Differential responses to the combination of navitoclax and venetoclax with doxorubicin in murine models of triple negative breast cancer

Therapy-induced Senescence (TIS) can potentially influence breast cancer treatment outcomes, in part by contributing to disease recurrence; hence, the utilization of senescence-eliminating agents (i.e., senolytics) is considered as a possibly adjuvant to both antitumor drugs and radiation. However, one of the most effective senolytic agents, navitoclax (ABT-263), is limited by its associated toxicities of thrombocytopenia and neutropenia. In contrast, venetoclax (ABT-199) is currently considered standard of care in CLL and AML. This study compared the senolytic potential of the BH3 mimetics, navitoclax and venetoclax, in targeting doxorubicin-induced senescence in two models of triple negative breast cancer (4T1 and E0771 cells). Senescence was cytochemically confirmed via Senescence-associated β-galactosidase upregulation (and quantified by flow cytometry), TP53 and p21Cip1 (CDKN1A) induction, and the senescence-associated secretory phenotype (SASP) expression (using qRT-PCR). Cell viability and the percentage of apoptotic cells were determined using MTT and Annexin V/7AAD assays, respectively. Responses differed in the two cell lines. Both navitoclax and venetoclax were effective as apparent senolytics in the E0771 cells. In contrast, only navitoclax was effective against the 4T1 cells. The in vitro findings in E0771 cells were validated through studies conducted in vivo in immunocompetent mice implanted with E0771-derived tumors where both drugs reduced tumor progression in sequential combination with doxorubicin. These findings suggest that administration of venetoclax has the potential to enhance suppression of chemotherapy-induced senescent cancer cells, and that it may not be necessary to use senolytic agents that target Bcl-xL. However, given the variable outcomes in the two triple negative breast tumor cell lines, it becomes incumbent to identify the factors that confer susceptibility to Bcl-2 targeting senolytics in anticipation of their potential utilization in the clinic for combination therapy in solid tumors. Citation Format: H. As Sobeai, K. Alhazzani, M. Almutairi, T. Saleh, B. Al-Ramadi, D. Gewirtz, H. Harada, M. Alotaibi. Differential Responses to the Senolytic Activities of Navitoclax and Venetoclax in Murine Models of Triple Negative Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-12-27.

BackgroundLeptin, an energy balance regulator secreted by adipocytes, increases metastatic potential of breast cancer cells. The impact on cancer cell metabolism remains unclear given that most studies of leptin and breast cancer cell metabolism utilize supraphysiological glucose concentrations.MethodsUsing two murine models of metastatic triple-negative breast cancer (TNBC) differing in genetic alterations (4T1: p53 and Pik3ca mutations; metM-Wntlung: increased Wnt signaling) and cultured in physiological (5 mM) glucose media, we tested the hypothesis that leptin increases migration of metastatic breast cancer cells through regulation of glucose metabolism.ResultsOur results showed that leptin treatment, compared with vehicle, increased cell migration in each cell line, with decreased leptin receptor (Ob-R) mRNA expression in 4T1, but not metM-Wntlung, cells. AMP-activated protein kinase (AMPK) was activated in 4T1 with leptin treatment but decreased in metM-Wntlung. Leptin decreased fatty acid synthase (Fasn) and carnitine palmitoyltransferase 1a (Cpt1a) mRNA expression in 4T1 cells but increased their expression in metM-Wntlung cells. Fatty acid oxidation was not necessary for leptin-induced migration in either cell line. Leptin increased palmitate synthesis from glucose in metM-Wntlung, but not 4T1 cells. Moreover, although leptin increased glucose transporter 1 (Glut1) mRNA expression in both cell lines and inhibition of glycolysis blocked leptin-induced migration in metM-Wntlung, but not 4T1 cells.ConclusionTaken together, these results demonstrate that at physiological glucose concentrations, leptin increases migration of 4T1 and metM-Wntlung cells via shared and distinct effects on energy metabolism, suggesting that the type of TNBC genetic alteration plays a role in differential metabolic regulation of leptin-induced migration.

