IntroductionTriple-negative breast cancer (TNBC) comprises a heterogeneous group of clinically aggressive tumors with high risk of recurrence and metastasis. Current pharmacological treatment options remain largely limited to chemotherapy. Despite promising results, the efficacy of immunotherapy and chemo-immunotherapy in TNBC remains limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, including g-secretase inhibitors (GSIs), are quite effective in preclinical models of TNBC. However, the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects, since Notch plays key roles in T-cell activation, including CD8 T-cells in tumors. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and ideally, do not affect tumor immunity. We identified sulindac sulfide (SS), the active metabolite of FDA-approved NSAID sulindac, as a potential candidate to replace GSIs.MethodsWe investigated the pharmacological and immunotherapeutic properties of SS in TNBC models in vitro, ex-vivo and in vivo.ResultsWe confirmed that SS, a known γ-secretase modulator (GSM), inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models tested. In a transplantable mouse TNBC tumor model (C0321), SS had remarkable single-agent anti-tumor activity and eliminated Notch1 protein expression in tumors. Importantly, SS did not inhibit Notch cleavage in T- cells, and the anti-tumor effects of SS were significantly enhanced when combined with a-PD1 immunotherapy in our TNBC organoids and in vivo.DiscussionOur data support further investigation of SS for the treatment of TNBC, in conjunction with chemo- or -chemo-immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be a cost-effective, rapidly deployable therapeutic option for a patient population in need of more effective therapies.

A critical feature of cancer is the ability to induce immunosuppression and evade immune responses. Tumor-induced immunosuppression diminishes the effectiveness of endogenous immune responses and decreases the efficacy of cancer immunotherapy. In this study, we describe a new immunosuppressive pathway in which adenosine promotes Casitas B-lineage lymphoma b (Cbl-b)-mediated Notch1 degradation, causing suppression of CD8+ T-cells effector functions. Genetic knockout and pharmacological inhibition of Cbl-b prevents Notch1 degradation in response to adenosine and reactivates its signaling. Reactivation of Notch1 results in enhanced CD8+ T-cell effector functions, anti-cancer response and resistance to immunosuppression. Our work provides evidence that targeting the Cbl-b-Notch1 axis is a novel promising strategy for cancer immunotherapy.

Background: Tumor-induced immunosuppression is a way in which cancers evade the host immune response. In recent years, a great effort has been made in designing immunotherapies that can boost the immune system response against cancer. However, not all tumors respond to these therapies. Therefore, there remains an unmet need for immunotherapies that are able to circumvent tumor-induced immunosuppression. Adenosine is an immunosuppressive metabolite which is overproduced in hypoxic tumor microenvironments and dampens T-cell anti-tumor immune responses. Adenosine A2A receptor (A2AR) activation was shown to downregulate Notch1, a key regulator of T-cell functions, in CD8+ T-cells, leading to immunosuppression. Notch1 signaling appears to protect T-cells against immunosuppressive signals, as Notch1 overexpressing T-cells were shown to be resistant to tumor-induced immunosuppression mediated by myeloid-derived suppressor cells (MDSC), as well as adenosine. We hypothesize that rescuing Notch1 from downregulation will make T-cells resistant to tumor-induced immunosuppression. We applied several functional assays in primary CD8+ T-cells and tumor-derived organoids to study how Notch1 is regulated by A2AR and explore strategies to target this pathway for cancer immunotherapy. Results: Our results indicate that the ubiquitin ligase Cbl-b, a negative regulator of T-cell functions, is responsible for the ubiquitination and degradation of Notch1 in CD8+ T-cells. A2AR signaling leads to Notch1 downregulation by promoting Cbl-b-mediated Notch1 degradation. Inhibition of Cbl-b using small molecule compounds restores Notch1 and T-cell functions in CD8+ T-cells in the presence of adenosine, making them resistant to A2AR-mediated immunosuppression. Cbl-b inhibitors show anti-tumor activity in tumor-derived organoids from pre-clinical models and enhance immune-checkpoint immunotherapy, by promoting T-cells anti-cancer responses. Conclusions: Our work suggests that promoting Notch1 signaling by blocking Cbl-b-mediated degradation results in increased T-cell responses and resistance to immunosuppression. Targeting Cbl-b-Notch1 axis represents a promising novel strategy to boost anti-cancer T-cell responses. Citation Format: Giulia Monticone, Fred Csibi, Silvana Leit, Jermaine E. Austin, Deniz A. Ucar, Fokhrul M. Hossain, Samarpan Majumder, Barbara A. Osborne, Christine Loh, Lucio Miele. Targeting Cblb-Notch1 axis as a novel strategy for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2064.

