Triple-negative Breast Cancer Xenograft Models Research Articles

ATR Inhibitor AZD6738 (Ceralasertib) Exerts Antitumor Activity as a Monotherapy and in Combination with Chemotherapy and the PARP Inhibitor Olaparib.

This detailed preclinical investigation, including pharmacokinetics/pharmacodynamics and dose-schedule optimizations, of AZD6738/ceralasertib alone and in combination with chemotherapy or PARP inhibitors can inform ongoing clinical efforts to treat cancer with ATR inhibitors.

Folate Receptor-Alpha Targeted 7x19 CAR-γδT Suppressed Triple-Negative Breast Cancer Xenograft Model in Mice.

Background Triple-negative breast cancer (TNBC) is the worst prognosis subtype of breast cancer due to lack of specific targets. Recent studies have shown that immunotherapy may solve that problem by targeting folate receptor-alpha (FRα). Methods Gene modified γδ T cells were manufactured to express FRa specific chimeric antigen receptor (FRa CAR) and secrete interleukin-7 (IL-7) and chemokine C–C motif ligand 19 (CCL19). CAR-γδT cells that secrete IL-7 and CCL19 (7 × 19 CAR-γδT) were evaluated for their antitumor activity both in vitro and in vivo. Results 7 × 19 CAR-γδT showed remarkable antitumor activity in vitro. Combined with PBMC, 7 × 19 CAR-γδT inhibited TNBC xenograft model growth superiorly compared with single-application or conventional CAR-γδT cells. Histopathological analyses showed increased DC or T cells infiltration to tumor tissues. Conclusion Taken together, our results showed that 7 × 19 CAR-γδT have remarkable anti-TNBC tumor activity and showed a broad application prospect in the treatment of incurable TNBC patients.

Abstract P5-05-08: CXCL16 as a potential therapeutic target of triple-negative breast cancer

Abstract CXCL16, as a chemotactic cytokine, promotes cancer proliferation, migration, and immune cell infiltration in tumor microenvironment. However, CXCL16 has been shown to play a conflicting role in breast cancer. The aim of this study was to investigate the therapeutic potential of CXCL16 in breast cancer. Breast cancer patients (n=1097) from the TCGA dataset were divided into two group according to the CXCL16 expressions, the CXCL16low (n=548) and the CXCLhigh (n=549) groups. Mean diagnostic ages were similar between groups (59 ± 13.5 vs 58 ± 12.9 years, P=0.233). The ratio of triple-negative breast cancer (TNBC) was significantly higher in the CXCLhigh than that of the CXCL16low groups (24.8 vs 6.8%, P<0.001), while the presence of distant metastasis or overall survivals were not different between groups. Interestingly, gene scoring analysis showed that oncogenic molecules including M2-macrophage- (r=0.36, P<0.001), efferocytosis- (r=0.42, P<0.001), and chemokine- (r=0.46, P<0.001) related gene sets showed significantly strong positive relations with the CXCL16 expressions in all patients including both TNBC (n=924) and non-TNBC (n=173). Additionally, the gene set score from PI3K/Akt pathway showed positive relation with the CXCL16 expressions in the non-TNBC (r=022, P<0.001) but not in the TNBC group. Next, a xenograft mouse model of TNBC was established with MDA-MB-231 cells, and anti-mouse CXCL16 antibody (designated as aCXCL16 group) or control IgG (designated as control group) were intravenously injected (25µg/100µl), twice per week. During 3 weeks, tumor growth was significantly delayed in the aCXCL16 than the control group by 37%. In conclusion, CXCL16 is highly expressed in TNBC and positively correlates with M2-macrophage related genes. Neutralizing CXCL16 by anti-CXCL16 shows therapeutic effects on the xenograft model of TNBC, suggesting that CXCL16 can be a potential therapeutic target for breast cancer. Citation Format: Sun Wook Cho, Mi Gyeong Jang, Hyun Jin Sun, Han Sai Lee, Young Shin Song, Seong Keun Kim. CXCL16 as a potential therapeutic target of triple-negative 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 P5-05-08.

Abstract OT2-01-01: Phase 1 study of ST101, a first-in-class peptide antagonist of CCAAT/enhancer-binding protein β (C/EBPβ), in patients with advanced solid tumors, with a phase 2 expansion in patients with hormone receptor positive breast cancer (HR+ BC)

