Triple-negative Breast Cancer Models Research Articles

Abstract PO1-19-08: Phase I trial of pegylated liposomal doxorubicin chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer

Abstract Background: Only a subset of patients with metastatic triple-negative breast cancers (TNBC) demonstrate response to FDA approved PD-1 immune checkpoint blockade (ICB), and few have durable responses. Data suggests that breast cancers have defects in antigen presentation and that antigen presenting cells especially the cDC1 subtype of dendritic cells (DCs) are required for response to ICB. CD40 agonists activate antigen presenting cells including DCs and B cells and repolarize macrophages to an anti-tumor phenotype. Flt3 ligand is a growth factor that increases differentiation and expansion of DCs. We recently demonstrated in pre-clinical TNBC models that the combination of liposomal doxorubicin chemotherapy, a CD40 agonist, and a Flt3 ligand improves outcomes compared to alternate combinations. Methods: This is a single arm phase I pilot study of liposomal doxorubicin, CDX-1140 (CD40 agonist monoclonal antibody), and CDX-301 (recombinant Flt3 ligand) combination therapy in patients with metastatic or unresectable locally advanced metastatic TNBC. Patients will be randomized to 3 lead-in arms (triplet therapy, doublet immunotherapy only, or liposomal doxorubicin only) for one cycle prior to receiving triplet therapy with tissue biopsies done before and after the lead-in treatment. CDX-301 will be given for only two cycles; liposomal-doxorubicin and CDX-1140 will be continued until disease progression or clinically limiting toxicities. Primary endpoint is determination of a recommended phase II dose based on treatment-related adverse events and dose-limiting toxicities. Secondary endpoints include anti-tumor immune response after triplet therapy, after immunotherapy alone, and after liposomal doxorubicin alone; median progression-free survival, overall response rate, duration of response, and clinical benefit rate. Key eligibility criteria are unresectable stage III or stage IV TNBC (ER ≤10%, PR ≤10%, HER2/neu negative), 1st to 3rd line treatment for metastatic disease (1st line patients need to be PD-L1 negative by 22C3 assay), measurable disease by RECIST 1.1 criteria, consent for pre-treatment and on-treatment biopsies of amenable soft tissue tumor lesions, no prior treatment with an anti-CD40 antibody or a Flt3 ligand, no anthracycline treatment in the metastatic setting, no prior progression while on anthracycline-based therapy or within 6 months of completing neoadjuvant chemotherapy, and no history of non-infectious pneumonitis or current pneumonitis. This trial will enroll up to 45 patients across multiple sites (NCT05029999) and is currently open at University of Texas Southwestern Medical Center, Texas Oncology, University of Chicago, University of Texas San Antonio, Sarah Cannon Research Institute, and Johns Hopkins. Citation Format: Sangeetha Reddy, Joyce O'Shaughnessy, Navid Sadeghi, Samira Syed, Cesar Santa-Maria, Virginia Kaklamani, Nan Chen, Denise Yardley, Yisheng Fang, Isaac Chan, Nisha Unni, Sarah Kashanian, Namrata Peswani, Shahbano Shakeel, Meredith Carter, Kelly Kyle, Farjana Fattah, Chul Ahn, Ina Patel, Joshua Gruber, Dawn Klemow, Glenda Delgado, Nicole Sinclair, Michael Yellin, Heather McArthur, Rita Nanda, Suzanne Conzen, Carlos Arteaga. Phase I trial of pegylated liposomal doxorubicin chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-19-08.