The epithelial to mesenchymal transition (EMT) is characterized by a loss of cell polarity, a decrease in the epithelial cell marker E-cadherin, and an increase in mesenchymal markers including the zinc-finger E-box bind-ing homeobox (ZEB1). The EMT is also associated with an increase in cell migration and anchorage-independent growth. Induction of a reversal of the EMT, a mesenchymal to epithelial transition (MET), is an emerging strategy being explored to attenuate the metastatic potential of aggressive cancer types, such as triple-negative breast can-cers (TNBCs) and tamoxifen-resistant (TAMR) ER-positive breast cancers, which have a mesenchymal phenotype. Patients with these aggressive cancers have poor prognoses, quick relapse, and resistance to most chemother-apeutic drugs. Overexpression of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 is associated with poor patient survival in breast cancer. Moreover, TNBC and tamoxifen resistant cancers are unresponsive to most targeted clinical therapies and there is a dire need for alternative therapies. In the current study, we found that MAPK3, MAPK1, and MAPK7 gene expression correlated with EMT mark-ers and poor overall survival in breast cancer patients using publicly available datasets. The effect of ERK1/2 and ERK5 pathway inhibition on MET was evaluated in MDA-MB-231, BT-549 TNBC cells, and tamoxifen-resistant MCF-7 breast cancer cells. Moreover, TU-BcX-4IC patient-derived primary TNBC cells were included to enhance the translational relevance of our study. We evaluated the effect of pharmacological inhibitors and lentivirus-induced activation or inhibition of the MEK1/2-ERK1/2 and MEK5-ERK5 pathways on cell morphology, E-cadherin, vimentin and ZEB1 expression. Additionally, the effects of pharmacological inhibition of trametinib and XMD8-92 on nuclear localization of ERK1/2 and ERK5, cell migration, proliferation, and spheroid formation were evaluated. Novel compounds that target the MEK1/2 and MEK5 pathways were used in combination with the AKT inhibitor ipatasertib to understand cell-specific responses to kinase inhibition. The results from this study will aid in the design of innovative therapeutic strategies that target cancer metastases.

Extracellular signal-regulated kinase (ERK5) is an essential regulator of cancer progression, tumor relapse, and poor patient survival. Epithelial to mesenchymal transition (EMT) is a complex oncogenic process, which drives cell invasion, stemness, and metastases. Activators of ERK5, including mitogen-activated protein kinase 5 (MEK5), tumor necrosis factor α (TNF-α), and transforming growth factor-β (TGF-β), are known to induce EMT and metastases in breast, lung, colorectal, and other cancers. Several downstream targets of the ERK5 pathway, such as myocyte-specific enhancer factor 2c (MEF2C), activator protein-1 (AP-1), focal adhesion kinase (FAK), and c-Myc, play a critical role in the regulation of EMT transcription factors SNAIL, SLUG, and β-catenin. Moreover, ERK5 activation increases the release of extracellular matrix metalloproteinases (MMPs), facilitating breakdown of the extracellular matrix (ECM) and local tumor invasion. Targeting the ERK5 signaling pathway using small molecule inhibitors, microRNAs, and knockdown approaches decreases EMT, cell invasion, and metastases via several mechanisms. The focus of the current review is to highlight the mechanisms which are known to mediate cancer EMT via ERK5 signaling. Several therapeutic approaches that can be undertaken to target the ERK5 pathway and inhibit or reverse EMT and metastases are discussed.

[This corrects the article DOI: 10.1371/journal.pone.0226464.].