Abstract Background: CCAAT/Enhancer Binding Protein Beta (C/EBPβ) is a transcription factor that is active during embryofetal development but held in an inactive state in most mature cells. C/EBPβ is upregulated or overactivated in multiple cancers, where it inversely correlates with disease prognosis. In breast cancer, C/EBPβ drives the expression of factors that promote tumor survival, proliferation and inhibit differentiation. ST101 is a cell-penetrating all D amino acid peptide antagonist of C/EBPβ. ST101 exposure inhibits C/EBPβ target gene expression, leading to selective tumor cell death in multiple human cancer cell lines, including hormone receptor positive breast cancer (HR+ BC) and triple negative breast cancer (TNBC), without impacting normal cell viability. In vivo, ST101 displays rapid uptake into multiple organs, the ability to cross the blood-brain barrier, and a long plasma half-life due to its resistance to proteolytic degradation. Potent ST101 anti-tumor activity, demonstrated by dose-dependent inhibition of tumor growth in subcutaneous HR+ and orthotopic TNBC xenograft models in vivo, supported advancing ST101 into clinical development.Trial design: This phase 1-2 study uses a standard 3+3 design with dose doubling for the first 4 dose levels then 50% escalations thereafter. The recommended phase 2 dose will be used in 4 expansion cohorts in specific tumor types, including HR+ BC. Patients receive intravenous ST101 once weekly.Eligibility criteria: The dose-escalation phase is enrolling patients ≥18 years of age with advanced, unresectable metastatic solid tumors refractory to or intolerant of other therapeutic options. In expansion, patients with HR+ BC must have progressed after 1-3 prior hormone-based therapies. Previous treatment with CDK 4/6 inhibitor, mTOR inhibitor, or chemotherapy is allowed as monotherapy or in combination. Specific aims: The primary objective of phase 1 is to evaluate safety and tolerability of ST101. Secondary objectives include the recommendation of a dose and regimen of ST101 for further evaluation, analysis of pharmacokinetics, assessment of several pharmacodynamic measures, and to assess preliminary efficacy. Statistical design: The recommended phase 2 dose will be used in a 15-30 patient HR+ BC expansion cohort, with a Simon 2-stage design, which requires one response to expand the cohort to 30 patients. Up to 120 patients are planned in a total of four expansion cohorts, which should be enrolling by Q3 2021.Accrual: We began recruitment in August 2020. Enrollment is ongoing, and by July 2021, 18 patients were recruited in five dose-escalation cohorts up to 6 mg/kg; a 6th cohort (9 mg/kg) is ongoing. Dose escalation should be complete by Sept 2021, and the phase 2 portion in the HR+ BC cohort will be underway (n=15-30). Please contact rob.michel@bexonclinical.com if you have a specific interest in this trial. Citation Format: Alice S Bexon, Hendrik-Tobias Arkenau, Jeff Evans, Gerald S Falchook, Stefan N Symeonides, Meredith A McKean, Elisa Fontana, Manojkumar Bupath, Alistair McLaren, Sreenivasa Chandana, Tze-en Ding, Emerson A Lim, Jim Rotolo, Gina Capiaux, Rob Michel, Stephen Kaesshaefer, Nehal J Lakhani. Phase 1 study of ST101, a first-in-class peptide antagonist of CCAAT/enhancer-binding protein β (C/EBPβ), in patients with advanced solid tumors, with a phase 2 expansion in patients with hormone receptor positive breast cancer (HR+ BC) [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 OT2-01-01.

PRMT1 Regulates EGFR and Wnt Signaling Pathways and Is a Promising Target for Combinatorial Treatment of Breast Cancer.

Simple SummaryPatients with triple-negative breast cancer (TNBC) respond well to chemotherapy initially but are prone to relapse. Searching for new therapeutic targets, we found that PRMT1 is highly expressed in TNBC tumor samples and is essential for breast cancer cell survival. Furthermore, this study proposes that targeting PRMT1 in combination with chemotherapies could improve the survival outcome of TNBC patients.Identifying new therapeutic strategies for triple-negative breast cancer (TNBC) patients is a priority as these patients are highly prone to relapse after chemotherapy. Here, we found that protein arginine methyltransferase 1 (PRMT1) is highly expressed in all breast cancer subtypes. PRMT1 depletion decreases cell survival by inducing DNA damage and apoptosis in various breast cancer cell lines. Transcriptomic analysis and chromatin immunoprecipitation revealed that PRMT1 regulates the epidermal growth factor receptor (EGFR) and the Wnt signaling pathways, reported to be activated in TNBC. PRMT1 enzymatic activity is also required to stimulate the canonical Wnt pathway. Type I PRMT inhibitors decrease breast cancer cell proliferation and show anti-tumor activity in a TNBC xenograft model. These inhibitors display synergistic interactions with some chemotherapies used to treat TNBC patients as well as erlotinib, an EGFR inhibitor. Therefore, targeting PRMT1 in combination with these chemotherapies may improve existing treatments for TNBC patients.

TOP1 inhibition induces bifurcated JNK/MYC signaling that dictates cancer cell sensitivity.