Our group has previously demonstrated, in in vitro and in vivo studies on triple-negative breast cancer, that morphine promoted breast cancer progression whereas naloxone was able to reduce it. In this subsequent investigation, we aimed to assess the combinatorial effects of these two drugs in an animal model of triple negative breast cancer. In order to evaluate the in vivo effects of the combination of morphine and naloxone in human breast cancer, a mouse model of human triple-negative breast cancer was generated by injecting the MDA-MB-231 cells subcutaneously in nude mice. Naloxone and morphine were daily intraperitoneally co-injected in mice for 4 weeks at two different doses. Micro-vessel formation was detected by fluorescein isothiocyanate-dextran (100 μl) injected into the lateral tail vein of mice and confirmed by immunohistochemistry for PECAM-1 on mice tumor sections. In vivo experiments showed that naloxone was able to counteract the promoting effects of morphine on tumor growth. No impairment of micro-vessel formation in tumors of mice treated with the two drugs was observed. Herein, we demonstrated that naloxone was able to counteract the promoting effects of morphine on tumor growth without impairing micro-vessel formation.

BackgroundThe main limits of current antitumor therapies are chemoresistance, relapses and toxic effects that impair a patient’s quality of life. Therefore, the discovery of therapeutic alternatives, such as adjuvants to...

<div>Abstract<p><b>Purpose:</b> The clinical success of arsenic trioxide (As<sub>2</sub>O<sub>3</sub>) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As<sub>2</sub>O<sub>3</sub> encapsulated in liposomal vesicles or “nanobins” [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As<sub>2</sub>O<sub>3</sub> would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas.</p><p><b>Experimental Design:</b> The cytotoxicity of NB(Ni,As), the empty nanobin, and free As<sub>2</sub>O<sub>3</sub> was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As<sub>2</sub>O<sub>3</sub> were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer.</p><p><b>Results:</b> The NB(Ni,As) agent was much less cytotoxic <i>in vitro</i> than free As<sub>2</sub>O<sub>3</sub> against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As<sub>2</sub>O<sub>3</sub> <i>in vivo</i> in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As).</p><p><b>Conclusions:</b> Nanobin encapsulation of As<sub>2</sub>O<sub>3</sub> improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent <i>in vivo</i>. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors. Clin Cancer Res; 16(14); 3607–17. ©2010 AACR.</p></div>

<div>Abstract<p><b>Purpose:</b> The clinical success of arsenic trioxide (As<sub>2</sub>O<sub>3</sub>) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As<sub>2</sub>O<sub>3</sub> encapsulated in liposomal vesicles or “nanobins” [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As<sub>2</sub>O<sub>3</sub> would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas.</p><p><b>Experimental Design:</b> The cytotoxicity of NB(Ni,As), the empty nanobin, and free As<sub>2</sub>O<sub>3</sub> was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As<sub>2</sub>O<sub>3</sub> were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer.</p><p><b>Results:</b> The NB(Ni,As) agent was much less cytotoxic <i>in vitro</i> than free As<sub>2</sub>O<sub>3</sub> against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As<sub>2</sub>O<sub>3</sub> <i>in vivo</i> in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As).</p><p><b>Conclusions:</b> Nanobin encapsulation of As<sub>2</sub>O<sub>3</sub> improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent <i>in vivo</i>. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors. Clin Cancer Res; 16(14); 3607–17. ©2010 AACR.</p></div>

The clinical success of arsenic trioxide (As(2)O(3)) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As(2)O(3) encapsulated in liposomal vesicles or "nanobins" [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As(2)O(3) would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas. The cytotoxicity of NB(Ni,As), the empty nanobin, and free As(2)O(3) was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As(2)O(3) were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer. The NB(Ni,As) agent was much less cytotoxic in vitro than free As(2)O(3) against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As(2)O(3) in vivo in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As). Nanobin encapsulation of As(2)O(3) improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent in vivo. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors.