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). Although MAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration (FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive breast cancers and TNBC patients are pathologically negative for estrogen receptor (ER−), progesterone receptor (PR−), and human epidermal growth factor receptor 2 (HER2−) amplification, which impedes the use of targeted therapies used in other breast cancer subtypes. Obesity is a chronic state of inflammation and is associated with increased secretion of pro-inflammatory cytokines, increased infiltration of immune cells and the development of a microenvironment that supports tumor growth. Several epidemiological studies support the association of obesity with TNBC; however, the underlining molecular mechanisms remain unknown. We developed an immunocompetent obese FVB (female) mouse model by feeding ‘Western diet' or control diet for four months and then injecting with syngeneic C0321 (mouse TNBC) cells to investigate the immune signatures and to study the role of obesity related factors in TNBC progression. We found significant body weight increases in ‘Western diet' fed mice group as expected. The percentage of CD8+ and CD4+ T cells, and macrophages in spleen, liver and peripheral blood in obese mice did not change significantly compared to lean mice. However, the percentages of immunosuppressive Myeloid Derived Suppressor cells (MDSCs), particularly Monocytic-MDSCs were elevated in liver and visceral fat tissue in obese mice. Importantly, tumors in ‘Western Diet' fed mice achieved significantly larger volumes. We found no noticeable difference in the percentage of CD8+ and CD4+ T cells, MDSC or macrophages in the spleen of tumor bearing obese mice. Interestingly, there was a decreasing trend in tumor infiltrating CD8+ and CD4+ T cells in tumor bearing obese mice. Further, tumor infiltrating myeloid cells, MDSCs and macrophages were significantly increased in obese mice, suggesting the formation of an immunosuppressive environment. More importantly, tumor infiltrating MDSCs from obese mice were more immunosuppressive than those from lean mice. In summary, our results reveal alterations in the number and function of tumor infiltrating immune cells in obese mice. RNA-Sequencing of lean vs obese tumors revealed significant changes in pathways related to Hypertrophic and Dilated Cardiomyopathy, Adrenergic signaling in Cardiomyocytes, as well as signaling pathways of Insulin, cGMP-PKG, Glucagon, Calcium and Adipocytokine and others. Our data warrants further investigation on the immunosuppressive tumor microenvironment in obese TNBC patients. Citation Format: Fokhrul Hossain, Deniz A Ucar, Maria Sanchez-Pino, Matthew Dean, Samarpan Majumder, Dorota Wyczechowska, Giulia Monticone, Rachel Sabol, Keli Xu, Luis D Valle, Jovanny Zabaleta, Bruce Bunnell, Lucio Miele. Investigating tumor infiltrating immune cells signature in obese triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3866.

Metaplastic breast carcinoma (MBC) is a clinically aggressive and rare subtype of breast cancer, with similar features to basal-like breast cancers. Due to rapid growth rates and characteristic heterogeneity, MBC is often unresponsive to standard chemotherapies; and novel targeted therapeutic discovery is urgently needed. Histone deacetylase inhibitors (DACi) suppress tumor growth and metastasis through regulation of the epithelial-to-mesenchymal transition axis in various cancers, including basal-like breast cancers. We utilized a new MBC patient-derived xenograft (PDX) to examine the effect of DACi therapy on MBC. Cell morphology, cell cycle-associated gene expressions, transwell migration, and metastasis were evaluated in patient-derived cells and tumors after treatment with romidepsin and panobinostat. Derivations of our PDX model, including cells, spheres, organoids, explants, and in vivo implanted tumors were treated. Finally, we tested the effects of combining DACi with approved chemotherapeutics on relative cell biomass. DACi significantly suppressed the total number of lung metastasis in vivo using our PDX model, suggesting a role for DACi in preventing circulating tumor cells from seeding distal tissue sites. These data were supported by our findings that DACi reduced cell migration, populations, and expression of mesenchymal-associated genes. While DACi treatment did affect cell cycle-regulating genes in vitro, tumor growth was not affected compared to controls. Importantly, gene expression results varied depending on the cellular or tumor system used, emphasizing the importance of using multiple derivations of cancer models in preclinical therapeutic discovery research. Furthermore, DACi sensitized and produced a synergistic effect with approved oncology therapeutics on inherently resistant MBC. This study introduced a role for DACi in suppressing the migratory and mesenchymal phenotype of MBC cells through regulation of the epithelial-mesenchymal transition axis and suppression of the CTC population. Preliminary evidence that DACi treatment in combination with MEK1/2 inhibitors exerts a synergistic effect on MBC cells was also demonstrated.