Rationale: Triple-negative breast cancer (TNBC) does not respond to anti-estrogen and anti-HER2 therapies and is commonly treated by chemotherapy. TNBC has a high recurrence rate, particularly within the first 3 years. Thus, there is an urgent clinical need to develop more effective therapies for TNBC. Topoisomerase I (TOP1) inhibitors cause DNA damage, making these drugs desirable for TNBC treatment since DNA repair machinery is defective in this subtype of breast cancer. Among the main molecular subtypes of breast cancer, the TNBC cell lines exhibited the highest TOP1 inhibition sensitivity. However, clinically used TOP1 inhibitors, such as topotecan and irinotecan, have shown limited clinical applications and the reasons remain unclear. Understanding the mechanism of differential responses to TOP1 blockade and identifying the predictive markers for cancer cell sensitivity will help further TOP1-targeted therapy for TNBC treatment and improve the clinical use of TOP1 inhibitors.Methods: Viability assays were used to evaluate breast cancer cell sensitivity to topotecan and other TOP1 inhibitors as well as TOP2 inhibitors. An in vitro-derived topotecan-resistant TNBC cell model and TNBC xenograft models were employed to confirm cancer cell response to TOP1 blockade. RNA-seq was used to identify potential predictive markers for TNBC cell response to TOP1 blockade. Western blotting and qRT-PCR were performed to measure the protein levels and RNA expression. ATAC-seq and luciferase reporter assays were used to examine MYC transcriptional regulations. The effects of MYC and JNK in cancer cell response to TOP1 inhibition were validated via loss-of-function and gain-of-function experiments.Results: We observed two distinct and diverging cancer cell responses - sensitive versus resistant to TOP1 inhibition, which was confirmed by TNBC xenograft mouse models treated by topotecan. TNBC cells exhibited bifurcated temporal patterns of ATR pathway activation upon TOP1 inhibitor treatment. The sensitive TNBC cells showed an “up then down” dynamic pattern of ATR/Chk1 signaling, while the resistant TNBC cells exhibited a “persistently up” profile. On the contrary, opposite temporal patterns of induced expression of MYC, a key regulator and effector of DNA damage, were found in TNBC cells treated by TOP1 inhibitors. Mechanistically, we showed that TOP1-induced JNK signaling upregulated MYC expression. Furthermore, pharmacological inhibition of ATR reversed TNBC cell resistance to topotecan, whereas MYC knockdown and JNK inhibition reduced cancer cell sensitivity.Conclusions: Dynamic temporal profiles of induced ATR/Chk1 and JNK activation as well as MYC expression, may predict cancer cell response to TOP1 inhibitors. JNK activation-mediated constitutive elevation of MYC expression may represent a novel mechanism governing cancer cell sensitivity to TOP1-targeting therapy. Our results may provide implications for identifying TNBC patients who might benefit from the treatment with TOP1 inhibitors.

Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as ‘vasculogenic mimicry’ (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody–endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear β-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases.

CDK4/6 Inhibition and Radiation as a Treatment Strategy to Improve Local Disease Control in Breast Cancers With Poor Prognoses

Locoregional control remains an issue in women with multi-node positive estrogen receptor-positive (ER+) breast cancer and many women with triple-negative breast cancer (TNBC). There is growing evidence that CDK4/6 inhibition (CDK4/6i) sensitizes breast cancer cells to ionizing radiation (RT) by suppressing the DNA damage response, but the role of RB is currently unclear. We sought to understand the role of RB and the implications of RB loss in ER+ and TNBC.Clonogenic survival assays were used to calculate radiosensitivity and calculate enhancement ratios (rER). Cellular viability was quantified 72 hours after drug addition to calculate half-maximal inhibitory concentrations (IC50s). Phospho- and total RB expression levels were assessed by immunoblotting. DNA repair was assessed with γH2AX and RAD51 immunofluorescence. GFP-based plasmid reporter systems were used to assess homologous recombination (HR) and non-homologous end joining (NHEJ) competency. Isogenic RB1 knockout cells were generated with CRISPR-Cas9, and siRNA was used for transient RB1 knockdown. G1 cell cycle arrest was quantified using propidium iodide-based flow cytometry. In vivo efficacy of CDK4/6 inhibition + RT was assessed using MDA-MB-231 xenografts.Four TNBC cell lines with intact RB expression were radiosensitized (rER 1.08 - 2.22) after CDK4/6i with palbociclib, ribociclib, or abemaciclib, but two RB null TNBC models were not radiosensitized with combination treatment (rER: 0.84 - 1.00). In ER+ and TNBC cell lines, response to CDK4/6i + RT was accurately predicted by the presence or absence of RB protein, and higher RB expression led to increased radiosensitization of breast cancer cell lines at lower doses. In addition, pRB expression decreased in wild type TNBC cell lines treated with CDK4/6i + RT. HR was suppressed with CDK4/6i in RB wild type - but not mutant - cell lines. NHEJ efficiency in RB wild type TNBC was unchanged with CDK4/6i. Genetic knockdown of RB1 led to the loss of radiosensitization in ER+ and TNBC cell lines (rER: 0.84 - 1.13) as well as a decrease in sensitivity to CDK4/6i monotherapy. Parental and Cas9 control TNBC cells arrested in G1 24 hours after CDK4/6i and remained arrested at 48 hours, but RB1 CRISPR TNBC cells did not arrest after drug treatment. In a TNBC xenograft model with wild type RB expression (MDA-MB-231), CDK4/6i + RT led to significant radiosensitization in vivo and delayed time to tumor doubling.RB loss mitigates CDK4/6 inhibitor-mediated radiosensitization of both ER+ and TNBC cells. Loss of RB expression prevents CDK4/6i-mediated radiosensitization, suggesting that RB expression may be a valuable clinical biomarker to predict efficacy of CDK4/6i + RT in future clinical trials. Our data also provide the preclinical rationale for CDK4/6i with RT not just in ER+ BC, but also in women with TNBC that express RB.

