Triple-negative Breast Cancer Models Research Articles

Abstract 6628: Mapping the spatial and molecular determinants of macrophage heterogeneity in breast cancer

Abstract Tumor-infiltrating macrophages support critical steps in tumor progression, and their accumulation in the tumor microenvironment (TME) is associated with adverse outcomes and therapeutic resistance across human cancers. In the TME, macrophages adopt diverse phenotypic alterations, giving rise to heterogeneous immune activation states and induction of cell cycle. While the transcriptional profiles of these activation states are well-annotated across human cancers, the underlying signals that regulate macrophage heterogeneity and accumulation remain incompletely understood. Here, we leveraged a novel ex vivo organotypic TME (oTME) model of breast cancer, in vivo murine models, and human samples to map the determinants of functional heterogeneity of TME macrophages. We identified a subset of F4/80high Sca-1+ self-renewing macrophages maintained by type-I interferon (IFN) signaling and requiring physical contact with cancer-associated fibroblasts. We discovered that the contact-dependent self-renewal of TME macrophages is mediated via Notch4, and its inhibition abrogated tumor growth of breast and ovarian carcinomas in vivo, as well as lung dissemination in a PDX model of triple-negative breast cancer (TNBC). Through spatial multi-omic profiling of protein markers and transcriptomes, we found that the localization of macrophages further dictates functionally distinct but reversible phenotypes, regardless of their ontogeny. Whereas immune-stimulatory macrophages (CD11C+CD86+) populated the tumor epithelial nests, the stroma-associated macrophages (SAMs) were proliferative, immunosuppressive (Sca-1+CD206+PD-L1+), resistant to CSF-1R depletion, and associated with worse patient outcomes. Notably, following cessation of CSF-1R depletion, macrophages rebounded primarily to the SAM phenotype, which was associated with accelerated growth of mammary tumors. Our work reveals the spatial determinants of macrophage heterogeneity in breast cancer and highlights the disruption of macrophage self-renewal as a potential new therapeutic strategy. Citation Format: Nir Ben Chetrit, Xiang Niu, Jesus Sotelo, Ariel Swett, Vinagolu Rajasekhar, Maria Jiao, Caitlin Stewart, Priya Bhardwaj, Sanjay Kottapalli, Saravanan Ganesan, Pierre-Louis Loyher, Catherine Potenski, Kristy Brown, Neil M. Iyengar, Dilip D. Giri, Scott W. Lowe, John H. Healey, Frederic Geissmann, Irit Sagi, Johanna A. Joyce, Dan A. Landau. Mapping the spatial and molecular determinants of macrophage heterogeneity in 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 6628.

Abstract 6624: MYC promotes lobuloalveologenesis and mammary tumorigenesis in male mice

Abstract Male breast cancer is a rare disease that is largely overlooked and clinically regarded to recapitulate female breast cancer biology. The worse overall outcome for male patients compared to females, however, underscores inadequate disease management and a sex-based divergence in disease biology. To gain more insight into male breast biology and pathology, we used mouse models of triple-negative breast cancer and 3D whole-mount confocal imaging techniques to evaluate both in vivo and ex vivo mammary gland development, tumor formation, and hormone signaling dependence. Our results show that Myc overexpression in collaboration with Trp53 deficiency drives mammary tumorigenesis in male mice. We show that MYC overexpression promotes estrogen-independent formation of lobuloalveolar structures, which progress into tumors with a similar histology and immune landscape as female mammary tumors but with significantly more genomic instability. In female mice, MYC overexpression bypasses the dependency on paracrine estrogen signaling in mammary tumorigenesis. These data provide robust evidence that MYC overrides mammary tumor estrogen dependence and abolishes the tumor-preventive effect of a low-estrogen environment. Supporting this notion, in vitro experiments in human female breast cancer cell lines indicate that MYC overexpression may functionally contribute to endocrine therapy resistance and therefore could possibly serve as a prognostic biomarker or clinically targetable pathway for treating both male and female breast cancers. Collectively, this preclinical work presents new insights into male mammary gland biology and mechanisms of cancer progression that might translate to a broader range of breast cancers in the clinic. Citation Format: Emilia M. Pulver, Eline van der Burg, Anne Paulien Drenth, Julia Yemelyanenko, Nils Ravensbergen, Roebi de Bruijn, Sjoerd Klarenbeek, Ji-Ying Song, Manon Boeije, Bjørn Siteur, Natalie Proost, Colinda Scheele, Peter Bouwman, Jos Jonkers. MYC promotes lobuloalveologenesis and mammary tumorigenesis in male mice [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 6624.

Abstract 3390: Development of hypoxia responsive targeted polymersomes drug therapy validated using patient derived xenograft of triple negative breast cancer