Entolimod, a clinical stage flagellin derivative, improves clinically relevant chemo-immunotherapy responses in pre-clinical triple negative breast cancer

Abstract Chemotherapy+anti-PD-1 is FDA approved and has improved outcomes for locally advanced and metastatic PD-L1+ triple negative breast cancer (TNBC). Unfortunately, a need still exists to improve this chemo-immunotherapy (CTx-I) platform especially in advanced disease. Entolimod, an agonist of the TLR5 and NAIP5 receptors, is our flagellin derivative which has successfully completed Phase I safety trials. We previously showed that systemically administered entolimod enhances durable antitumor immunity against metastasis of 4T1, a preclinical PD-L1+ TNBC model. However, the antimetastatic activity of single-agent entolimod was not completely effective, highlighting the need to combine entolimod with additional immune-based modalities such as CTx-I to further improve tumor control. Here, we used Paclitaxel+anti-PD-1 as a clinically oriented CTx-I platform in pre-clinical metastatic TNBC models. CTx-I+entolimod effectively controlled primary tumors and reduced lung metastatic burden more so than the single or double agent treatments. Consistent with our prior work that neutrophils express functional TLR5, depletion abrogated CTx-I+entolimod efficacy. Collectively, the immune enhancing effects of entolimod can be leveraged to boost urgently needed CTx-I outcomes in TNBC. Because entolimod has successfully completed Phase I clinical trials, this preclinical work supports the rationale design of a clinical trial that combines CTx-I with entolimod as an effective immunotherapy platform.

Activation of the Anaphase Promoting Complex Restores Impaired Mitotic Progression and Chemosensitivity in Multiple Drug-Resistant Human Breast Cancer.

The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy.

Priming with oncolytic adenovirus followed by anti-PD-1 and paclitaxel treatment leads to improved anti-cancer efficacy in the 3D TNBC model

Triple-negative breast cancer (TNBC) is considered one of the most incurable malignancies due to its clinical characteristics, including high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Therefore, it remains a critical unmet medical need. On the other hand, poor delivery efficiency continues to reduce the efficacy of anti-cancer therapeutics developed against solid tumours using various strategies, such as genetically engineered oncolytic vectors used as nanocarriers. The study was designed to evaluate the anti-tumour efficacy of a novel combinatorial therapy based on oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with an anti-PD-1 (pembrolizumab) and paclitaxel (PTX). Here, we first tested the antineoplastic effect in two-dimensional (2D) and three-dimensional (3D) breast cancer models in MDA-MB-231, MDA-MB-468 and MCF-7 cells. Then, to further evaluate the efficacy of combinatorial therapy, including immunological aspects, we established a three-dimensional (3D) co-culture model based on MDA-MB-231 cells with peripheral blood mononuclear cells (PBMCs) to create an integrated system that more closely mimics the complexity of the tumour microenvironment and interacts with the immune system. Treatment with OV as a priming agent, followed by pembrolizumab and then paclitaxel, was the most effective in reducing the tumour volume in TNBC co-cultured spheroids. Further, T-cell phenotyping analyses revealed significantly increased infiltration of CD8+, CD4+ T and Tregs cells. Moreover, the observed anti-tumour effects positively correlated with the level of CD4+ T cell infiltrates, suggesting the development of anti-cancer immunity. Our study demonstrated that combining different immunotherapeutic agents (virus, pembrolizumab) with PTX reduced the tumour volume of the TNBC co-cultured spheroids compared to relevant controls. Importantly, sequential administration of the investigational agents (priming with the vector) further enhanced the anti-cancer efficacy in 3D culture over other groups tested. Taken together, these results support further evaluation of the virus in combination with anti-PD-1 and PTX for the treatment of triple-negative breast cancer patients. Importantly, further studies with in vivo models should be conducted to better understand the translational aspects of tested therapy.