Periostin Exon-21 Antibody Neutralization of Triple-Negative Breast Cancer Cell-Derived Periostin Regulates Tumor-Associated Macrophage Polarization and Angiogenesis.

Simple SummaryDespite remarkable advances in breast cancer treatment, few strategies other than standard cytotoxic chemotherapy are available for patients with triple-negative breast cancer (TNBC) due to the lack of therapeutic target molecules. TNBC is still the most aggressive subtype, with a high risk of recurrence and metastasis within 2 years after initial treatment. Thus, there is an unmet medical need to develop new treatments for metastatic and recurrent TNBC patients. In this study we tested a new antibody, targeting extracellular periostin protein alternative splicing variants, which are induced by conventional chemotherapy or during the process of endothelial mesenchymal transition. This antibody reduced periostin-secreting TNBC in a mouse xenograft model, accompanied by a decrease in the number of M2 tumor-associated macrophages and tumor vessels. Periostin alternative splicing variants might be a specific and safe therapeutic target in patients with TNBC.Periostin (Pn) is involved in multiple processes of cancer progression. Previously, we reported that Pn expression is correlated with mesenchymal tumor markers and poor prognosis in triple-negative breast cancer (TNBC). In the TNBC xenograft model, chemotherapy increased expression of a Pn alternative splicing variant (ASV) with exon 21, and administration of the neutralizing antibody against Pn with exon 21 (Pn-21 Ab) overcame chemoresistance with a reduction in the mesenchymal cancer cell fraction. In the present study, the role of Pn ASV with exon 21 in TNBC progression has been addressed. We first established a stable cell line carrying a fluorescence-based splicing reporter. Pn-positive TNBC has higher expression of genes related to tumor-associated macrophage (TAM) recruitment and ECM-receptor interaction than Pn-negative cells. In a xenograft model, only Pn-positive cells initiated tumor formation, and the Pn-21 Ab suppressed tumor cell growth, accompanied by decreased M2 TAM polarization and the number of tumor vessels. These data suggest that cancer cell-derived Pn ASV educates TAMs and regulates angiogenesis, which in turn establishes a microenvironmental niche that is supportive of TNBC.

In Vivo Delivery of siRNAs Targeting EGFR and BRD4 Expression by Peptide-Modified Redox Responsive PEG-PEI Nanoparticles for the Treatment of Triple-Negative Breast Cancer.

The study aims to investigate the in vivo distribution, antitumor effect, and safety of cell membrane-penetrating peptide-modified disulfide bond copolymer nanoparticles loaded with small-interfering RNA (siRNA) targeting epidermal growth factor receptor (EGFR) and bromodomain-containing protein 4 (BRD4) in triple-negative breast cancer (TNBC). Polyethylene glycol disulfide bond-linked polyethylenimine (PEG-SS-PEI) was modified with peptides GALA and CREKA and used as vectors to prepare siRNA nanoparticles. The GALA- and CREKA-modified PEG-SS-PEI nanoparticles (GC-NPs) were prepared by mixing siEGFR and siBRD4 (1:1) with GALA-PEG-SS-PEI and CREKA-PEG-SS-PEI (1:1) in an aqueous solution at an N/P ratio of 30:1. Nanoparticles loaded with scrambled siRNA were prepared with the same method. The gene silencing effect on EGFR and BRD4 in vitro was evaluated by Western blotting analysis. TNBC xenograft models were established by subcutaneous injection of MDA-MB-231 cells into female nude mice. At 1, 3, 6, 12, and 24 h after administration of five formulations of Cy5-siRNA (133 μg/10 g) via the tail vein, the mice were observed and imaged for a biodistribution study using an in vivo imaging system. In the pharmacodynamics experiment, tumor-bearing mice were treated with respective siRNA preparations at a dose of 133 μg/10 g for 18 days, and the body weight and tumor size were recorded every other day. The protein expression levels of EGFR, p-EGFR, PI3K, p-PI3K, Akt, p-Akt, BRD4, and c-Myc were determined using Western blotting analysis. Hematological and serum biochemical parameters, organ indices, and HE staining results for the heart, liver, spleen, lung, and kidney were analyzed to evaluate the safety of the nanoparticles. GC-NPs loaded with siEGFR and siBRD4 significantly inhibited the expression of EGFR and BRD4 in vitro. The strongest fluorescence signals were observed in the GC-NP group, especially in tumors, indicating the excellent tumor-targeted delivery of GC-NPs we constructed. Tumor growth was significantly inhibited in the GC-NP-treated group, and the expression of EGFR, p-EGFR, PI3K, p-PI3K, Akt, p-Akt, BRD4, and c-Myc in the tumors decreased by 71%, 68%, 61%, 68%, 48%, 58%, 59%, and 74% compared to the control group, respectively. There was no significant change in hematological parameters, biochemical indices, or tissue morphology in GC-NP-treated mice. SiRNA cotargeting EGFR and BRD4 delivered by GALA- and CREKA-modified PEG-SS-PEI had favorable antitumor effects in vivo toward TNBC with tumor-targeting efficacy and good biocompatibility.