Abstract Introduction: Eliminating triple-negative breast cancer (TNBC) resistance to neoadjuvant chemotherapy is a critical unmet clinical need. TNBC accounts for 20% of breast cancer and is more aggressive due to increased metastasis, high recurrence rate, and significant resistance to chemotherapy (e.g., Doxorubicin, DOX). Napabucasin (NAPA, a small organic molecule) is recognized to kill cancer stem cells by targeting the transcription factor STAT3 pathway and is currently in clinical trials. Our previous in vitro results showed significant effects with hypoxia-responsive targeted polymersomes compared to control in MDA-MB-231 and Patient-Derived Xenograft (PDX) TNBC cells. Further, we observed increased NRP-1 expression in TNBC patient-derived xenograft (PDX) cells under hypoxic (0.2% oxygen) conditions compared to normoxia (21% oxygen). Hence, we hypothesize that targeted hypoxia-responsive polymersome drug therapy would reduce the tumor growth of the PDX mouse model of triple-negative breast cancer in vivo. Methods: We have established the TNBC PDX mouse model with two major phases, initial propagation (F1-F3) and final study phase (F3-F6), for in vivo study (TNBC tumor samples received from Sanford Broadway Clinic, Fargo, IRB approved). We utilized 3-6 months old female adult NSG mice (Jackson Lab) weighing 18-25 g. PDX tumor-bearing female NSG mice were administered polymersome-encapsulated drug (doxorubicin and other groups) by intravenous injection twice a week for 4 weeks and then observed the animals for an additional week. The percentage of tumor volume growth was calculated on these treatment groups and compared to vehicle. The tumors from all the groups were excised and dissected after the treatment and fixed in 10% formalin for histological evaluation. Tumors from all the groups after treatment were stained using hematoxylin and eosin to analyze the tumor core necrosis, and the average area of necrosis was compared among all the groups. Results: The TNBC PDX mouse model treated with DOX alone and a combination of encapsulated DOX with NAPA showed significant weight loss compared to the control (over 4 weeks). DOX-alone treated mice have shown reduced tumor growth and exhibited a significant difference compared to the control group. Notably, the targeted DOX-encapsulated polymersome demonstrated a much lower tumor volume and displayed a marked difference compared to non-targeted vehicles, free-DOX, and saline. In the initial analysis, we observed a higher necrosis in the tumor of the targeted DOX-encapsulated polymersome compared to the control. Summary and Conclusion: Overall, the hypoxia-responsive targeted polymersomes showed potent antitumor activity in the novel TNBC PDX animal model. With further developments, the targeted polymersomes might have translational potential as drug carriers for treating TNBC. Citation Format: Yogaraj S Ramakrishnan, Shubhashri Ambhore, Connor Edvall, Jiyan Mohammed, Sanku Mallik, Daniel M Tuvin, Venkatachalem Sathish. Development of hypoxia responsive targeted polymersomes drug therapy validated using patient derived xenograft 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 3390.

Abstract 2616: BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts

Abstract Metastatic triple-negative breast cancer (TNBC) patients have poor overall survival, highlighting the need for novel treatments. Although the TROP2-targeting ADC sacituzumab govitecan recently received accelerated approval from the FDA for the treatment of patients with metastatic TNBC, the on-target toxicity of this single-target agent has limited clinical efficacy. We sought to improve the specificity and efficacy of future TNBC-targeted therapies by generating a bispecific antibody-drug conjugate (bsADC) targeting TROP2 and PTK7, another tumor-associated antigen (TAA) that is highly expressed in TNBC and correlates with poor prognosis and metastatic disease. We previously generated fully human antibodies targeting PTK7 and TROP2 from Biocytogen’s fully human, common light chain antibody transgenic RenLite® mice, which were selected such that their monovalent antibodies exhibited reduced internalization profiles for improved tumor selectivity. These antibodies were then constructed into a bispecific antibody targeting PTK7 × TROP2, which demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including TNBC, with high affinity. PTK7 × TROP2 bsAb also showed enhanced internalization in vitro compared with parental monovalent antibodies. Next, the PTK7 x TROP2 bsAb was conjugated to vcMMAE with a drug-to-antibody ratio (DAR) of 4 to generate a bsADC (BCG033). BCG033-vcMMAE demonstrated superior activity to benchmark and parental ADCs in a cell line-derived xenograft (CDX) TNBC model with low PTK7 expression. Here, we demonstrate that BCG033-vcMMAE ADCs also exhibited superior efficacy to benchmark ADCs in PDX models, including TNBC xenografts and NSCLC xenografts. We next tested the efficacy of the PTK7 x TROP2 bsAb conjugated to BLD1102, Biocytogen’s novel, proprietary linker/payload composed of a DNA Topo I inhibitor payload and a highly hydrophilic protease-cleavable linker, with a DAR of 8. BCG033-BLD1102 showed potent activity in a colorectal cancer PDX model in which benchmark ADCs were inactive, even at high doses. In summary, BCG033 conjugates demonstrate promising preclinical efficacy in vivo, which ultimately could provide a new treatment option for TNBC and other solid tumors expressing PTK7 and TROP2. Citation Format: Sufei Yao, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frk an, Yi Yang. BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts [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 2616.

Abstract 4612: Inhibition of BCL2 and XPO1 demonstrates synergistic effects in triple-negative breast cancer cell lines