Synergistic Action of Benzyl Isothiocyanate and Sorafenib in a Nanoparticle Delivery System for Enhanced Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) presents a therapeutic challenge due to its complex pathology and limited treatment options. Addressing this challenge, our study focuses on the effectiveness of combination therapy, which has recently become a critical strategy in cancer treatment, improving therapeutic outcomes and combating drug resistance and metastasis. We explored a novel combination therapy employing Benzyl isothiocyanate (BITC) and Sorafenib (SOR) and their nanoformulation, aiming to enhance therapeutic outcomes against TNBC. Through a series of in vitro assays, we assessed the cytotoxic effects of BITC and SOR, both free and encapsulated. The BITC-SOR-loaded nanoparticles (NPs) were synthesized using an amphiphilic copolymer, which demonstrated a uniform spherical morphology and favorable size distribution. The encapsulation efficiencies, as well as the sustained release profiles at varied pH levels, were quantified, revealing distinct kinetics that were well-modeled by the Korsmeyer-Peppas equation. The NP delivery system showed a marked dose-dependent cytotoxicity towards TNBC cells, with an IC50 of 7.8 μM for MDA-MB-231 cells, indicating improved efficacy over free drugs, while exhibiting minimal toxicity toward normal breast cells. Furthermore, the NPs significantly inhibited cell migration and invasion in TNBC models, surpassing the effects of free drugs. These findings underscore the potential of BITC-SOR-NPs as a promising therapeutic approach for TNBC, offering targeted delivery while minimizing systemic toxicity.

Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts the Coactivator Med25 Protein-Protein Interactions.

Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (LPPM-8) increases the affinity for the coactivator Med25 >20-fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein-protein interactions (PPIs). The lipid structure, the peptide sequence, and the C-terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM-8 and its effectiveness as an inhibitor. LPPM-8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, LPPM-8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes.

Structure-Guided Discovery and Preclinical Assessment of Novel (Thiophen-3-yl)aminopyrimidine Derivatives as Potent ERK1/2 Inhibitors.

The RAS-RAF-MEK-ERK signaling cascade is abnormally activated in various tumors, playing a crucial role in mediating tumor progression. As the key component at the terminal stage of this cascade, ERK1/2 emerges as a potential antitumor target and offers a promising therapeutic strategy for tumors harboring BRAF or RAS mutations. Here, we identified 36c with a (thiophen-3-yl)aminopyrimidine scaffold as a potent ERK1/2 inhibitor through structure-guided optimization for hit 18. In preclinical studies, 36c showed powerful ERK1/2 inhibitory activities (ERK1/2 IC50 = 0.11/0.08 nM) and potent antitumor efficacy both in vitro and in vivo against triple-negative breast cancer and colorectal cancer models harboring BRAF and RAS mutations. 36c could directly inhibit ERK1/2, significantly block the phosphorylation expression of their downstream substrates p90RSK and c-Myc, and induce cell apoptosis and incomplete autophagy-related cell death. Taken together, this work provides a promising ERK1/2 lead compound for multiple tumor-treatment drug discovery.

A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer.

New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and DI) domains. In proof-of-concept studies, we demonstrated that a monoclonal antibody (mAb) that simultaneously inhibits both domains represents a promising therapeutic approach. Here, we screened a hybridoma library using a multistep selection strategy, including flow cytometry for Ab binding to native conformation protein and in vitro cell-based functional assays to isolate a novel panel of highly specific human ADAM8 dual MP and DI inhibitory mAbs, called ADPs. The screening of four top candidates for in vivo anti-cancer activity in an orthotopic MDA-MB-231 TNBC model of ADAM8-driven primary growth identified two lead mAbs, ADP2 and ADP13. Flow cytometry, hydrogen/deuterium exchange-mass spectrometry (HDX-MS) and alanine (ALA) scanning mutagenesis revealed that dual MP and DI inhibition was mediated via binding to the DI. Further testing in mice showed ADP2 and ADP13 reduce aggressive TNBC characteristics, including locoregional regrowth and metastasis, and improve survival, demonstrating strong therapeutic potential. The continued development of these mAbs into an ADAM8-targeted therapy could revolutionize TNBC treatment.

EMP2 Serves as a Functional Biomarker for Chemotherapy-Resistant Triple-Negative Breast Cancer.