The FHP01 DDX3X Helicase Inhibitor Exerts Potent Anti-Tumor Activity In Vivo in Breast Cancer Pre-Clinical Models.

Simple SummaryDDX3X has been repeatedly proven to exert pro-tumorigenic effects in breast cancer. An inhibitor of DDX3X helicase function, FHP01, exhibited very effective antiproliferative and tumor killing activity in vitro and in vivo against several breast cancer cells, yet showed no systemic toxicity but good bioavailability in vivo. FHP01 may therefore represent a novel therapeutic approach with high potential as a personalized strategy for breast cancer, confirming DDX3X as a new actionable molecular target in these tumors.Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies.

Exploring Marine-Derived Ascochlorins as Novel Human Dihydroorotate Dehydrogenase Inhibitors for Treatment of Triple-Negative Breast Cancer.

Human dihydroorotate dehydrogenase (hDHODH) is an attractive tumor target essential to de novo pyrimidine biosynthesis. Novel potent hDHODH inhibitors with low toxicity are urgently needed. Herein, we demonstrate the isolation of 25 ascochlorin (ASC) derivatives, including 13 new ones, from the coral-derived fungus Acremonium sclerotigenum, and several of them showed pronounced inhibitions against hDHODH and triple-negative breast cancer (TNBC) cell lines, MDA-MB-231/-468. Interestingly, we found that hDHODH is required for proliferation and survival of TNBC cells, and several ASCs significantly inhibited TNBC cell growth and induced their apoptosis via hDHODH inhibition. Furthermore, the novel and potent hDHODH inhibitors (1 and 21) efficiently suppressed tumor growth in patient-derived TNBC xenograft models without obvious body weight loss or overt toxicity in mice. Collectively, our findings offered a novel lead scaffold as the hDHODH inhibitor for further development of potent anticancer agents and a potential therapeutic strategy for TNBC.

Structure-Guided Optimization Provides a Series of TTK Protein Inhibitors with Potent Antitumor Activity.

TTK is an essential spindle assembly checkpoint enzyme in many organisms. It plays a central role in tumor cell proliferation and is aberrantly overexpressed in a wide range of tumor types. We recently reported on a series of potent and selective TTK inhibitors with strong antiproliferative activity in triple negative breast cancer (TNBC) cell lines (8: TTK IC50 = 3.0 nM; CAL-51 IC50 = 84.0 nM). Inspired by previously described potent tricyclic TTK inhibitor 6 (TTK IC50 = 0.9 nM), we embarked on a structure-enabled design and optimization campaign to identify an improved series with excellent potency, TTK selectivity, solubility, CYP inhibition profile, and in vivo efficacy in a TNBC xenograft model. These efforts culminated in the discovery of 25 (TTK IC50 = 3.0 nM; CAL-51 IC50 = 16.0 nM), which showed significant single-agent efficacy when dosed iv in a TNBC xenograft model without body weight loss.

Abstract 1270: The novel PARP1-selective inhibitor, AZD5305, is efficacious as monotherapy and in combination with standard of care chemotherapy in the in vivo preclinical models