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, accounting for about 15-20% of all breast cancers. Characterized by a lack of estrogen and progesterone receptors and non-amplified HER2, treatment options are limited compared to other breast cancer subtypes. The aim of this study was to evaluate eltanexor (Elta), an XPO1 inhibitor, in combination with venetoclax (Ven), a Bcl-2 inhibitor to promote apoptosis in TNBC models. Methods: CellTiter-Glo Cell Viability Assay was utilized to determine cell viability in 6 TNBC cell lines after 72-hour exposure to no drug, Elta, Ven or the combination. Synergy scores were determined using the Bliss model in SynergyFinder+, scores 10 or above indicating synergy. Apoptosis (by Annexin V) was assessed after 24- and 48-hour exposure to drug via flow cytometry. Immunoblots were used to assess the mechanism of action in cell lines treated for 24 hours. Results: CellTiter Glo displayed greater anti-proliferative activity in combination versus single agent exposure. Synergy was observed in CAL-120, BT-20, and MDA-MB-468 cell lines, with synergy score values ranging from -13.22 - 25.07. Additive effects were observed in other cell lines. An increase in apoptosis was observed in CAL-51 and MDA-MB-231 cells after 24- and 48-hour treatment that was greater with the combination. CAL-51 cells exposed to single agent Ven or Elta [0.5 µM] for 24 hours had significantly less apoptosis than those exposed to the combo (p = <0.0001). After a 48-hour exposure, CAL-51 cells exposed to single agent Ven had less apoptosis than in combination with Elta [0.1 µM] (p = 0.0431) or Elta [0.5 µM] (p = 0.0036 µM]. Additionally, single agent Elta [0.5 µM] resulted in less apoptosis than the combination (p = 0.0067). Similar results were observed in MDA-MB-231 cells. We observed an increase in apoptotic markers with the combination in all cell lines treated. In all cell lines, DNA-damage marker pH2Ax increased with Ven and the combination. In the CAL-51 cells, p53 increased following Elta or combination exposure. In the MDA-MB-231 cells, a decrease in the anti-apoptotic protein MCL1 was observed with Elta or combination. Conclusion: The combination of Elta and Ven resulted in increased apoptosis and antiproliferative activity in TNBC cell lines. Combined, the two drugs synergistically functioned to increase DNA damage and promote apoptotic cell death. This work supports the continued evaluation of Elta and Ven in TNBC. Citation Format: Marilyn Marie Jackson, Stephen G. Smoots, Adrian T. Dominguez, Evan Dus, Stacey Bagby, Cameron Binns, Todd M. Pitts, Jennifer R. Diamond. Inhibition of BCL2 and XPO1 demonstrates synergistic effects in triple-negative breast cancer cell lines [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 4612.

Abstract 7591: Tumor Treating Fields (TTFields) show efficacy in Triple-Negative breast cancer (TNBC) cells alone and in combination with PARP inhibitor Talazoparib

Abstract Breast cancer is the most common cancer in women worldwide, and the fifth leading cause of cancer-related deaths globally. Triple-negative breast cancer (TNBC), the most aggressive form of breast cancer, is usually treated with surgery, chemotherapy, and radiation. Tumor Treating Fields therapy are FDA-approved for the treatment of glioblastoma (GBM) and malignant pleural mesothelioma (MPM).TT Fields use alternating electric fields to inhibit tumor growth through mitotic disruption, cellular damage, integrated stress response activation, and immune mechanisms and inhibit cell proliferation, induce cell death, and cause cell cycle arrest.TT Fields can induce BRCAness and when combined with PARP inhibition, this combination may result in synthetic lethality. We explored the effect of TTFields on two TNBC cell lines MDA-MB436 (BRCA1-mutant, p53-null) and MDA-MB468 (BRCA1 wild-type, p53-mutant) treated with TTFields (150 kHz) for 24 hours. Western blot analysis showed elevation of p21 independent of p53 mutational status. We examined the combination of PARP inhibitor (Talazoparib) with TTFields in MDA-MB468 cells to determine the effect of the combination in BRCA wild-type cancer cells. Western blot analysis showed p21 upregulation after 24 hours in combination without an increase in p53. p21 acts as a G1-checkpoint protein and loss of p21 expression has been observed in a high percentage of human breast cancer cells. Our results suggest a rational and potentially effective therapy that could be further developed and used for the treatment of breast cancer. Our ongoing studies are exploring potential mechanisms of synergy between PARP inhibitor Talazoparib and TTFields using various TNBC models, and the effect of the combination on cell cycle arrest in cell culture and in vivo. Citation Format: Maryam Ghandali, Praveen Srinivasan, Yutong Xia, Eric T. Wong, Robert W. Sobol, Lanlan zhou, Benedito A. Carneiro, Stephanie L. Graff, Wafik S. El-Deiry. Tumor Treating Fields (TTFields) show efficacy in Triple-Negative breast cancer (TNBC) cells alone and in combination with PARP inhibitor Talazoparib [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 7591.

Abstract 7592: Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC)