Breast cancer (BC) remains among the most commonly diagnosed cancers in women worldwide. Triple-negative BC (TNBC) is a subset of BC characterized by aggressive behavior, a high risk of distant recurrence, and poor overall survival rates. Chemotherapy is the backbone for treatment in patients with TNBC, but outcomes remain poor compared to other BC subtypes, in part due to the lack of recognized functional targets. In this study, the expression of the tetraspan protein epithelial membrane protein 2 (EMP2) was explored as a predictor of TNBC response to standard chemotherapy. We demonstrate that EMP2 functions as a prognostic biomarker for patients treated with taxane-based chemotherapy, with high expression at both transcriptomic and protein levels following treatment correlating with poor overall survival. Moreover, we show that targeting EMP2 in combination with docetaxel reduces tumor load in syngeneic and xenograft models of TNBC. These results provide support for the prognostic and therapeutic potential of this tetraspan protein, suggesting that anti-EMP2 therapy may be beneficial for the treatment of select chemotherapy-resistant TNBC tumors.

Chitin-mediated blockade of chitinase-like proteins reduces tumor immunosuppression, inhibits lymphatic metastasis and enhances anti-PD-1 efficacy in complementary TNBC models

BackgroundChitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1).MethodsFemale BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling.ResultsChitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α+ T-cell infiltration and activation in primary tumor and lymphoid organs.ConclusionsChitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.

Targeting CXCR4/CXCL12 axis via [177Lu]Lu-DOTAGA.(SA.FAPi)2 with CXCR4 antagonist in triple-negative breast cancer

PurposeRadiopharmaceutical therapies targeting fibroblast activation protein (FAP) have shown promising efficacy against many tumor types. But radiopharmaceuticals alone in most cases are insufficient to completely eradicate tumor cells, which can partially be attributed to the protective interplay between tumor cells and cancer-associated fibroblasts (CAFs). The C-X-C chemokine receptor type 4/C-X-C motif chemokine 12 (CXCR4/CXCL12) interaction plays an important role in orchestrating tumor cells and CAFs. We hereby investigated the feasibility and efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2, a FAP-targeting radiopharmaceutical, in combination with AMD3100, a CXCR4 antagonist, in a preclinical murine model of triple-negative breast cancer (TNBC).MethodsPublic database was first interrogated to reveal the correlation between CAFs’ scores and the prognosis of TNBC patients, as well as the expression levels of FAP and CXCR4 in normal tissues and tumors. In vitro therapeutic efficacy regarding cell proliferation, migration, and colony formation was assessed in BALB/3T3 fibroblasts and 4T1 murine breast cancer cells. In vivo therapeutic efficacy was longitudinally monitored using serial 18F-FDG, [18F]AlF-NOTA-FAPI-04, and [68Ga]Ga-DOTA-Pentixafor PET/CT scans and validated using tumor sections through immunohistochemical staining of Ki-67, α-SMA, CXCR4, and CXCL12. Intratumoral abundance of myeloid-derived suppressive cells (MDSCs) was analyzed using flow cytometry in accordance with the PET/CT schedules. Treatment toxicity was evaluated by examining major organs including heart, lung, liver, kidney, and spleen.ResultsCAFs’ scores negatively correlated with the survival of TNBC patients (p < 0.05). The expression of CXCR4 and FAP was both significantly higher in tumors than in normal tissues. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 significantly suppressed cell proliferation, migration, and colony formation in cell culture, and exhibited synergistic effects in 4T1 tumor models along with a decreased number of MDSCs. PET/CT imaging revealed lowest tumor accumulation of 18F-FDG and [18F]AlF-NOTA-FAPI-04 on day 13 and day 14 after treatment started, both of which gradually increased at later time points. A similar trend was observed in the IHC staining of Ki-67, α-SMA, and CXCL12.ConclusionThe combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 is a feasible treatment against TNBC with minimal toxicity in main organs.