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown efficacy in homologous recombination deficient (HRD) tumours, such as those with BRCA mutations (BRCAm). In this setting PARPi treatments lead to accumulation of DNA damage and cancer cell death. PARPi currently in clinical use inhibit both PARP1 and PARP2, as well as other members of the PARP family. Here, we report for the first time in vivo profiling of AZD5305, a potent and highly selective PARP1 inhibitor and trapper, currently in Ph1 clinical trials. Dose response efficacy of AZD5305 was evaluated in the BRCA1m triple-negative breast cancer (TNBC) xenograft model MDA-MB-436. AZD5305 dosed at 0.1mg/kg QD or higher for 35 days delivered about 90% regression, compared with 83% regression caused by treatment with 100mg/kg QD olaparib. Anti-tumour effects of AZD5305 continued after cessation of treatment and complete responses were achieved which were sustained for the whole duration of the study, over 100 days after treatment withdrawal, in contrast to the olaparib-treated group where regrowth of tumours was observed from day 63 after treatment withdrawal. Investigation of the PK/PD/efficacy relationship in MDA-MB-436 showed that maximum efficacy of AZD5305 was achieved when unbound plasma concentrations were maintained above the IC95 estimated from an in vitro DLD-1 BRCA2-/- cell growth assay. Similar results were obtained in a BRCA1m patient-derived explant (PDX) model, HBCx-17. Anti-tumour efficacy of AZD5305 was also tested in the DLD-1 BRCA2-/- and wild-type (WT) isogenic xenograft models. In the DLD-1 BRCA2-/- model, AZD5305 dosed at 10mg/kg QD and 1mg/kg QD delivered 78% and 63% tumour regression, respectively. AZD5305 at 0.1mg/kg QD resulted in responses similar to those observed in the olaparib 100mg/kg QD group (40-54% tumour growth inhibition, TGI). As expected, AZD5305 and olaparib showed no anti-tumour efficacy in the DLD-1 WT tumour model. Due to improved PARP1 selectivity, AZD5305 has the potential to show improved efficacy and tolerability in combination with standard of care chemotherapy when compared to non-selective PARPi. Hence, we investigated the anti-tumour effects of AZD5305 in combination with carboplatin or paclitaxel in a BRCA1m TNBC xenograft, SUM149PT, and BRCA WT TNBC PDX model, HBCx-9. In both models, combination of AZD5305 with carboplatin was well tolerated and demonstrated clear benefit compared to each monotherapy treatment. The effects of adjusted dosing and scheduling of the combination on the anti-tumour efficacy will be presented. Citation Format: Anna D. Staniszewska, James W. Yates JWT, Andy Pike, Christine Fazenbaker, Kimberly Cook, Emily Bosco, Aaron Smith, Joanne Wilson, Elisabetta Leo. The novel PARP1-selective inhibitor, AZD5305, is efficacious as monotherapy and in combination with standard of care chemotherapy in the in vivo preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1270.

Abstract 1038: PMD-026, a first in class oral RSK inhibitor, demonstrates synergy when combined with standard of care in breast cancer tumor models

Abstract PMD-026 is a first in class, reversible, oral small molecule inhibitor of p90 ribosomal S6 kinase (RSK), a kinase family activated by the MAPK and PDK-1 pathways, which regulate substrates involved in cancer cell proliferation and drug resistance. Specifically, RSK2 has been identified as a major driver in breast cancer (BC). In preclinical studies and a Phase I clinical trial in metastatic BC, PMD-026 demonstrated a good safety profile, making it an attractive candidate for combinations with standard of care therapies. Here in we show that RSK2 is activated in 87% of triple negative breast cancer (TNBC), as well as in other subtypes (80% of ER+/PR+ and 81% of HER2+). In a screen of 28 BC cell lines across a broad mutational spectrum, PMD-026 induced apoptosis in most of the models. Next, to determine the role of PMD-026 in the context of conventional BC treatment, we combined it with standard of care chemotherapies fulvestrant, paclitaxel, or doxorubicin, in vitro and in vivo. PMD-026 synergized with fulvestrant, with combination drug index (CDI) values ranging from 0.73 - 0.92 in the HR+ cell line MCF-7. Likewise, PMD-026 synergized with paclitaxel and doxorubicin in TNBC cell lines with CDI values ranging from 0.40 - 0.84 and 0.58 - 0.92 for paclitaxel and doxorubicin, respectively. Consistent with in vitro screening data, PMD-026 synergized with paclitaxel in vivo in the SUM149PT TNBC xenograft model after 38 days of treatment. The combination inhibited tumor growth by 66% (P < 0.0001), whereas paclitaxel and PMD-026 as single agents inhibited tumor growth by 22% (P = 0.3051) and 41% (P = 0.0041), respectively. The synergy of this combination was further supported by a CDI value of 0.75. Treatment among the groups was well tolerated with no changes in body weight observed. Similarly, the combination of PMD-026 and paclitaxel was synergistic in SUM149PT(48 days, CDI: 0.78) and MDA-MB-231 (28 days, CDI: 0.66) xenografts and additive in a PDx model of metastatic TNBC (18 days, CDI: 0.96), resulting in improved survival. To address the safety of combining PMD-026 and paclitaxel, drug-drug interactions (DDI) were assessed. In cytochrome P450-mediated in vitro metabolism assays, PMD-026 showed weak inhibition of Cyp2C8 and Cyp3A4, the main enzymes responsible for paclitaxel metabolism. To understand whether PMD-026 might alter the metabolism of paclitaxel, this potential DDI was assessed in vivo. Pharmacokinetic analysis of PMD-026 (7 days repeat dosing) combined with paclitaxel (8 mg/kg IV dose Day 1 and Day 7) was evaluated in CD-1 mice, however, PMD-026 did not change the absorption, distribution, or blood levels of paclitaxel. Together, these data support adding PMD-026 to standard of care therapies in breast cancer, as they have demonstrated synergy. In particular, PMD-026 is synergistic with paclitaxel in multiple TNBC models, in which it improves efficacy without added toxicity. Citation Format: Aarthi Jayanthan, My-my Huynh, Jangsoon Lee, Gerrit Los, Lambert Yue, Mary Rose Pambid, Naoto T. Ueno, Sandra E. Dunn. PMD-026, a first in class oral RSK inhibitor, demonstrates synergy when combined with standard of care in breast cancer tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1038.