Abstract Triple-negative breast cancers (TNBCs) constitute approximately 12% of all breast cancer cases and are approximately twice as prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.3% in 2017), and New Orleans has among the highest incidences of TNBC in the country. Louisiana patients also have a high incidence of co-morbidities that affect breast cancer biology and outcomes, including type 2 diabetes and obesity. TNBCs have an aggressive clinical presentation due to high rates of metastasis, recurrence and chemoresistance. There are currently no clinically approved targeted therapies for TNBC; cytotoxic chemotherapy is the first-line treatment for TNBC, and recurrent, chemoresistant cancers are usually fatal. TNBCs are molecularly heterogeneous, consisting of at least four molecular subgroups, and immunologically heterogeneous. Both molecular and immunologic properties are associated with clinical outcomes and are seriously understudied in patients under-represented in biomedical research. Patient-derived xenografts (PDXs), as well as patient-derived organoids (PDO), are currently the best model for translational oncology therapeutic research because they accurately recapitulate the complex architecture and heterogenous genetic and molecular composition of solid cancers. To date, the majority of TNBC research has been based on Caucasian patients, although incidence rates of TNBC are higher in African-American cohorts. Our collaborative team aims to overcome this obstacle by establishing and characterizing TNBC PDX models that represent this understudied cohort. We currently have ten TNBC PDX models representing different patient ethnicities, responsiveness to chemotherapies, as well as different TNBC molecular subtypes and metastatic behavior. We dissect and evaluate the various individual components (tumor cell biology, stroma, immune, extracellular matrix) of TNBC tumors. We utilize these models in vivo, ex vivo and in vitro to examine how unique kinases and targeted inhibitors affect the distinct tumor characteristics. In addition to in vivo treatment studies, we generated cell lines and PDOs and we utilize novel techniques such as tissue decellularization to examine extracellular matrix components. We also analyze mechanistically relevant transcript (qRT-PCR) and protein (Western blot, immunohistochemistry) expression patterns that are unique to each PDX model to evaluate the effects of targeted therapies. We work with surrounding laboratories in the greater New Orleans area (Tulane, LSU, Xavier) that are also focused on therapeutic discovery of TNBC in a collaborative effort to provide translational models for their projects. Our aim is to leverage novel PDX models from understudied patients with a range of clinical and molecular presentations to guide the selection of therapeutically targetable pathways and therapeutic agents in specific molecular subtypes of TNBC. Citation Format: Margarite D. Matossian, Steven Elliott, Hope E. Burks, Maryl Wright, Rachel A. Sabol, Van T. Hoang, Deniz A. Ucar, Alex Alfortish, Jovanny Zabaleta, Fokhrul Hossain, Tiffany Chang, Henri Wathieu, Nicholas Pashos, Bruce Bunnell, Krzysztof Moroz, Arnold Zea, Adam Riker, Steven D. Jones, Elizabeth C. Martin, Lucio Miele, Bridgette M. Collins-Burow, Matthew E. Burow. Applications of patient-derived triple-negative breast cancer xenografts that represent understudied patients in Louisiana in targeted therapeutic research [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C110.