Lung-derived soluble factors support stemness/plasticity and metastatic behaviour of breast cancer cells via the FGF2-DACH1 axis

Patients with triple-negative breast cancer (TNBC) have an increased propensity to develop lung metastasis. Our previous studies demonstrated that stem-like ALDHhiCD44+ breast cancer cells interact with lung-derived soluble factors, resulting in enhanced migration and lung metastasis particularly in TNBC models. We have also observed that the presence of a primary TNBC tumor can ‘prime’ the lung microenvironment in preparation for metastasis. In this study, we hypothesized that soluble lung-derived factors secreted in the presence of a primary TNBC tumor can influence stemness/plasticity of breast cancer cells. Using an ex vivo pulmonary metastasis assay (PuMA), we observed that the lung microenvironment supports colonization and growth of ALDHhiCD44+ TNBC cells, potentially via interactions with lung-derived FGF2. Exposure of TNBC cells to lung-conditioned media (LCM) generated from mice bearing TNBC primary tumors (tbLCM) significantly enhanced the proportion of ALDHhiCD44+ cells compared to control or LCM from tumor-naïve mice (tnLCM). Further analysis using a human cancer stem cell qPCR array revealed that, relative to tnLCM or control, exposure of TNBC cells to tbLCM leads to downregulation of the transcription factor and putative tumor suppressor Dachshund homolog 1 (DACH1), a downstream regulator of FGF2. In addition, inhibition of DACH1 using siRNA or treatment with recombinant FGF2 enhanced the ALDHhiCD44+ phenotype. Taken together, our findings suggest that the FGF2-DACH1 signaling axis supports stemness/plasticity of TNBC cells in the lung microenvironment and lays the foundation for future evaluation of FGF2 as a potential novel therapeutic target for treatment or prevention of breast cancer metastasis to the lung.

Nanofluidic delivery implant sustains localization and maximizes efficacy of intratumoral immunotherapy

Intratumoral immunotherapy holds promise for improving cancer treatment efficacy. However, rapid clearance from the tumor upon bolus volume injections limits efficacy and impedes assessment of dose-dependent effects on the tumor immune microenvironment (TIME). To this end, we developed a drug-agnostic nanofluidic implant to deliver immunotherapy within the tumor, providing a mechanism for sustained and controlled intratumoral dosing. Diffusive drug elution from the implant reservoir is controlled by a nanofluidic membrane, which abrogates rapid drug elimination from the tumor and maximizes immunotherapy localization. Here we first achieve in vitro sustained release of agonist CD40 monoclonal antibody (mAb), anti-programmed death ligand-1 (PDL1) mAb, and immune-cell targeted polymeric prodrugs of Resiquimod (toll-like receptor 7/8,TLR 7/8) and a small molecule STING agonist. We then demonstrated in vivo sustained intratumoral drug localization of agonist CD40 Ab and anti-PDL1 Ab in the 4T1 murine model of triple-negative breast cancer (TNBC). Further, we show highly effective anti-tumor efficacy with radiation therapy and sustained intratumoral co-delivery of agonist CD40 Ab and anti-PDL1 Ab in the EMT6 TNBC murine model. Finally, we demonstrate the versatility of this implant by showing that sustained intratumoral delivery of the STING or Resiquimod polymeric prodrugs strongly inhibits 4T1 tumor growth. Collectively, these results support our nanofluidic system as a therapeutic platform technology to investigate sustained intratumoral dosing of immunotherapy.

Oncolytic adenovirus encoding apolipoprotein A1 suppresses metastasis of triple-negative breast cancer in mice