Abstract 1924: Therapeutic potential of inhibitor of apoptosis protein (IAP) inhibitor APG-1387 combined with DR5 agonist monoclonal antibody (mAb) CTB-006 in preclinical models of solid tumors

Abstract TNF-related apoptosis-inducing ligand (TRAIL) receptor (DR4 or DR5) agonists are promising agents for cancer therapy because they induce apoptosis selectively in cancer cells. However, their clinical effect is hampered by either primary or acquired resistance in cancer cells. Second mitochondria-derived activator of caspase (Smac) mimetics that antagonize the IAPs potently sensitize cancer cells to TRAIL-induced apoptosis in a caspase-8-dependent manner. We evaluated antitumor effects of small-molecule IAP antagonist APG-1387combined with an agonist mouse mAb directed against TRAIL death receptor type 5 (DR5) termed CTB-006 in the preclinical setting. Both agents are in phase I/II clinical development for patients with solid tumors. In vitro the combination treatment showed synergy in a dose-dependent manner across various cancer cell lines, as evidenced by synergistic antiproliferative activity and enhanced induction of caspase-3 cleavage. Potential synergistic in vivo antitumor activity of the combination was then assessed in human triple negative breast cancer (TNBC) xenograft models in mice. Specifically, in MDA-MB-231 and 2LMP TNBC xenograft models, either APG-1387 or CTB-006 single agent exhibited limited antitumor activity, with T/C (%) values of >35% (T, tumor volume in the treatment group; C, tumor volume in the vehicle control group). Only one tumor showed partial regression (PR) by DR5 treatment. Addition of APG-1387 potentiated this effect, with the combination treatment leading to T/C values of 3% to 15%, and complete regression, PR, or stable disease in all treated tumors. In summary, our results suggest great potential of combination therapy with APG-1387 and CTB-006 for solid tumor therapy and deserves further clinical investigation. Citation Format: Qiang Li, Douglas D. Fang, Zhe Li, Dajun Yang, Fengqi Cao, Yifan Zhai. Therapeutic potential of inhibitor of apoptosis protein (IAP) inhibitor APG-1387 combined with DR5 agonist monoclonal antibody (mAb) CTB-006 in preclinical models of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1924.

MiR-34a-Mediated Survivin Inhibition Improves the Antitumor Activity of Selinexor in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Here, we pursued a combinatorial therapeutic approach to enhance the activity of selinexor, the first-in-class XPO1 inhibitor, by miR-34a ectopic expression in human TNBC experimental models. Anti-proliferative activity induced by selinexor and miR-34a expression, singly and in combination, was evaluated by MTS assay and cell counting. The effect of treatments on survivin and apoptosis-related proteins was assessed by western blotting and ELISA. The antitumor and toxic effects of individual and combined treatments were evaluated on TNBC orthotopic xenografts in SCID mice. Selinexor consistently showed anti-proliferative activity, although to a variable extent, in the different TNBC cell lines and caused the impairment of survivin expression and intracellular distribution, accompanied by apoptosis induction. Consistent with in vitro data, the XPO1 inhibitor variably affected the growth of TNBC orthotopic xenografts. miR-34a cooperated with selinexor to reduce survivin expression and improved its anti-proliferative activity in TNBC cells. Most importantly, miR-34a expression markedly enhanced selinexor antitumor activity in the less sensitive TNBC xenograft model, in absence of toxicity. Our data form a solid foundation for promoting the use of a miR-34a-based approach to improve the therapeutic efficacy of selinexor in TNBC patients.

Pre-clinical development of an anti-cancer drug ENZ302, a promising candidate for multiple cancer therapeutics.