Abstract The LAR subtype of TNBC is enriched for targetable biomarkers such as androgen receptor (AR) expression, PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combination of CDK4/6, AR, and PI3K-AKT inhibitors in preclinical models of LAR TNBC. The MDAMB453 and MFM223 (AR positive/Rb intact/PIK3CAmutant) LAR TNBC cells were treated with each inhibitor alone or in combination. Drug sensitivity was assessed using colony formation, cell viability, and live-cell analysis. The Loewe additivity equation was used to measure the additive drug effects. Immunoblot analysis was used to evaluate cell cycle and PI3K-AKT signaling and ARE luciferase assay was used to assess AR transcriptional activity. MDAMB453 and MFM223 cells were sensitive to the single agents palbociclib, alpelisib, and capivasertib (IC50, ~500 nM), which inhibited CDK4/6, PI3Kα, and AKT, respectively. Enzalutamide, an AR antagonist, exerted no growth inhibitory activity (IC50, 15-25 μM). Treatment with palbociclib, alpelisib, or capivasertib did not alter the AR protein levels or transcriptional activity. The combination of palbociclib+capivasertib synergistically inhibited the proliferation of MDAMB453 and MFM223 cells (synergistic scores 7.34, p=5.81-11, and 4.78, p=0.012, respectively) better than palbociclib+alpelisib. Enzalutamide did not enhance either combination. Palbociclib+capivasertib inhibited PI3KCA-mutant and Rb-intact LAR TNBC PDX growth more potently than palbociclib+alpelisib (p=0.046). Treatment of LAR TNBC cells with palbociclib suppressed Rb phosphorylation and resulted in adaptive activation of P-AKTS473/T308 and the AKT substrates GSK3β, PRAS40, and FoxO3a after 24 h. Capivasertib inhibited the phosphorylation of AKT and AKT substrates, following CDK4/6 blockade with palbociclib, more potently than alpelisib. PI3Kβ inhibitors (TGX221, GSK2636771) did not block AKT substrate upregulation, suggesting PI3Kβ does not mediate adaptive responses. Rictor knockdown failed to reduce AKT activation following palbociclib treatment, suggesting an mTORC2-independent mechanism. Human Phospho-kinase Array of MDA-MB-453 cells treated with palbociclib showed time dependent upregulation of GSK3βS9, STAT3Y727 and PDFGRβY751. PDGFRβ activation after palbociclib treatment was confirmed in MFM223 cells. PDGFRβ siRNAs in MDAMB453 cells blocked P-AKTS473 upregulation after palbociclib treatment. Treatment with palbociclib and the PDGFRβ small-molecule antagonist CP673451 arrested LAR TNBC growth similar to palbociclib+capivasertib, suggesting an adaptive response involving PDGFRβ signaling. In conclusion, this study supports the clinical testing of the palbociclib and capivasertib combination for LAR TNBC patients and reveals adaptive responses after palbociclib treatment through PDGFRβ signaling. Citation Format: Rosario Chica-Parrado, Gun Min Kim, Yasuaki Uemoto, Dan Ye, Fabiana Napolitano, Chang-Ching Lin, Emmanuel Bikorimana, Kyung-min Lee, Saurabh Mendiratta, Ariella B. Hanker, Carlos L. Arteaga. Combined inhibition of CDK4/6 and AKT is highly active against the luminal androgen receptor (LAR) subtype of triple negative breast cancer (TNBC) [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 7592.

Abstract 4726: Identification of efficacy biomarkers for therapies targeting miR-10b

Abstract Breast cancer stemness is one of the drivers of metastatic spread and presents a key target in the battle against metastatic disease, especially in triple-negative breast cancer (TNBC). Existing treatment options for TNBC are limited due to recurrence and resistance, representing a major unmet clinical need. MicroRNA-10b (miR-10b) is one of the major drivers of breast cancer cell migration and invasion. In our earlier studies, we discovered that miR-10b is also responsible for metastatic cell viability. With the goal of translation of this finding to the clinic, we developed a novel therapeutic, or “nanodrug,” that targets miR-10b, consisting of antisense anti-miR-10b oligonucleotides conjugated to magnetic nanoparticles (MN) that serve as delivery vehicles. Administration of the nanodrug in mice bearing metastatic MDA-MB-231 TNBC tumors prevents the development of metastases and stops the growth of pre-existing metastases. Additionally, combination treatment with adjuvant doxorubicin induced a stable regression of metastases in immunocompromised and immunocompetent mouse models of metastatic TNBC. A limitation of our previous studies is the lack of understanding of the molecular basis for the therapeutic effect of MN-anti-miR10b. To address this gap in knowledge, we performed RNA sequencing on MDA-MB-231 cells treated with the nanodrug or controls. Functional enrichment analysis of differentially expressed genes (DEGs) between cells treated with the nanodrug versus controls (PBS or bare MN) identified a significant overrepresentation of genes associated with cell differentiation. It has been suggested that miR-10b confers stem cell-like properties such as self-renewal and drug resistance onto cancer cells. Our RNA sequencing findings led us to hypothesize that MN-anti-miR10b functions by inhibiting these stem-like cancer cell (CSC) features that are critical to cancer survival. Here, we present preliminary data showing that miR-10b is overexpressed in MDA-MB-231 cells expressing a CSC-associated surface marker phenotype (CD44+/CD24-, compared to CD44-/CD24- cells) and that this trend is not universal among other miRNAs. Furthermore, we found that the nanodrug interferes with the ability of cells to assemble as spheroids, a property associated with CSCs. With our recent observation that the nanodrug inhibits miR-10b in metastases in vivo in as little as one dose after 72 hours, our next steps are to determine whether the cell differentiation-associated DEGs identified in RNA sequencing results can be used as biomarkers of nanodrug efficacy in vivo. Understanding the changes in gene expression induced by MN-anti-miR10b as a function of time will be critical for guiding treatment regimens in future preclinical and clinical trials. Citation Format: Alan Halim, Nasreen Al-Qadi, Sujan K. Mondal, Elizabeth Kenyon, N. Anna Savan, Hussein Mackie, Anna Moore. Identification of efficacy biomarkers for therapies targeting miR-10b [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 4726.