BackgroundDysregulation of cholesterol metabolism is associated with the metastasis of triple-negative breast cancer (TNBC). Apolipoprotein A1 (ApoA1) is widely recognized for its pivotal role in regulating cholesterol efflux and maintaining cellular cholesterol homeostasis. However, further exploration is needed to determine whether it inhibits TNBC metastasis by affecting cholesterol metabolism. Additionally, it is necessary to investigate whether ApoA1-based oncolytic virus therapy can be used to treat TNBC.MethodsIn vitro experiments and mouse breast cancer models were utilized to evaluate the molecular mechanism of ApoA1 in regulating cholesterol efflux and inhibiting breast cancer progression and metastasis. The gene encoding ApoA1 was inserted into the adenovirus genome to construct a recombinant adenovirus (ADV-ApoA1). Subsequently, the efficacy of ADV-ApoA1 in inhibiting the growth and metastasis of TNBC was evaluated in several mouse models, including orthotopic breast cancer, spontaneous breast cancer, and human xenografts. In addition, a comprehensive safety assessment of Syrian hamsters and rhesus monkeys injected with oncolytic adenovirus was conducted.ResultsThis study found that dysregulation of cholesterol homeostasis is critical for the progression and metastasis of TNBC. In a mouse orthotopic model of TNBC, a high-cholesterol diet promoted lung and liver metastasis, which was associated with keratin 14 (KRT14), a protein responsible for TNBC metastasis. Furthermore, studies have shown that ApoA1, a cholesterol reverse transporter, inhibits TNBC metastasis by regulating the cholesterol/IKBKB/FOXO3a/KRT14 axis. Moreover, ADV-ApoA1 was found to promote cholesterol efflux, inhibit tumor growth, reduce lung metastasis, and prolonged the survival of mice with TNBC. Importantly, high doses of ADV-ApoA1 administered intravenously and subcutaneously were well tolerated in rhesus monkeys and Syrian hamsters.ConclusionsThis study provides a promising oncolytic virus treatment strategy for TNBC based on targeting dysregulated cholesterol metabolism. It also establishes a basis for subsequent clinical trials of ADV-ApoA1 in the treatment of TNBC.

477 Evaluating the therapeutic efficacy of combination IL-12 and trabectedin for the treatment of triple negative breast cancer

OBJECTIVES/GOALS: IL-12 has potent immune effects but the presence of myeloid-derived suppressor cells (MDSC) can inhibit IL-12-induced NK cell cytotoxicity. Thus, we hypothesized that combining IL-12 with trabectedin, an immunosuppressive myeloid cell depleting agent, would improve its therapeutic efficacy in triple negative breast cancer (TNBC). METHODS/STUDY POPULATION: Combination IL-12 and trabectedin was tested in the 4T1 mouse model of TNBC. 4T1 cells were injected into the mammary fat pad of female BALB/cj mice. When tumors reached 50 mm3, mice were randomly divided into 4 groups and treated with PBS, IL-12 (0.5 μg/mouse 3x/wk), 0.15 mg/kg trabectedin weekly or the combination. Tumor volumes were measured by calipers. Mass cytometry was performed on spleens and tumors using a 35-antibody panel. Plasma IFN-γ levels were measured by ELISA. The role of NK cells was evaluated via depletion with anti-asialo-GM1. The Luminex Discovery Assay was used to measure plasma cytokines and immunohistochemistry was performed for CD4 and CD8a. Linear/nonlinear mixed effects modeling was used for in vivo data analysis and applicable t- or ANOVA tests were used for in vitrodata analysis. RESULTS/ANTICIPATED RESULTS: Combination IL-12 and trabectedin led to a significant reduction in tumor burden compared to single-agent IL-12, trabectedin and control treatments (all p&lt;0.001), as well as higher levels of IFN-γ (all p&lt;0.04). One combination treated mouse had complete tumor regression. Splenic MDSC were significantly decreased in combination treated mice. NK depletion abrogated the effects of combination therapy. Compared to mice receiving a control antibody, NK depletion of combination treated mice led to lower levels of CCL5 (p&lt;0.01) and CXCL10 (p&lt;0.001) and significantly higher tumor burden (p=0.001). CD8+T cell levels were significantly higher in combination treated mice compared to those receiving IL-12 (p&lt;0.01), and these levels were decreased when mice were depleted of NK cells (p=0.01). DISCUSSION/SIGNIFICANCE: TNBC represents 15% of all breast cancer diagnoses and is associated with a worse prognosis compared to other subtypes. Black women are twice as likely to be diagnosed with TNBC and more likely to die from disease than White women. Thus, there is an increasing need to develop additional therapeutic options for this disease.