Obesity is an established risk factor for breast cancer. Dietary and surgical weight loss interventions reduce breast cancer risk but have significant limitations to their widespread use as anticancer strategies. Incretin-based pharmaceuticals safely and effectively promote weight loss, but whether these drugs exert similar protective effects against cancer is unknown. Here we test whether weight loss induced by the glucagon-like peptide 1 (GLP-1)/glucose-dependent insulinotropic polypeptide (GIP) receptor agonist tirzepatide limits tumor growth in an orthotopic mouse model of triple-negative breast cancer. Ten-week-old female C57BL/6NCrl mice were randomized to either a 10 kcal% low-fat diet (control; n=14) or 60 kcal% high-fat diet to promote diet-induced obesity (DIO; n=43). After 18 weeks on diet, DIO mice were randomized by body weight to either i) remain on the high-fat diet (DIO; n=15); ii) lose weight with tirzepatide (TZP; n=13); or iii) lose weight with 30% daily calorie restriction relative to control mice (CR; n=15). All mice were subcutaneously injected with either TZP or vehicle every other day for 13 weeks with the following escalating dosing schedule: 3 nmol/kg TZP in week 1; 10 nmol/kg TZP in weeks 2-3; 20 nmol/kg TZP in weeks 4-7; 30 nmol/kg TZP in weeks 8-11; and 40 nmol/kg TZP in weeks 12-13. Fasting blood glucose and body composition was assessed after 9 weeks of weight loss, and 35,000 E0771 cells were orthotopically transplanted into the 4th mammary fat pad. Tumors growth was monitored for 4 weeks. TZP-treated mice lost on average >20% weight loss by week 4 and remained at or below 20% weight loss for the remainder of the study. Directly prior to E0771 tumor cell injections, TZP-treated mice had lost 23.6±8.9% body weight, versus 39.7±3.6% in CR mice. Relative to DIO mice, blood glucose control was restored in both weight loss groups (DIO: 149.5±18.5 mg/dl; TZP: 124.5±15.5 mg/dl; CR: 107.6±18.8 mg/dl) and >87.5% of the lost weight was attributed to fat mass, rather than lean mass, for TZP-treated and CR mice. Notably, tirzepatide suppressed tumor growth relative to DIO, but the CR group had a greater anticancer effect than TZP. Reduced tumor mass in both weight loss groups was accompanied by a significant reduction in circulating IGF-1, insulin, and leptin relative to DIO mice. These findings are timely and important because i) obesity is a highly prevalent metabolic disease that increases risk and progression of breast cancer and >13 other cancers; ii) incretin therapies, which show promise as highly effective weight loss drugs, are increasingly being prescribed; and iii) the tumor suppressive effects of tirzepatide described herein newly suggest incretin agonism as another propitious option for breaking the obesity-cancer link. Citation Format: Elaine M. Glenny, Alyssa N. Ho, Violet A. Kiesel, Fangxin Chen, Claire E. Gates, Evan M. Paules, Ruihan Xu, C. Alex Holt, Michael F. Coleman, Stephen D. Hursting. Tirzepatide mitigates obesity-associated metabolic dysregulation and tumor progression in a mouse model of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2189.

Background: Programmed death-ligand 1 (PDL1) expression on tumor and other cells dampens T cell activation and promotes exhaustion. Pyruvate carboxylase (PC) converts pyruvate to oxaloacetate which in T cells and tumor associated macrophages supports antitumor immunity. PC is required for lung metastasis in several models of triple negative breast cancer (TNBC); however the role of PC expression in primary tumor growth is less well understood. Suppression of PC in tumor associated macrophages and T cells blunts antitumor immunity. We have shown that suppression of PC in tumor cells is sufficient to promote tumor progression and immune evasion. Here we investigated in murine models of TNBC whether and how PC suppression might alter the expression of PDL1. Methods: PC was stably suppressed in M-Wnt, metM-Wntlung and E0771 cells using shRNA, and transiently suppressed using siRNA. C57BL6NCrl mice were orthotopically injected with shPC and scram M-Wnt (n=8/group) or E0771 (n=5/group) cells. Transcriptomic analysis of E0771 cells was performed using Clariom D microarray. PC and PDL1 expression was determined in M-Wnt and metM-Wntlung cells by qPCR. Cell signaling was assayed by western blot using pERK and pAKT antibodies. Statistical analysis was performed using two-sided students t-test, with Welch’s correction where variance was not equal. Results: Suppression of PC in M-Wnt and E0771 cells promotes tumor growth. In vitro, suppression of PC promoted PDL1 expression in E0771 cells. We confirmed similar effects in M-Wnt and metM-Wntlung cells stably expressing shRNA directed against PC using qPCR. Finally we confirmed that transient transfection of siRNA directed against PC similarly induced PDL1 expression. Basal AKT and ERK signaling was induced by stable suppression of PC in M-Wnt cells. Conclusions: PC suppression in several murine cell models of TNBC promotes PDL1 expression and reveals an underexplored interaction between tumor cell anaplerosis and immune evasion. Through suppression of PC we have identified a reciprocal relationship between PC and PDL1 expression, with a concomitant induction of both AKT and ERK signaling. A better understanding of the mechanisms through which PC-related metabolic reprogramming modulates antitumor immunity will identify new targets for inhibiting TNBC progression. Future studies will address whether targeted restoration of anaplerosis or overexpression of PC can suppress PDL1 and promote antitumor immunity. Citation Format: Numair Attaar, Alexander J. Pfeil, Eylem K. Cotul, Dorothy Teegarden, Michael K. Wendt, Michael F. Coleman, Stephen D. Hursting. Suppression of pyruvate carboxylase drives PDL1 expression in mouse models of TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1267.