Triple negative breast cancers (TNBCs) are a clinically and biologically aggressive breast cancer (BC) subtype; TNBC tumors have higher rates of metastasis, relapse and acquired/inherent drug resistance. Incidence and mortality rates of TNBC are stratified based on patient ethnicity - patients with African ancestry have higher mortality rates and diagnoses of invasive cancers compared to patients representing other ethnicities. Louisiana has a high proportion of African-American residents (32.7% in 2018), and New Orleans has among the highest incidences of TNBC in the country. Many of our patients present with TNBC tumors that are partially or completely resistant to neoadjuvant chemotherapies. There are currently no clinically approved targeted therapies for TNBC. Current therapeutic discovery focused TNBC research does not aptly address the knowledge gap regarding ethnic disparity in TNBC incidence/mortality rates and TNBC biology. To date, most TNBC-related research and knowledge has been acquired from Caucasian patients, although patients with African and Hispanic ancestries represent the majority of TNBC cases. Patient-derived xenografts (PDXs) are extensively used in BC research, as they mimic complex microanatomy, oncoarchitecture, and cell-cell/cell-stroma interactions of tumors. Here, we demonstrated the unique composition of PDX tumors is not dramatically affected by serial transplantation in mice, based on molecular phenotypes (examined using qRT-PCR and RNA sequencing) and the oncoarchitecture of the extracellular matrix (based on cryogenic scanning electron microscopy). Using these models in basic research facilitates translation of laboratory findings to the clinical setting, and dramatically enhanced drug discovery research. We have established over twelve TNBC PDX models, 90% of which represent patients of African ancestry, and most of which are resistant to neoadjuvant regimens. We focus on dissecting and evaluating kinase inhibitor/targeted drug response to various individual components (tumor cell biology, stroma, immune, extracellular matrix) of chemotherapy resistant TNBC tumors. Histone deacetylase inhibitors (DACi) are a promising therapeutic agent in TNBC systems; they have been shown to suppress tumorigenesis and metastasis in TNBC through suppression of the mesenchymal phenotype in cell line-based studies. In this study we utilized various TNBC PDX models (TU-BcX-2K1, -2O0, 4IC, -4M4, -4QAN, -4QX) to assess these findings in more translational systems. Interestingly, we showed that DACi effect on tumorigenesis and metastasis varied depending on specific TNBC PDXs utilized. These data implicate specific genes/signaling pathways exist in individual patient tumors that can predict tumor responsiveness to DACi. Preliminary data using the NCI oncology drug set implicated the MEK1/2 pathway contributed to sensitization of TNBC cells. Furthermore, we found a disconnect in gene expressions that were previously shown to be affected by DACi therapy (CDH1, VIM, ZEB1, ZEB2) in various derivations of PDX models (cells, PDX-Os, ex vivo, in vivo). These findings demonstrate that testing various derivations of PDX models is crucial to parsing out specific mechanisms of targeted therapies. Our methods presented here to assess targeted drug response and drug resistance using PDX models can be applied to any area of cancer research and is not limited to breast cancer. Citation Format: Margarite Matossian, Steven Elliott, Maryl Wright, Tiffany Chang, Madlin Alzoubi, Henri Wathieu, Rachel Sabol, Alex Alfortish, Hope Burks, Van Hoang, Deniz Ucar, Gabrielle Windsor, Thomas Yan, Jovanny Zabaleta, Fokhrul Hossain, Bruce Bunnell, Krzysztof Moroz, Arnold Zea, Adam Riker, Steven Jones, Elizabeth Martin, Lucio Miele, Bridgette Collins-Burow, Matthew Burow. Effect of histone deacetylase inhibitors on patient-derived neoadjuvant chemotherapy resistant triple negative breast cancer xenografts that represent understudied patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-17.

Conventional mitogen-activated protein kinase (MAPK) family members regulate diverse cellular processes involved in tumor initiation and progression, yet the role of ERK5 in cancer biology is not fully understood. Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. ERK5 signaling contributes to drug resistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). More recently a role for ERK5 in regulation of the extracellular matrix (ECM) has been proposed, and here we investigated the necessity of ERK5 in TNBC tumor formation. Depletion of ERK5 expression using the CRISPR/Cas9 system in MDA-MB-231 and Hs-578T cells resulted in loss of mesenchymal features, as observed through gene expression profile and cell morphology, and suppressed TNBC cell migration. In vivo xenograft experiments revealed ERK5 knockout disrupted tumor growth kinetics, which was restored using high concentration Matrigel™ and ERK5-ko reduced expression of the angiogenesis marker CD31. These findings implicated a role for ERK5 in the extracellular matrix (ECM) and matrix integrity. RNA-sequencing analyses demonstrated downregulation of matrix-associated genes, integrins, and pro-angiogenic factors in ERK5-ko cells. Tissue decellularization combined with cryo-SEM and interrogation of biomechanical properties revealed that ERK5-ko resulted in loss of key ECM fiber alignment and mechanosensing capabilities in breast cancer xenografts compared to parental wild-type cells. In this study, we identified a novel role for ERK5 in tumor growth kinetics through modulation of the ECM and angiogenesis axis in breast cancer.