Abstract 3187: Enhancing STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy

Abstract Background: Activating the innate immune pathway of stimulator of interferon genes (STING) can elicit potent anti-tumor immunity via the production of type-I interferons (IFN-I). However, cyclic-dinucleotide (CDN) STING agonists have undesirable pharmacological properties. Most CDNs in clinical development are administered intratumorally, thus precluding their utility against advanced cancer. Here, we report the development of a new nanoparticle system for the systemic delivery of CDN STING agonists with potent efficacy, favorable pharmaceutical properties, and acceptable safety profiles. Methods: Various metal ions were screened for synergy with CDNs for IFN-I response from bone marrow-derived dendritic cells. Lipid nanoparticles carrying CDN and manganese ions (termed SNP) were synthesized. Mice bearing syngenetic tumors (including CT26 and B16F10) as well as genetically engineered mouse models were treated by intravenous administration of SNP, followed by tumor monitoring and immune profiling of the tumor microenvironment. White New Zealand rabbits bearing VX2 tumors as well as healthy mongrel dogs were treated with SNP and evaluated. Lastly, human tumor biopsies derived from head and neck squamous cell carcinoma (HNSCC) patients were incubated with SNP or soluble STING agonists and analyzed for immune activation. Results: We identified that Mn2+ achieved strong synergy with CDN STING agonists for inducing IFN-I production. SNP carrying CDN and Mn2+ was dosed intravenously in mice, leading to robust IFN-I response, remodeling of the immunosuppressive tumor microenvironment, and expansion of anti-tumor CD8+ T cells. Intravenous SNP therapy resulted in robust anti-tumor efficacy in multiple murine tumor models, including CT26, B16F10, and a genetically engineered mouse model of MMTV-PyMT triple-negative breast cancer. We have also observed robust anti-tumor efficacy of SNP against VX2 squamous carcinomas in rabbits and have demonstrated the safety of intravenous SNP therapy in healthy dogs. Mechanistically, the efficacy of SNP therapy was dependent on the host STING expression but independent of the tumor STING expression. In addition, the anti-tumor efficacy of SNP was decreased in mice lacking Ifnar1 or Ifngr1 expression in the B16F10 melanoma model, showing the crucial effects of IFN-I and IFN-II responses. Moreover, SNP treatment led to robust IFN-I responses from fresh human tumor biopsies derived from HNSCC patients. Conclusions: SNP empowers highly effective systemic cancer immunotherapy via nanotechnology. It also underscores the potential of utilizing metal ions for immune-mediated disease treatment. Citation Format: Xingwu Zhou, Xiaoqi Sun, Wang Gong, Ziye Wan, Teresa Krieger-Burke, Vilma Yuzbasiyan-Gurkan, Yu Leo Lei, James Moon. Enhancing STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy [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 3187.

Abstract 5735: Efficient and safe delivery of doxorubicin by DNA fragments: A full preclinical study

Abstract The clinical uses of anthracyclines (e.g., doxorubicin) are hampered especially by dose-dependent cardiac toxicity, resulting in a narrow therapeutic window. Doxorubicin formulations have marginally improved its therapeutic efficacy while suffering from difficult and costly manufacturing, poor characterization and quality control, limited scalability in production, and disappointingly low drug loading. This study employs DNA fragments extracted from the salmon sperm cells, which is known for high biocompatibility in various biomedical application, to self-assemble via intercalation by doxorubicin. DNA/DOX nanocomplexes are easy in manufacturing, high scalability, straightforward characterization and quantification, and efficiency in drug loading. Doxorubicin and DNA fragments were dissolved in water and mixed at the optimized 1:6 weight ratio, followed by stirring and incubation for 30 min. DNA/DOX nanocomplexes were characterized for doxorubicin loading and release, size, surface charge, and morphology by UV/fluorescence spectroscopy, stable isotope tracer ultrafiltration assay (SITUA), dynamic light scattering particle analysis, and transmission electron microscopy. The anticancer efficacy of the DNA/DOX nanocomplexes were tested in vitro and in vivo using syngeneic and PDX tumor models. Along with PD/PK studies, DNA/DOX nanocomplexes were also evaluated for their systemic and cardiac toxicity. Characterization and quantification studies revealed DNA/DOX nanocomplexes to be relatively monodisperse with a size of 40 nm in diameter, efficient for drug encapsulation (~100%) and drug loading (14.3%, w/w), and stable for storage while readily releasing doxorubicin in a cell. The self-assembled nanocomplexes were formed when doxorubicin inter- and intra-molecularly chelated DNA fragments. DNA/DOX nanocomplexes were more efficient in killing cancer cells in vitro than both doxorubicin and the liposomal doxorubicin Doxil. PD/PK studies demonstrated that DNA/DOX nanocomplexes circulated longer with less accumulation in the heart than doxorubicin, which altogether lower toxicity. A repeated administration of the DNA/DOX nanocomplexes to Sprague-Dawley rats exhibited a substantially lower cardiotoxicity score than that of doxorubicin. The improved anti-tumor efficacy of the DNA/DOX nanocomplexes were finally confirmed using a syngeneic lymphoma mouse model and PDX mouse models of triple-negative breast and ovarian cancers. Complexing doxorubicin with DNA fragments addresses the current technological challenges in achieving an efficient and safe therapy for a broad range of cancers. DNA/DOX nanocomplexes are amenable to rigorous chemistry, manufacturing, and controls (CMC) evaluation with proven anti-cancer efficacy and improved safety profiles. Clinical trials on DNA/DOX nanocomplexes for cancer chemotherapy are warranted to fully validate their clinical utilities. Citation Format: Minhyeong Park, Youngwoo Kim, Hyunchu Cho, Saad Mohammad, Yeon Su Choi, Hyunjoo Lee, Seonah Lee, Edward Cedrone, Barry Neun, Maria Dobrovolskaia, David Fruman, Juwan Kim, Young Jik Kwon. Efficient and safe delivery of doxorubicin by DNA fragments: A full preclinical study [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 5735.