Pharmacological suppression of the OTUD4/CD73 proteolytic axis revives antitumor immunity against immune-suppressive breast cancers.

Despite widespread utilization of immunotherapy, treating immune-cold tumors remains a challenge. Multiomic analyses and experimental validation identified the OTUD4/CD73 proteolytic axis as a promising target in treating immune-suppressive triple negative breast cancer (TNBC). Mechanistically, deubiquitylation of CD73 by OTUD4 counteracted its ubiquitylation by TRIM21, resulting in CD73 stabilization inhibiting tumor immune responses. We further demonstrated the importance of TGF-β signaling for orchestrating the OTUD4/CD73 proteolytic axis within tumor cells. Spatial transcriptomics profiling discovered spatially resolved features of interacting malignant and immune cells pertaining to expression levels of OTUD4 and CD73. In addition, ST80, a newly developed inhibitor, specifically disrupted proteolytic interaction between CD73 and OTUD4, leading to reinvigoration of cytotoxic CD8+ T cell activities. In preclinical models of TNBC, ST80 treatment sensitized refractory tumors to anti-PD-L1 therapy. Collectively, our findings uncover what we believe to be a novel strategy for targeting the immunosuppressive OTUD4/CD73 proteolytic axis in treating immune-suppressive breast cancers with the inhibitor ST80.

Abstract 679: TXB-001 is an upgraded conjugated anthracycline that targets tumors with reduced risk of cardiotoxicity, hand-foot syndrome and alopecia

Abstract TXB-001 is a well-optimized N-(2-hydroxypropyl)methacrylamide copolymer-based conjugated pirarubicin (THP) and a novel candidate for developing anticancer drugs. It exhibits tumor-specific accumulation due to the enhanced permeability and retention effects and prolonged circulation time compared with free THP. Its hydrazone bond is dissociated under lower pH conditions, providing selective and rapid release of THP in acidic tumor environments. Therefore, TXB-001 is expected to have high efficacy and low toxicity. Anthracycline anticancer drugs have been widely used for the treatment of several cancers, but their use is limited by several adverse effects (AEs), including cardiotoxicity, which is a life-threatening AE. Furthermore, the maximum lifetime cumulative dose is decided for each anthracycline in clinical use, which leads to discontinuation of the medication. Hand-foot syndrome (HFS) is a dose-limiting AE of DOXIL®, a liposomal formulation of doxorubicin (DOX), and although it is not a life-threatening condition, it significantly deteriorates quality of life (QOL). Alopecia is another common AE, which is relatively noninvasive but adversely affects mental health and reduces QOL. In this study, we confirmed the efficacy of TXB-001 in a mice triple-negative breast cancer (TNBC) model and evaluated the cardiotoxicity-, HFS- and alopecia-inducing effects of TXB-001 in mice and rats to compare the severity with existing anthracycline anticancer drugs, including DOXIL®. The pharmacokinetic (PK) analysis in several tissues, including plasma, tumor, heart, skin of the chest, and palmar and plantar areas, was also performed after a single intravenous administration in rodents. As a result, TXB-001 showed an equivalent or greater antitumor effects than other anthracyclines in the TNBC model, and the AEs were scarcely observed. TXB-001 did not decrease left ventricular ejection fraction, with no or little HFS-like changes compared with DOXIL®, which showed significant histopathological changes. TXB-001 also showed weaker alopecia-inducing effects than DOX, DOXIL®, and THP. In the PK analysis, TXB-001 was distributed in the tumor at a higher concentration than in other tissues, except for plasma. Hence, most of the THP, the active ingredient, was released in the tumor. The exposure of the heart in the TXB-001-treated group was one-tenth of that in the DOXIL®-treated group. Moreover, accumulation in the skin, especially in palmar and plantar areas, was observed for DOXIL®, but no accumulation in the skin was observed for TXB-001. This PK profile consistently demonstrated the antitumor activity of TXB-001, without AEs. TXB-001 reduces the chance of dose-limiting and QOL-threatening AEs, and thus, cancer patients can continue treatment, making it a more convenient anticancer drug in clinical use with a wide safety margin. Citation Format: Keiyu Oshida, Mikito Hirakata, Emi Tomikawa, Chizuka Sakai, Masashi Uchida, Rieko Shimozono, Akiko Izawa, Yoko Koga, Tsubasa Okano, Hideki Narumi, Lisa Munakata, Ryo Suzuki, Miki Nonaka, Yasuhito Uezono. TXB-001 is an upgraded conjugated anthracycline that targets tumors with reduced risk of cardiotoxicity, hand-foot syndrome and alopecia [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 679.