Abstract One of the hallmarks of cancer is the ability to evade the host immune system and this is achieved through different mechanisms. Several studies have showed that the accumulation of adenosine in the tumor microenvironment suppresses T-cell functions, thus helping the tumor to evade the immune system. Finding a way to counteract adenosine-mediated immune suppression might greatly enhance the host endogenous immune response against cancer and the efficacy of adoptive immunotherapies. To achieve this, we need to understand how adenosine regulates T-cell functions. In a collaborative project, our group recently showed that stimulation of adenosine A2A receptor (A2AR) reduces T-cell receptor (TCR)-signaling and consequently Notch1 activation and expression in CD8+ T-cells, which are critical to cancer immunity. This suggests that A2AR stimulation suppresses CD8+ T-cell function through inhibition of TCR-induced Notch1, which is required for the activation and function of CD8+ T-cells. Based on these observations, we hypothesized that while stimulation of A2AR suppresses CD8+ T-cells, inhibition of A2AR should protect CD8+ T-cells from the adenosine-mediated immune suppression. Notch1 is likely to be critical in this process since it was shown that ectopic expression of Notch1 intracellular domain prevents the adenosine-mediated immune suppression in CD8+ T-cells. Therefore, we aim to investigate the effect of A2AR inhibition on TCR activation and Notch1 to evaluate the A2AR-Notch axis as a novel immunotherapeutic target. Our data show that pharmacological inhibition of A2AR with a selective antagonist induces tumor cell death and increases the number of CD8+ T-cells in tumor-derived spheroids from a mouse triple-negative breast cancer (TNBC) model. The effect of A2AR inhibition appears to be immune-mediated since the same agent did not induce cell death in tumor-derived spheroids from immunocompromised mice. Along the same lines, we show that, inhibition of A2AR restored Notch1 activation and proliferation in primary murine CD8+ T-cells, but did not affect Notch1 and proliferation in TNBC cell lines. This effect is likely to be strictly dependent on Notch since A2AR inhibition failed to rescue CD8+ T-cell proliferation from the suppressive effect of gamma-secretase inhibition. It is not yet known how A2AR regulates Notch1, however, our latest preliminary results suggest that A2AR stimulation might promote the endosomal degradation of Notch1, whereas, A2AR inhibition might switch Notch1 fate from endosomal degradation to activation. Further investigation is needed to establish how A2AR regulates Notch1 and whether A2AR inhibition can protect CD8+ T-cell function in vivo in the tumor microenvironment. Overall our current data provides a rationale for the evaluation of A2AR antagonists as a Notch-modulating immunotherapy. Citation Format: Giulia Monticone, Fokhrul M. Hossain, Deniz A. Ucar, Samarpan Majumder, Claudia Sorrentino, Paulo C. Rodriguez, Rosa A. Sierra, Antonio Pannuti, Stephen Hatfield, Barbara A. Osborne, Lisa M. Minter, Silvana Morello, Lucio Miele. Targeting Notch1 via adenosine A2A receptor to modulate tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4517.

Abstract Solid tumors are composed of heterogeneous cell populations that support rapid growth of cancer cells and suppress immune cell responses. Unleashing the body's own immune response to eradicate cancer cells is one of the most promising ways to treat cancer patients. However, cancer immunotherapy is not without challenges. Some of these include: 1) Phenotypic plasticity and clonal selection in a highly dynamic, heterogeneous and mutable cell population, which leads to de novo and acquired resistance; 2) Immune editing by cancer cells; 3) Expression of immune check point ligands and receptors to suppress immune response locally within the tumor microenvironment, and systemically using exosomes as immune checkpoint carriers; 4) Cross-talk between cancer stem cells and the immune microenvironment; 5) Feasibility of developing personalized treatment strategies, such as cost vs benefit of CAR-T-cell therapy and time constraint, and humanized antibody production; 6) Systemic toxicity of current immunotherapy strategies; To address these challenges, we propose to use a recombinant adeno-associated virus (rAAV) platform to simultaneously enhance tumor immunity and target cancer stem cells by intra-tumoral administration. We express programmed cell death ligand-1 (PD-L1), and B7-H3 (CD276), in combination with a Notch1 decoy. The latter was chosen because: 1) Notch is a crucial signaling component of cancer stem cells (CSCs) maintenance and resistance and 2) Studies have shown that within the tumor, regulatory T-cells (Treg) are activated by OX40 and Notch ligand Jagged1. Blocking Notch signaling would have dual advantages: blocking Notch signaling in CSCs, and preventing activation of immunosuppressive Tregs. Based on current clinical trials and our experimental results, we hypothesized that intra-tumoral injection of rAAV viral particles engineered to express soluble PD-1, B7-H3 and Notch1 decoys in combination with lipopolysaccharide (LPS) is a promising strategy. To test our hypothesis, we used two mouse triple negative breast (TNBC) cancer models: C0321 in the FVB background and M-Wnt in the C57/Bl6 background; both are aggressive, highly metastatic, and similar to human TNBC. Using tumor spheroids in vitro, in the presence of soluble decoys of PD-1, B7-H3 and Notch1 synergistically induced the most effective tumor cell killing, compared to single decoy application. Importantly, intra-tumoral injection of PD-1, B7-H3 and Notch1 decoys in combination with LPS induced necrosis of tumors in vivo in our preliminary findings. Citation Format: Deniz A. Ucar, Giulia Monticone, Fokhrul Hossain, Samarpan Majumder, Dorota Wyczechowska, Matthew J. Dean, Luis Del Valle, Jovanny Zabaleta, Yong Ran, Sudarvili Shanthalingam, Abraham Bert Chabot, Bridgette M. Collins-Burow, Matthew E. Burrow, Barbara Osborne, Todd Eliot Golde, Lucio Miele. Delivering intra-tumoral immune modulators and targeting cancer stem cells using recombinant- AAVs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6691.