Abstract 4208: In-vitro modeling of Sonic Hedgehog signaling identifies the extracellular matrix as a regulator of tumor invasion outcomes

Abstract Preclinical models of Sonic Hedgehog (SHH)-driven tumors support the therapeutic benefit of pharmacological inhibitors, yet clinical outcomes are conflicted due to biphasic tumor responses that include complete remission or faster disease progression. In such studies, the activity of the SHH pathway in the tumor-adjacent stroma supported or restrained tumor progression. Our prior studies show that altered SHH ligand levels can lead to faster tumor growth during pharmacological inhibition suggesting a role for the SHH pathway strength in the biphasic tumor responses of a triple-negative breast cancer (TNBC) model. In this study, the role of SHH pathway strength altered by tumor-fibroblast proximity was examined on the tumor growth, invasion, and transcriptional activity using in vitro and in vivo models. Tumor spheroid models composed of TNBC cell lines and fibroblasts were used to monitor SHH signal strength as a function of the culture modality and SHH ligand levels. In vitro, SHH signal strength was altered by tumor-fibroblast proximity and ligand concentration. Mixed cultures exhibited significantly higher SHH activity and reduced invasion than adjacent cultures. This effect was reversed by pharmacological inhibition of SMO, confirming the involvement of an SHH-dependent mechanism. In a tumor xenograft model, tumor metastasis outcomes were suppressed by pharmacological inhibition of the SHH pathway, but opposite results were observed in tumor xenografts composed of co-injected with fibroblasts indicating that the biphasic tumor response can be driven by the fibroblasts. Spatial transcriptional analysis identified extracellular matrix (ECM) activities in fibroblasts as the top altered bioprocesses in both in vitro and in vivo. This finding was confirmed in cultures, where fibroblast-derived decellularized matrix stimulated tumor invasion of single TNBC spheroids at levels observed in co-cultures. Further analysis of differentially expressed genes (DEGs) identified a small leucine-rich repeat proteoglycan (SLRP) as the primary ECM target altered by variations in SHH signal strength in fibroblasts. Furthermore, we confirmed the role of SLRP cues in governing tumor invasion potency within spheroid cultures embedded in a collagen hydrogel matrix. While high levels of SLRP in the matrix inhibited spheroid invasion, low SLRP matrix promoted spheroid invasion and increased secondary invasion sites. These findings agreed with the Vimentin expression levels observed in TNBC cells and confirmed the potency of SLRP in regulating tumor invasion outcomes. In summary, our study underscores the significance of ECM remodeling activities in fibroblasts and the role of SLRPs in governing tumor invasion, with implications for understanding the impact of altering SHH signaling activity in TNBC. Citation Format: Emanuel A. Carrasquillo-Dones, Heizel M. Rosado-Galindo, Ana M. Reyes-Ramos, Wandaliz Torres-Garcia, Said Cifuentes, Jan P. Rios-Grant, Gabriela Ortiz-Soto, Natalia A. Ramos-Acevedo, Miosotis Acevedo-Esquilin, Monica Colon-Vargas, Israel Almodovar-Rivera, Camilo Mora, Michelle M. Martinez-Montemayor, Maribella Domenech. In-vitro modeling of Sonic Hedgehog signaling identifies the extracellular matrix as a regulator of tumor invasion outcomes [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 4208.

Abstract 275: Deciphering biphasic responses to Sonic Hedgehog pathway inhibition in breast cancer: Insights from a tumor-fibroblast spheroid model and PDXs

Abstract A significant subset (30-40%) of triple-negative breast cancer (TNBC) cases exhibit hyperactivation of the sonic hedgehog (SHH) pathway, correlating with poor clinical outcomes. However, the therapeutic efficacy of SHH inhibitors in TNBC has yielded inconsistent results, showcasing biphasic tumor responses going from complete remission in some patients to faster disease progression in others. Such tumor response discrepancies underscore the need for more relevant culture models to elucidate how stromal cells respond to SHH signals and impact tumor cell reprogramming.We have developed a tumor-fibroblast spheroid model of robust paracrine SHH signaling to examine alterations in aggressive tumor cell phenotypes resulting from altered SHH pathway activity levels to understand better how SHH therapy leads to biphasic effects in tumor tissues. Specifically, tumor stemness alterations were examined as a function of SHH signal strength altered by tumor-fibroblast proximity, and findings were confirmed in two PDX models of TNBC of divergent sensitivity to pharmacological SHH pathway inhibition. In vitro, results show that tumor stemness potency is enriched by tumor-fibroblast proximity and inversely correlates with SHH pathway activity levels. Tumor stemness potency was determined via a mammosphere forming assay and resistance to chemotherapy using docetaxel as treatment. CD44+ sorted tumor cells derived from adjacent fibroblast cultures showed higher mammosphere forming efficiency and docetaxel sensitivity than those from mixed cultures. These effects correlated with differences in SHH pathway activity strengths and were reverted by pharmacological inhibition of SHH confirming pathway activity involvement. In two PDX models, both exhibiting high SHH pathway activity and similar tumor vascularization, we observed variable response to pharmacological SHH inhibition. PDX2 displayed favorable responses with minimal alpha-smooth muscle actin-positive (SMA+) fibroblast infiltration, while PDX1 exhibited no benefit from SHH therapy, accompanied by a high level of SMA+ fibroblasts. Spatial transcriptomic analysis confirmed stemness enrichments in the PDX1 model treated with SHH inhibitor- a scenario our mixed co-culture model resembled. Stemness-enriched tumor regions were positive for the Ki67 proliferation marker and confined to areas of high SMA+ cell abundance. Plotting Ki67 vs SMA+ cell levels revealed a positive correlation, suggesting that SMA levels may predict tumor response to SHH pathway inhibition.In summary, our studies demonstrate that pharmacological SHH inhibition can induce altered tumor plasticity by favoring stemness phenotypes. Strategies targeting tumor stemness may mitigate biphasic effects in response to SHH inhibitors, offering insights for optimizing TNBC therapeutic approaches. Citation Format: Heizel M. Rosado-Galindo, Miosotis Acevedo-Esquillin, Wandaliz Torres-García, Ana M. Reyes-Ramos, Gabriela Ortiz-Soto, Michelle M. Martínez-Montemayor, Maribella Domenech. Deciphering biphasic responses to Sonic Hedgehog pathway inhibition in breast cancer: Insights from a tumor-fibroblast spheroid model and PDXs [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 275.