Abstract 4098: A citrullinated-ENO1 peptide vaccine delayed tumor growth and markedly improved overall survival in a mouse model of triple-negative breast cancer

Abstract Purpose: To evaluate the impact of a novel citrullinated-enolase 1 (cit-ENO1) peptide-based vaccine on the immune response, tumor growth and overall survival in a mouse model of triple-negative breast cancer (TNBC). Background: Cancer vaccines have consisted primarily of mutated or overexpressed proteins in cancer. We have uncovered dysregulated protein citrullination by peptidyl arginine deiminases (PADI) as a feature of triple negative breast cancer inducing immunogenicity to citrullinated proteins. On the basis of these findings, we investigated a novel vaccine based on cancer-selective cit-ENO1 peptides that we identified from mass spectrometry analysis of the surfaceome and immunopeptidome in TNBC. We assessed the extent to which the vaccine elicited an immune response and delayed tumor growth and improved overall survival in a syngeneic mouse model of TNBC. Experimental Design: We immunized female B6129SF1/J mice with cit-ENO1 peptides or corresponding unmodified ENO1 (unm-ENO1) peptides as antigens, together with the TLR3 agonist poly I:C as an adjuvant, once a week, for three weeks. PBS or poly I:C alone were used as controls. One week post the last round of immunization, draining lymph nodes (LN) and immunized skin were harvested, and T cell phenotypes evaluated using flow cytometry. To assess anti-cancer efficacy, mice were immunized with cit-ENO1 or unm-ENO1 peptides, followed by orthotopic implantation with murine BRCA1co/co; MMTV-Cre; p53+/- TNBC cells. Tumor growth and overall survival were assessed. Results: Cit-ENO1 peptide vaccination induced a statistically significantly greater percentage of activated CD8+ PD-1+ effector T cells as well as CD8+ CD44+ CD62L- effector memory T cells in the LNs compared with either the control groups or the unm-ENO1 peptide. CD8+ PD-1+ T-cells were also significantly elevated in immunized skin of cit-ENO1 vaccinated mice. Both cit-ENO1 and unm-ENO1 vaccine increased CD4+ PD-1+ T cells from the LNs and immunized skin. Remarkably, the cit-ENO1 vaccine but not the um-ENO1 vaccine significantly delayed tumor growth and markedly improved overall survival compared to respective controls. Conclusion: Cancer-selective cit-ENO1 peptides induced a potent anti-cancer immune response and significantly delayed tumor initiation and progression in a preclinical, immune competent mouse model of TNBC. Citation Format: Ricardo A. Leon Letelier, Hiroyuki Katayama, Yihui Chen, Yinning Cai, Fuchung Hsiao, Banu Arun, Samir Hanash. A citrullinated-ENO1 peptide vaccine delayed tumor growth and markedly improved overall survival 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 4098.

Abstract 2189: Tirzepatide mitigates obesity-associated metabolic dysregulation and tumor progression in a mouse model of triple-negative breast cancer

Abstract 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 &amp;gt;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 &amp;gt;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 &amp;gt;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.