Triple negative breast cancer (TNBC) is defined as pathologically negative for estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 amplification (HER2-). TNBCs are a heterogeneous group of clinically aggressive cancers with high risk of recurrence and metastasis, and current treatment options remain limited. Immunotherapy with checkpoint inhibitors shows promise. However, recent data show that crosstalk between cancer stem cells (CSC) and the immune microenvironment leads to immunotherapy resistance, while myeloid-derived suppressor cells (MDSC) promote CSC survival via Notch signaling. Strong evidence supports the involvement of Notch, a prominent CSC pathway, in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, Including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC. However the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSIs. We confirmed that SS has Gamma Secretase Modifier (GSM) activity, in addition to cyclo-oxygenase (COX) inhibition. SS inhibits Notch1 cleavage in TNBC cells, but not in murine T-cells. SS significantly inhibited mammospheres growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In C0321 tumors in mice, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors without intestinal toxicity. SS caused an increase in intra-tumoral CD11c+ dendritic cells and CD8 cells. SS did not affect the numbers of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. RNA-Sequencing of SS-treated tumors revealed significant reduction of CXCL14, EGR1, HOXC6, MAGI2, NCAM1, APOE, CLU (a Wnt target), DTX4 (an E3-ligase positive regulator of Notch activation), and TGFB3 genes and upregulation of CCL17, EPCAM, FABP4, C4A, LTF, ZBTB16, INADL, and FGFR2 genes. Importantly, SS enhanced the antitumor effect of a-PDL1 immunotherapy in our 0321 TNBC mouse model. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be significantly easier and more cost-effective than developing unproven investigational agents. Citation Format: Fokhrul Hossain, Deniz A Ucar, Samarpan Majumder, Margarite Matossian, Giulia Monticone, Keli Xu, Yong Ran, Lisa Minter, Yaguang Xi, Matthew Burow, Todd Golde, Barbara Osborne, Lucio Miele. Sulindac sulfide as a non-immune suppressive gamma secretase modifier to target triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-19.

Breast cancer affects women globally; the majority of breast cancer-related mortalities are due to metastasis. Acquisition of a mesenchymal phenotype has been implicated in the progression of breast cancer cells to an invasive, metastatic state. Triple-negative breast cancer (TNBC) subtypes have high rates of metastases, recurrence, and have poorer prognoses compared to other breast cancer types, partially due to lack of commonly targeted receptors. Kinases have diverse and pivotal functions in metastasis in TNBC, and discovery of new kinase targets for TNBC is warranted. We previously used a screening approach to identify intermediate-synthesis nonpotent, nonselective small-molecule inhibitors from the Published Kinase Inhibitor Set that reversed the mesenchymal phenotype in TNBC cells. Two of these inhibitors (GSK346294A and GSK448459A) are structurally similar, but have unique kinase activity profiles and exhibited differential biologic effects on TNBC cells, specifically on epithelial-to-mesenchymal transition (EMT). Here, we further interrogate these effects and compare activity of these inhibitors on transwell migration, gene (qRT-PCR) and protein (western blot) expressions, and cancer stem cell-like behavior. We incorporated translational patient-derived xenograft models in these studies, and we focused on the lead inhibitor hit, GSK346294A, to demonstrate the utility of our comparative analysis as a screening modality to identify novel kinase targets and signaling pathways to pursue in TNBC. This study introduces a new method for discovering novel kinase targets that reverse the EMT phenotype; this screening approach can be applied to all cancer types and is not limited to breast cancer.