Abstract 1120: CD8 ImmunoPET imaging identifies an immunogenically active tumor microenvironment following radiotherapy in primary TNBC

Abstract Background: Tumor immune composition has been shown to drive therapy response in triple negative breast cancer (TNBC). Radiation is a component of standard-of-care treatments for TNBC, however immune populations have not been studied in the context of radiation resistance in breast cancer. Positron emission tomography (PET) allows for quantifying molecular signatures of the tumor microenvironment, which can precede anatomical changes in tumor volume. Recently, development of immune-targeted imaging approaches has allowed for noninvasive quantification of immune cells, allowing for quantification and long-term therapy response. Preclinical studies have demonstrated that combining therapies, such as radiotherapy and immunotherapy, could be beneficial in treating cancer. The goal of this study is to quantify changes in immune infiltration with [89Zr]-CD8 ImmunoPET following radiation therapy to determine variations in immune activation that can affect therapeutic response in radiation sensitive or radiation resistant tumors. Methods: Syngeneic radiation sensitive 4T1 or a radiation-resistant 4T1 tumors were treated with fractionated radiation (2 Gy/day) from day 0-5, injected with [89Zr]-CD8 minibody on day 6, and imaged with [89Zr]-CD8 PET on day 7. Following imaging, tumors were excised for biological assays (N = 20) or monitored for longitudinal changes in tumor volume (N = 15). Biological assays included CD8 immunofluorescence or assessment of immune population differences with flow cytometry against T-cells (CD3, CD4, CD8, IFN-gamma), macrophages (CD45, F4/80, CD86 and CD206), and natural killer cells (CD56 and CD16). A non-parametric T-test was used to assess for statistical differences. Results: In radiation sensitive tumors, [89Zr]-CD8 PET indicated that radiation significantly increases CD8 immune infiltration (p = 0.02) compared to controls, which was correlated with immunofluorescence (p < 0.01). Immune infiltration was increased in radiation sensitive tumors when treated with radiation therapy (4.6-fold increase in CD86+ M1-like macrophages, 4.7-fold increase in CD206+ M2-like macrophages, 5.3-fold increase in CD8 T-cells and 4.3-fold increase in CD4 T-cells) compared to control tumors. Radiation was observed to increase cytotoxic CD8 immune infiltration in radiation sensitive tumors. In radiation resistant tumors, no significant difference was observed in either CD8 immune infiltration or tumor volume in treated tumors compared to controls (p = 0.63). Conclusions: [89Zr]-CD8 ImmunoPET reveals significant increases in CD8 immune infiltration in a radiation sensitive model of TNBC and identifies an immune altered tumor immune microenvironment. Fractionated radiotherapy was also observed to significantly increase the infiltration of innate and adaptive immune cells. Citation Format: Patrick N. Song, Shannon E. Lynch, Chloe T. DeMellier, Alessandro Mascioni, Jia Fang, Anna G. Sorace. CD8 ImmunoPET imaging identifies an immunogenically active tumor microenvironment following radiotherapy in primary TNBC [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 1120.

Abstract 3198: An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting of triple-negative breast cancer

