7320 improved the safety profile of capivasertib/AZD-5363 by attenuating Akt/mTOR inhibitor-induced hyperglycemia, providing further support for the clinical exploration of SDX-7320 in combination with Akt/mTOR inhibitors in Her2+ breast cancer. Citation Format: Peter Cornelius, Benjamin Mayes, Pierre Dufour, Mark Kalinich, Jonathan Wang, Sara Little, Bradley Carver, James Shanahan. Inhibition of HER2+ tumor growth with SDX-7320, a novel MetAP2 inhibitor, alone and in combination with capivasertib/AZD-5363: Reduced expression of hypoxia-inducible and innate-immune system genes [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 P5-05-04.

Abstract P4-02-11: A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer

Abstract BACKGROUND: Estrogen Receptor (ER) + breast cancers (BC) comprise over 70% of BC cases and can be targeted via ER modulated therapies. Despite this, ER+BC patients can experience recurrence within 20 years and the majority of BC related deaths can be attributed to metastatic ER+BC. These distant metastases commonly become diagnosed as endocrine therapy resistant. Thus, there is an unmet need to identify novel biomarkers for treating ER+ patients with metastasis. We have identified a tumor suppressor, Singleminded 2s (SIM2s), expressed in breast epithelial cells that inhibits EMT and metastasis, and is downregulated in the progression of breast disease. Our previous studies found that loss of SIM2s expression results in downregulation of ESR1 and increased basal markers in the MCF7 (ER+BC) cell line. Likewise, Semaphorin 7a (SEMA7A) expression is associated with decreased overall survival in ER+BC patients. Patients with SEMA7Ahi tumors treated with Tamoxifen exhibit shortened relapse free survival. SEMA7A is a downstream target of both NFKB and PTGS2/COX-2; and our previously published results establish a cross-talk between NFKB and SIM2s to regulate PTGS2 via interactions with the AKT pathway. Moreover, COX-2 inhibition is associated with better prognosis for breast cancer patients. Therefore, we are investigating how SIM2s may repress AKT signaling in SEMA7A driven ER resistance and metastasis. Methods: We performed in silico analysis of the SEMA7A promoter using the ConTra web server. For in vitro assays, we examined estrogen dependent differences in SIM2s, ER, and SP1 binding utilizing luciferase reporter assays using intact promoter and SIM2s site deletion mutants (Invitrogen) in MCF7 cells. Using immunoblotting and qPCR analysis we also assessed expression of SEMA7A, ESR1, PI3K/AKT signaling genes and EMT associated genes in Crispr/Cas9 SIM2s knockout (SIM2KO) MCF7 cells. Treatment of MCF7 cells with exogenous SEMA7A was used to observe possible changes in SIM2s expression and SIM2s promoter activity. For in vivo assays, we used immunohistochemistry (IHC) to assess SIM2s in SEMA7A low and high expressing MCF7 xenografts. To examine the relationship between SIM2s and ER, SEMA7A-/- (KO) mice were crossed with C57/BL6 MMTV-PYMT mice. Results: In silico analysis of the SEMA7A promoter via ConTra revealed four potential SIM2 response elements containing the SIM2s central midline elements (CME) binding motif and highly conserved estrogen response element half-sites adjacent to two specificity protein 1 consensus bindings sites. Luciferase reporter assays in MCF7 cells confirmed SIM2s represses basal and estrogen induced-SEMA7A promoter activity. Moreover, SIM2s was unable to repress the SEMA7A promoter activity with CME mutation. MCF7 SIM2KO cells exhibit increased SEMA7A promoter activity, SEMA7A, AKT signaling, EMT signatures and decreased ESR1, CDH1, and PTEN protein and mRNA expression. Both SIM2s expression and SIM2s promoter activity are significantly decreased with the addition of exogenous SEMA7A. Moreover, MCF7 xenografts reveal reciprocal expression of SIM2s and SEMA7A. Additionally in MMTV-PyMT mice, SIM2s expression is lost as tumors transition from pre-malignant to invasive phenotypes, yet we observe maintenance of SIM2s and ER in SEMA7A-/-;PyMT mice. Conclusion: These findings establish a regulatory relationship between SIM2s and SEMA7A in ER+BC. SIM2s functions to downregulate SEMA7A mediated pro-tumor signaling and maintain ER expression, but can be turned off to allow for resistance in ER+BC. Given the observed reciprocal expression, our study suggests a SIM2s/SEMA7A switch which may confer resistance in ER+BC via AKT signaling through downregulation of PTEN. Thus, a SIM2s/SEMA7A switch may act as a prognostic indicator to provide therapeutic advantages in resistant ER+BC metastasis. Citation Format: Garhett L Wyatt, Lyndsey S Crump, Traci R Lyons, Weston W Porter. A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer [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 P4-02-11.