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive breast cancers. TNBC patients have a high risk of recurrence and metastasis, and current treatment options remain limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Emerging evidence suggests that cancer stem-like cells (CSCs) that escape chemo or radiation therapy in TNBC are often Notch-dependent. At the same time, there is evidence that active tumor immunity predicts good response to neo-adjuvant chemotherapy in TNBC. Notch inhibitors, including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC, where they eliminate CSCs resistant to chemotherapy. However, the success of GSIs in clinical trials is limited by their intestinal toxicity and adverse immunological effects. CD4 and CD8 T-cells, necessary to adaptive tumor immunity, require Notch1 for activation. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSI. SS has Gamma Secretase Modifier (GSM) activity. We confirmed that SS inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In contrast, SS did not inhibit Notch expression or cleavage in murine T cells. In C0321 tumors, which recapitulate human mesenchymal TNBC, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors. SS caused an increase in intra-tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD-1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the number of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. We are currently investigating the mechanisms of this anti-tumor activity. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy agents. Repurposing an FDA-approved, safe agent for the treatment of TNBC would be significantly easier and more cost-effective than developing unproven investigational agents. Citation Format: Hossain F, Ucar D, Majumder S, Xu K, Ran Y, Minter L, Xi Y, Burow M, Golde T, Osborne B, Miele L. Repurposing sulindac sulfide as a notch inhibitor to target cancer stem-like cells in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-22-01.

Triple negative breast cancer (TNBC) is defined as pathologically negative for estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 amplification (HER2-). TNBCs are a heterogeneous group of clinically aggressive breast cancers with high risk of recurrence and metastasis, but the current treatment options remain limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, Including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC. However the success of GSIs in clinical trials is limited by their intestinal toxicity and adverse immunological effects. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSIs. SS has documented Gamma Secretase Modifier (GSM) activity, in addition to cyclo-oxygenase (COX) inhibition. We confirmed that SS inhibits Notch1 cleavage in TNBC cells, but not in murine T-cells. SS significantly inhibited mammospheres growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In C0321 tumors, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors. SS caused an increase in intra-tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD-1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the numbers of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. RNA-Sequencing of SS-treated tumors revealed significant reduction of CXCL14, EGR1, HOXC6, MAGI2, NCAM1, APOE, CLU (a Wnt target), DTX4 (an E3-ligase positive regulator of Notch activation), and TGFB3 genes and upregulation of CCL17, EPCAM, FABP4, C4A, LTF, ZBTB16, INADL, and FGFR2 genes. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be significantly easier and more cost-effective than developing unproven investigational agents.Citation Format: Fokhrul Hossain, Deniz A Ucar, Samarpan Majumder, Margarite Matossian, Keli Xu, Yong Ran, Lisa Minter, Yaguang Xi, Matthew Burow, Todd Golde, Barbara Osborne, Lucio Miele. Sulindac sulfide as a gamma secretase modifier to target triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3439.

Cancer immunotherapy, which stimulates or augments host immune responses to treat malignancies, is the latest development in the rapidly advancing field of cancer immunology. The basic principles of immunotherapies are either to enhance the functions of specific components of the immune system or to neutralize immune-suppressive signals produced by cancer cells or tumor microenvironment cells. When successful, these approaches translate into long-term survival for patients. However, durable responses are only seen in a subset of patients and so far, only in some cancer types. As for other cancer treatments, resistance to immunotherapy can also develop. Numerous research groups are trying to understand why immunotherapy is effective in some patients but not others and to develop strategies to enhance the effectiveness of immunotherapy. The Notch signaling pathway is involved in many aspects of tumor biology, from angiogenesis to cancer stem cell maintenance to tumor immunity. The role of Notch in the development and modulation of the immune response is complex, involving an intricate crosstalk between antigen-presenting cells, T-cell subpopulations, cancer cells, and other components of the tumor microenvironment. Elegant studies have shown that Notch is a central mediator of tumor-induced T-cell anergy and that activation of Notch1 in CD8 T-cells enhances cancer immunotherapy. Tumor-infiltrating myeloid cells, including myeloid-derived suppressor cells, altered dendritic cells, and tumor-associated macrophages along with regulatory T cells, are major obstacles to the development of successful cancer immunotherapies. In this article, we focus on the roles of Notch signaling in modulating tumor-infiltrating myeloid cells and discuss implications for therapeutic strategies that modulate Notch signaling to enhance cancer immunotherapy.