Increased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistance in the Metastatic 4T1 Breast Cancer Model

Synergistic Anti-Tumor Effects of Newcastle Disease Virus and Doxorubicin: Evidence from A Murine Breast Cancer Model

BackgroundOncolytic peptides are attractive tools for the development of novel anticancer regimens [1]. LTX-315 is a synthetic peptide with a marked capacity to elicit tumor-targeting immunity in preclinical cancer models...

PurposeExposure to the polyphenolic plant lignan secoisolariciresinol diglucoside (SDG) and its metabolite enterolactone (ENL) has been associated with reduced breast cancer progression, particularly for estrogen receptor alpha (ERα)-negative disease, and decreased preclinical mammary tumor growth. However, while preclinical studies have established that SDG and ENL affect measures of progression in models of triple-negative breast cancer (TNBC, a subset of ERα-negative disease), the molecular mechanisms underlying these effects remain unclear.MethodsC57BL/6 mice were fed a control diet (control, 10% kcal from fat) or control diet + SDG (SDG, 100 mg/kg diet) for 8 weeks, then orthotopically injected with syngeneic E0771 mammary tumor cells (a model of TNBC); tumor growth was monitored for 3 weeks. The role of reduced NF-κB signaling in SDG’s anti-tumor effects was explored in vitro via treatment with the bioactive SDG metabolite ENL. In addition to the murine E0771 cells, the in vitro studies utilized MDA-MB-231 and MCF-7 cells, two human cell lines which model the triple-negative and luminal A breast cancer subtypes, respectively.ResultsSDG supplementation in the mice significantly reduced tumor volume and expression of phospho-p65 and NF-κB target genes (P < 0.05). Markers of macrophage infiltration were decreased in the distal-to-tumor mammary fat pad of mice supplemented with SDG relative to control mice (P < 0.05). In vitro, ENL treatment inhibited viability, survival, and NF-κB activity and target gene expression in E0771, MDA-MB-231, and MCF-7 cells (P < 0.05). Overexpression of Rela attenuated ENL’s inhibition of E0771 cell viability and survival.ConclusionsSDG reduces tumor growth in the E0771 model of TNBC, likely via a mechanism involving inhibition of NF-κB activity. SDG could serve as a practical and effective adjuvant treatment to reduce recurrence, but greater understanding of its effects is needed to inform the development of more targeted recommendations for its use.

Metastatic breast cancer is a deadly disease which requires new therapeutic strategies. Endogenous TNF-related apoptosis-inducing ligand (TRAIL) functions as a metastasis suppressor by activating proapoptotic TRAIL receptors (TRAIL-R1/DR4 and/or TRAIL-R2/DR5) in transformed cells, making it an attractive pathway for antimetastatic therapies. However, it is unclear whether TRAIL-R1 or TRAIL-R2 is a better therapeutic target in metastatic breast cancer. Several metastatic, triple (estrogen receptor, progesterone receptor, and HER2)-negative cancer cell lines were treated with human agonistic monoclonal antibodies targeting TRAIL-R1 (mapatumumab) or TRAIL-R2 (lexatumumab). The effects on cell viability, apoptosis, and caspase-8 activation were determined. An orthotopic model of triple-negative breast cancer in which fluorescently labeled breast cancer cells metastasize from the mammary gland to lymph nodes and lung was utilized to evaluate the effects of mapatumumab, lexatumumab, or doxorubicin on primary and metastatic tumor burden in vivo. Lexatumumab was more effective than mapatumumab in activating caspase-8, inducing apoptosis and inhibiting long-term survival of metastatic cancer cells, which expressed both TRAIL-R1 and TRAIL-R2. Human mammary epithelial cells transformed by oncogenic Ras were more sensitive to lexatumumab than nontransformed cells. Lexatumumab inhibited lymph node and lung metastases more robustly than mapatumumab in an orthotopic model of triple-negative breast cancer; both agents inhibited mammary tumor growth. In addition, lexatumumab was more effective than doxorubicin at suppressing metastases at doses of doxorubicin that were associated with toxicity, even though doxorubicin reduced primary tumor burden more robustly than lexatumumab. Targeting TRAIL-R2 receptor may be an effective therapeutic strategy for metastatic breast cancer.