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.

Abstract P4-02-04: Endocrine therapy treatment radiosensitizes estrogen receptor-positive breast cancers

Abstract Purpose: Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of breast cancer (BC) pathogenesis and therefore a target of first-line therapies for ER-positive (ER+) BC patients. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of ER+ BC, the sequencing of therapy and the effect of ET on tumor radiosensitization remain unclear. Recently, this question has become much more clinically relevant when many physicians started offering ET as a bridging strategy to surgery and RT during the COVID-19 pandemic. Here we assessed the efficacy and mechanism of ER inhibition in ER+ BC in combination with RT in preclinical models. Methods: Clonogenic survival assays were used to assess radiosensitization. Inhibition of ER signaling was accomplished by treating ER+ MCF-7 and T47D cells with the selective ER modulator (SERM), tamoxifen, or the selective ER degrader (SERD), fulvestrant. The ER-negative SUM-159 cells were used as a negative control. DNA damage was assessed by the neutral comet assay. Efficiency of homologous recombination (HR) was measured by Rad51 foci or a GFP reporter system. Non-homologous end joining (NHEJ) efficiency was assessed with a pEYFP reporter. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow cytometry. Senescence was measured using β-galactosidase staining. Western blotting was used to quantify expression of proteins and phospho-proteins involved in cell cycle and apoptosis. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo. Synergy was determined using the fractional tumor volume (FTV) method. Results: ER inhibition with tamoxifen radiosensitized ER+ MCF-7 (10-250 nM, enhR: 1.14-1.50) and T47D (500 nM-2.0 µM, enhR: 1.33-1.60) cells but not ER-negative SUM-159 cells (500 nM-2.0 µM, enhR: 0.99-1.02). ER degradation with fulvestrant had similar radiosensitization effects in MCF-7 (1-25 nM, enhR: 1.33-1.76) and T47D cells (0.5-5 nM, enhR: 0.97-2.81) with no radiosensitization observed in SUM-159 cells (1-25 nM, enhR: 1.01-1.03). MCF-7 cells treated with 500 nM tamoxifen and 4 Gy RT had an increase in dsDNA breaks compared to RT alone as measured by the comet assay (p<0.05), and there was a decrease in NHEJ-mediated repair with tamoxifen treatment (p<0.05). No changes were observed in HR-mediated repair by Rad51 foci or an HR reporter (p=NS). RT alone and in combination with tamoxifen and fulvestrant induced similar levels of cell cycle arrest, suggesting that radiosensitization with the combination therapy is a cell-cycle independent effect. In addition, there were no significant changes in apoptosis in MCF-7 or T47D cells with endocrine therapy, RT, or the combination (p=NS). Although treatment with ET did induce senescence in ER+ MCF-7 and T47D cells, the combination treatment of ET with RT induced senescence to a much greater level suggesting this mechanism may contribute to radiosensitization (p<0.05). In vivo, combination RT and tamoxifen led to a significant delay in time to tumor doubling (17 days in control, 40 days with tamoxifen alone, 32 days with RT alone, and undefined with combination; p<0.0001) and a significant difference in tumor growth between mice treated with tamoxifen or RT alone compared to mice treated with tamoxifen and RT with synergy noted with combination treatment (FTV 1.297). Conclusion: Our data suggest that ET can radiosensitize ER+ breast tumors, and ET with RT may be more effective for radiosensitization. Ongoing studies will address concurrent versus sequential ET with RT. This work also supports further clinical investigation of the timing of RT for patients receiving ET, especially as ET prior to RT is increasingly used as a bridging therapy during the COVID-19 pandemic. Citation Format: Anna R Michmerhuizen, Lynn Lerner, Andrea M Pesch, Connor Ward, Rachel Schwartz, Kari Wilder-Romans, Meilan Liu, Bryan Wharram, Alexis Harold, Ruth Azaria, Nicole Zambrana Garcia, Daniel F Hayes, James M Rae, Lori J Pierce, Corey W Speers. Endocrine therapy treatment radiosensitizes estrogen receptor-positive breast cancers [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 P4-02-04.