e13053 Background: Vernonia cinerea extract has been known to be used for various haematological disorders including cancer. In the current study, using state-of-the-art extraction and bioanalytical tools we have identified an active compound having anti-tumor properties from the same plant Vernonia cinerea. The crude extract of Vernonia cinerea as well as the active molecule obtained through the chemical route (ENZ302) showed anti-tumor activity against various cancer models including CML, AML, ALL, HER2+ breast cancer, colon cancer, etc. Methods: Various methods including RP-HPLC, XRD, NMR, IR, cell based assay and LC-MS were used to elucidate the molecular structure of ENZ302. MTD, DRF, PK, and Xenograft study etc were carried out in murine model during the preclinical development of ENZ302. Results: ENZ302 resulted significant cytotoxic effect within 8 hrs of treatment in TNBC (Triple Negative Breast Cancer) model, MDA-MB-468 cells when compared with a known inhibitor Lapatinib. However, ENZ302 treatment in primary cells demonstrates no anti-proliferation activity, indicating the specificity of it’s mode of action. Interestingly, a dose-dependent decrease in tumor volumes was observed for ENZ302 in a xenograft model of TNBC while administered orally. ENZ302 did not cause any adverse changes even at 2gm/Kg dose, indicating the safety of the compound. Although a single-dose PK study showed that ENZ302 has a half-life of ̃40 mins, however, in vitro metabolism of the compound in mouse, rat, dog, and human hepatocytes/microsomes showed the molecule is stable in hepatocyte with very low clearance in microsomes (half-life > 30 min), indicating most of the clearance occurring through the kidney. Conclusions: Our preliminary data suggest that ENZ302 is a specific tyrosine kinase (e.g. Src) inhibitor and a promising therapeutic agent against several cancers. A comprehensive proteomic analysis is ongoing to understand the mechanism of action of ENZ302 that makes it effective against several cancers. The discovery of ENZ302 holds ample opportunities to develop a safe and efficacious treatment regimen against several cancers including TNBC.

Regulation of Breast Cancer Progression by Small G Proteins

Breast cancer (BC) is the second leading cause of cancer-associated mortality among U.S. women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due to the lack of targeted treatment options. Management remains fixed in time with toxic chemotherapy developed in the 1960s as the only systemic treatment. An urgent and unmet need exists to identify new molecular targets for the treatment of TNBC. We identified RALA as a gene of interest and potential therapeutic target. Analysis of gene expression data and patient tissue arrays indicate elevated RALA is significantly associated with poor patient outcome. RalA is a small GTPase implicated in tumor proliferation, survival, and metastasis in a variety of cancers, although little is known of its role in BC. The necessity of RalA in TNBC progression was evaluated by its depletion in murine, MVT1, and human, MDA-MB-231, TNBC cell lines. In vitro, cell migration, proliferation, and nonadherent cell growth assays validated a pro-tumorigenic role for RalA in TNBC. Knockdown of RalA inhibited orthotopic primary tumor growth and metastasis of both MVT1 and MDA-MB-231 cells in vivo. To explore the potential of drug intervention, MVT1 and MDA-MB-231 were treated with BQU57, a small molecule Ral inhibitor. In vitro treatment hindered growth and a greater than additive effect was observed in combined treatment with paclitaxol. Further, BQU57 was effective in delaying orthotopic mammary tumor growth as well as lung metastasis and hindered the subcutaneous growth of a patient-derived xenograft model of metastatic TNBC. Together, these data demonstrate an important role for RalA in the pathogenesis of TNBC, exhibit the potential of Ral inhibitors as treatments for advanced TNBC, and advocate further investigation of RalA as a target for the precise treatment of metastatic TNBC. Ongoing investigations seek to determine the molecular mechanisms behind RalA's tumor and metastasis-promoting functions and the development of Ral inhibitors unique to RalA.

Ultrasound-Targeted Microbubble Destruction Enhances Inhibitory Effect of Apatinib on Angiogenesis in Triple Negative Breast Carcinoma Xenografts.

Ultrasound-targeted microbubble destruction (UTMD) has been proven as an effective technique to assist drugs to cross the vascular wall and cell membrane. This study was aimed at evaluating the synergistic antiangiogenic and growth-inhibiting effects of apatinib (APA) and UTMD on the triple negative breast cancer (TNBC). The TNBC xenograft model was established in nude mice (n = 40) which were then randomly divided into the APA plus UTMD (APA-U) group, UTMD group, APA group, and model control (M) group (n = 10 per group). Corresponding treatment was done once daily for 14 consecutive days. The general condition and body weight of tumor-bearing nude mice were monitored. Routine blood test and detection of liver and kidney function were done after treatments. The tumor size and microcirculation were examined by two-dimensional ultrasonography (2DUS) and contrast-enhanced ultrasonography (CEUS), respectively. Then, the tumor tissues were harvested for the detection of vascular endothelial growth factor (VEGF) by immunohistochemistry and for CD31-PAS double staining to assess microvessel density (MVD) and heterogeneous vascular positivity rate. After treatments, the tumor growth and angiogenesis were significantly inhibited in the APA group and the APA-U group, and these effects were more obvious in the APA-U group. The tumor volume, CEUS parameters, VEGF expression, and MVD in the APA-U group were significantly lower than those in the APA group (P < 0.05), while there were no marked differences in the heterogeneous vascular positivity rate, body weight, and blood parameters between the two groups (P > 0.05). In the UTMD group, the tumor growth and angiogenesis were not significantly inhibited, and all the parameters were similar to those in the M group (P > 0.05). During the experiment, all mice survived and generally had good condition. In conclusion, APA combined with UTMD may exert synergistic antiangiogenic and growth-inhibiting effects on the TNBC and not increase the heterogeneous vasculature and the severity of APA-related systemic side effects.