Although effective therapies exist for BRAF-mutant metastatic melanomas (MM) and ER+/PR+/HER2+ breast cancers, fewer options are available for the more aggressive triple negative breast cancers (TNBC) and Ras-mutant MM. Immune infiltration is frequently observed in patient subsets with MM or TNBC. An anti-tumor host immune response may be restrained by the expression of immune checkpoint proteins, such as the programmed death 1 (PD1) protein. We evaluated mDX400, a murine version of the anti-PD-1 antibody MK-3475 that is currently in human clinical trials, in genetically engineered murine models (GEMMs) of melanoma and breast cancer (BC). PD1 antibody was used alone and in combination. Models studied were for basal-like, TNBC (C3TAg), Claudin-Low breast cancer p53null, luminal BC (MMTV-Her2/Neu), RAS-driven melanoma (Tyr-H-Ras and null for Ink4a/Arf (TRIA)) and BRaf-mutant, Pten-deficient melanoma (BRaf/Pten). Mice were housed, treated, and evaluated in the UNC Lineberger Comprehensive Cancer Center Mouse Phase I Unit (MP1U). mDX400 and isotype antibody (10mg/kg IP qw) were supplied by Merck &amp; Co., Inc. Murine cohorts were assessed weekly and therapeutic intervention began once tumors reached 40-64mm3. TNBC models were treated with Carboplatin (C, 50mg/kg IP qwk) in combination with mDX400. mDX400 was administered as a single agent in Melanoma GEMMs. Endpoints were maximal response at ≥21 days and median overall survival (OS). In the C3TAg BC model, mDX400 did not exhibit single-agent activity, but substantial synergy was observed in combination with C [partial response (PR) or CR in 12 of 15 vs. 0 of 19 in non-treated animals, Fisher's exact p=0.006 or vs. C alone 5 of 13 animals, p=0.05] and prolonged OS (41 vs. 28 days in NT, log-rank p=0.006 or C only 28 days, p=0.006) in the C3TAg model. Treatment with single-agent mDX400 induced a marked response in the RAS-driven TRIA melanoma model that is resistant to multiple systemic treatments. mDX400-treated TRIA mice enjoyed an almost tripling of OS [median 56 vs. 21 days, p=0.006]. BRaf/Pten melanomas did not respond to mDX400. Likewise mDX400 plus C provided no benefit to the p53null or MMTV-Her2/Neu BC GEMMs vs. C alone. Our data show differential response of PD-1 antibody therapies to various melanoma and BC models, the latter in combination with C. The responsiveness of a RAS-driven melanoma model to mDX400 is consistent with earlier reports about the clinical benefit of patients with NRAS-mutant melanoma to Ipilimumab, and the lack of PD-L1 expression and immune infiltrates of PTEN-deficient melanomas. At the meeting we will report expression of PD-L1 and other immune checkpoint proteins by cancer cells. The basis whereby carboplatin potentiates the activity of anti-PD1 in TNBC is an area of ongoing study. Citation Format: David Darr, Kelly S. Clark, Joseph H. Phillips, Elaine Pinheiro, Venkataraman Sriram, Jessie Xiong, Jamie Jordan, Norman E. Sharpless, Charles Perou, Stergios Moschos. mDX400, the murine analog against the anti-PD1 antibody MK-347 is active in immunocompetent, autochthonous murine models of melanoma and breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5024. doi:10.1158/1538-7445.AM2014-5024