Abstract P5-16-10: RLY-2608: The first allosteric mutant- and isoform-selective inhibitor of PI3Kα, is efficacious as a single agent and drives regressions in combination with standard of care therapies in PIK3CA mutant breast cancer models

Abstract Inhibition of CDK4/6 combined with the estrogen receptor (ER) degrader fulvestrant significantly improves progression free survival and overall survival in advanced hormone receptor positive (HR+) breast cancer patients and is now the standard of care (SOC) in this disease. Up to 40% of HR+ breast cancers harbor PIK3CA mutations leading to activation of phosphoinositide 3-kinase alpha (PI3Kα), which has been associated with resistance to CDK4/6 inhibitors and fulvestrant. Therefore, PI3Kα inhibitor combinations with CDK4/6 inhibitors and/or fulvestrant are of high interest in HR+, PIK3CA mutant breast cancer. The therapeutic index of active site (orthosteric) inhibitors of PI3Kα has been limited by the dual issues of no clinically meaningful selectivity for mutant versus wild-type (WT) PI3Kα and off-isoform inhibitory activity. Alpelisib, the only approved orthosteric PI3Kα inhibitor, is emblematic of the class with toxicity related to inhibition of wild type PI3Kα and other PI3K isoforms resulting in sub-optimal inhibition of mutant PI3Kα, frequent discontinuation, and challenges in combining with CDK4/6 inhibitors. To overcome these limitations, we designed RLY-2608, the first allosteric, mutant, and isoform-selective inhibitor of PI3Kα. We solved the full-length cryo-EM structure of PI3Kα, performed long time-scale molecular dynamic simulations to elucidate conformational differences between WT and mutant PI3Kα, and leveraged these insights to enable the design of RLY-2608. RLY-2608 does not compete with orthosteric inhibitors for binding and associates 8x faster with mutant PI3Kα relative to WT PI3Kα. In biochemical assays, RLY-2608 inhibits kinase domain (H1047R) and helical domain (E542K, and E545K) mutant PI3Kα activity, demonstrating <10nM potency with 8-12x selectivity relative to WT. RLY-2608 is > 1000-fold selective over the β, δ, and γ PI3K isoforms in biochemical assays and demonstrates exquisite selectivity across a panel of 322 kinases, with no other kinases showing >50% inhibition. We performed in vitro combinations in two HR+ PIK3CA mutant cell lines (MCF7: E545K; T47D: H1047R) and observed synergy between RLY-2608 and fulvestrant or CDK4/6 inhibitors. In vivo, we tested combinations of RLY-2608 with fulvestrant and/or the CDK4/6 inhibitor abemaciclib in the MCF7 xenograft model. Oral administration of RLY-2608 in combination with fulvestrant led to improved efficacy compared to either agent alone in a dose-dependent manner, with regressions observed in the combination arms at all doses. Furthermore, the triple combination of RLY-2608, fulvestrant, and abemaciclib resulted in superior efficacy compared to either the RLY-2608 + fulvestrant or RLY-2608 + abemaciclib doublets, with deep regressions observed in the triple combination arm. In addition, in vivo combination efficacy with fulvestrant and CDK4/6 inhibitors (palbociclib or abemaciclib) was assessed in patient-derived xenografts harboring the PIK3CA H1047R or E545K mutation along with a second site PIK3CA minor mutation. In these studies, combination benefit was observed with doses of RLY-2608 significantly lower than the dose required for maximum efficacy as a single agent. RLY-2608 synergizes in vitro with both anti-estrogen and CDK4/6 inhibitors in cell models of HR+/PIK3CA mutant breast cancer. RLY-2608 can be combined with fulvestrant and CDK4/6 inhibitors in vivo with tumor regressions observed in both cell- and patient-derived xenograft models. The pre-clinical profile of RLY-2608 supports the clinical development of RLY-2608 both in single agent and combination clinical trials in patients with PIK3CA mutant tumors, including HR+/PIK3CA mutant breast cancer. Citation Format: Ermira Pazolli, Randy Kipp, Alessandro Boezio, Hakan Gunaydin, Amanda Iskandar, Matthew Zubrowski, Bret Williams, Kelley Shortsleeves, Alexandre Larivee, Tom McLean, Klaus Michelsen, Hongtao Zeng, Jonathan LaRochelle, Joe Manna, Lucian DiPietro, Andre Lescarbeau, Mary Mader, Bindu Bennet, Jeremy Wilbur, Qi Wang, Levi Pierce, Iain Martin, James Watters, Pascal Fortin, Donald Bergstrom. RLY-2608: The first allosteric mutant- and isoform-selective inhibitor of PI3Kα, is efficacious as a single agent and drives regressions in combination with standard of care therapies in PIK3CA mutant breast cancer models [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 P5-16-10.

Abstract P5-06-08: Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis

Abstract Background: Metastatic disease is the foremost cause of breast cancer (BC) related mortality in women. One of the crucial challenges in reducing metastasis-related mortality is in identifying and understanding why certain BCs metastasize and recur. Although cancer cell-intrinsic factors are a key determinant in BC metastasis, host-intrinsic factors like the cells of the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs) are what might be driving certain BCs to metastasize and recur. We recently showed that CAFs from the primary (TME) enter into circulation as cCAFs, form heterotypic clusters with tumor cells, and arrive at metastatic sites. This interaction between CAFs and tumor cells is crucial in furthering the metastatic cascade. However, the functional heterogeneity of the different CAF phenotypes and how that impacts BC metastasis is unknown. Stromal derived factor-1 (SDF-1/CXCL12) is an important chemokine that is known to be involved in promoting tumor cell invasion and metastasis. In this study, we examine the functional differences between CAF isolated from different molecular subtypes of BC. Furthermore, we elucidate the role of CAF secreted SDF-1 as being one of the mechanisms by which a subset of CAF cells permanently reprograms poorly-metastatic MCF-7 cells to augment EMT-driven genes and become metastatic in vivo. Methods: We used primary CAF cell lines derived from BCs of different molecular subtypes and examined their global gene expression profiles using RNAseq. We used NSG mice xenografted with MCF-7 BC cells and primary CAF cells to evaluate the contribution of two different CAF phenotypes in promoting BC progression and metastasis. We developed dissociated tumor cell lines from CAF co-injected MCF-7 xenografts to determine the molecular changes that CAFs impart to MCF-7 cells in vivo. Using limiting dilution tumor xenograft assays and gene expression profiles we examined the molecular pathways, BC stemness markers, and tumor-initiating capacity of the dissociated tumor cell lines. By long-term SDF-1 treatment of MCF-7 cells in vitro and using these cells in various RNASeq, in vitro, and in vivo assays we determined the role of SDF-1 in altering the phenotype of MCF-7 cells. Results: We found that CAFs from different molecular subtypes have a differential effect on tumor initiation, progression, and metastasis in xenograft assays. We also found that a subset of CAFs has the ability to make the poorly-metastatic MCF-7 cells metastatic in vivo. We also found that this subset of CAF cells has a higher expression of SDF-1 and the tumors formed with the co-injection of MCF-7 cells with this subset of CAF cells are enriched in cancer stem cell-like metastasis initiating cells. We also found that upon serial transplant, the tumors formed by CAF-reprogramed MCF-7 cells have a gene expression profile that shows enrichment for EMT-driven genes and these tumors are robustly metastatic. Furthermore, we found that the SDF-1/CXCR4 axis is one of the critical mechanisms by which this subset of CAF cells permanently reprograms the phenotype of the MCF-7 cells. Conclusions: CAFs are a highly heterogeneous population of cells in the breast TME. This study examines the contribution of the functional heterogeneity of the CAF cells in BC metastasis. We have found that a unique subset of CAF cells has the ability to reprogram a poorly metastatic BC cell to not only become metastatic but induce a permanent EMT-driven phenotypic shift in the BC cells. SDF-1 may be one of the mechanisms that drive this CAF-induced change and understanding the CAF functional landscape and its role in BC metastasis might be crucial to developing new therapeutic modalities to abrogate BC metastasis. Citation Format: Utsav Sharma, Jun Sun, Kelsie Medina-Saenz, Susan Bare, Philip Miller, Manuel Picon-Ruiz, Joyce Slingerland, Dorraya El-Ashry, Marc Lippman. Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis [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 P5-06-08.