Abstract Background: Every breast cancer diagnosis is unique in its own way. Triple-negative breast cancer (TNBC), has a highly aggressive phenotype characterized by a rapid growth rate profile, increased risk of metastasis and recurrence. TNBC is devoid of epidermal growth factor receptor 2, estrogen, and progesterone receptors. All three are commonly present in other forms of breast cancer disease. The aim of this study was to develop a cell membrane lipid-extracted nanoliposomes (CLENs) for selective targeting compared to conventional nanoliposomal varieties. An additional objective was to investigate the role of microenvironment on targeting using cell model of TNBC, and representations of non-target tissue environments for comparison. Method: Lipid extracts (LE) obtained from 4T1, a murine mammary carcinoma cell line from a BALB/cfC3H mouse was used to formulate the CLENs (cell membrane lipid-extracted nanoliposomes) with different ratios of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a phospholipid used in most conventional drug delivery liposomes. Different lipid compositions and ratios were employed to evaluate cellular uptake in target, non-target and off-target cells. The target cell line, 4T1, off-target and non-target cell populations included CRL-2089, normal breast fibroblasts, SKBR3 a (human breast cancer), and A549 a (lung cancer) cell lines. Other studies involved determination of molecular charge and zeta potential of various nanoliposomal preparations and corresponding fluorescence studies in vitro. Results: The physicochemical properties of CLENs such as particle size and surface charge characteristics were vital when assessing benefit of the lipid-based preparations. The size of the CLENs typically ranged between 130 - 165 nm and zeta potential values were negatively-charged. The inclusion of 70 mol% of LE in preparation of 4T1-CLENs demonstrated increased cellular uptake and binding by the 4T1 target cells compared to the control DOPC (100%). The highest degree of selectivity was observed in early cell binding events, and with studies involving more extended incubation time points under different experimental conditions. Off-target cells demonstrated reduced uptake by comparison. Overall, 4T1-CLENs was most efficient when applied against intended target 4T1 cells, when compared to relevant control cell populations. In conclusion, studies performed to date suggest the notion that lipid extracts derived from TNBC improves targeting. Citation Format: Christiana Ayertey, Parmida Amid, Mohammed H. Almozain, Robert B. Campbell. An analysis of breast cancer membrane lipid-modified nanoliposomes for enhanced targeting 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 3198.

Abstract 7120: Investigating PARP1 selective inhibitor DSB1559 efficacy in BRCA1-associated triple negative breast cancer

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) have improved outcomes of BRCA-associated breast cancer. However, toxicity can limit patient response as well as the ability to be combined with other therapeutics. Current PARPi broadly inhibit members of the PARP protein family, however only inhibition of PARP1 is needed for PARPi-mediated cell death. As inhibition of PARP2 is associated with hematological toxicity seen in PARPi patients, selectively inhibiting PARP1 may reduce PARPi toxicity and lead to better outcomes for patients. Here we characterize and test the efficacy of the PARP1 selective inhibitor (PARP1i) DSB1559 compared to Olaparib in BRCA1-associated triple negative breast cancer (TNBC). Methods: NanoBRET, PARylation, and pharmacokinetic/pharmacodynamic assays were performed to examine DSB1559 potency, PARP family selectivity, and in vivo behavior. Immunocompetent FVB/n mice bearing BRCA1-deficient TNBC (CMV-Cre;Brca1f/f;Trp53f/f) tumors were treated daily with PARPi (Olaparib) or PARP1i (DSB1559) for 60 days and followed for survival. CellTiter-Glo and qPCR assays were used to examine DSB1559 effect on cell viability and STING activation, respectively. Results: DSB1559 is a novel and potent PARP1 inhibitor which has superior selectivity for PARP1 compared to Olaparib and PARP1i AZD5305. DSB1559 has highly desirable physico-chemical and in vitro ADME properties, which translate to high oral bioavailability and moderate clearance in rodent PK studies. Additionally, DSB1559 demonstrated superior tumor residence time and tumor/plasma ratio compared to AZD5305. Compared to Olaparib, DSB1559 significantly increased STING activation and reduced cell viability in BRCA1-associated murine TNBC cells. BRCA1-associated TNBC-bearing immune-competent mice treated with DSB1559 had significantly reduced tumor growth and increased overall survival (median survival 74 days) compared to Olaparib (median survival 18 days). Analysis of changes in the tumor microenvironment are ongoing. Conclusions: Taken together, DSB1559 is a highly selective PARP1 inhibitor that demonstrates superior efficacy compared to Olaparib in a BRCA1-associated immune-competent TNBC model. Citation Format: Adam Nelson, Kelly F. Zheng, Carlos Wanderley, Daniel E. Michaud, Phillip M. Cowley, Gillian M. Campbell, Barry E. McGuinness, Alan Wise, Jennifer Guerriero. Investigating PARP1 selective inhibitor DSB1559 efficacy in BRCA1-associated 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 7120.

Abstract 4216: Indication agnostic evaluation of CAR T cell function using patient-derived immunocompetent tumoroid modelling platform

Abstract Introduction: Engineered T cells redirected towards tumor-specific cytotoxicity is a promising immunotherapeutic modality that has been well characterized for efficacy against B cell malignancies and evaluated in multiple tumor types. However, the development and optimization of CAR T cell therapies requires a rigorous preclinical model for the evaluation of CAR T cell function, tumor killing, cytokine production, and memory responses. Experimental Procedure: We are evaluating our model platform Custom Organoid Modelling Platform for Accurate and Speedy SolutionS (COMPASS 2) as a comprehensive platform for testing developmental and functional stages of CAR T cells including activation, memory, and exhaustion. We are performing qualitative and quantitative analyses of surface markers to recapitulate the complexity of human cancer. Additionally, since CAR T cells mediate crosstalk with other immune components in the TME, we also evaluate and monitor the presence of B cells, NK cells, macrophages, and endogenous T cells. Triple Negative Breast Cancer (TNBC); Immunophenotyping (IP); Immunofluorescence (IF); Fluorescence Activated Cell Sorting (FACS); T regulatory (Treg). Summary of Results: Our evaluation of COMPASS 2 for functional testing of CAR T cells demonstrates the feasibility of the platform in preclinical CAR T cell evaluation. We showed T cell infiltration and analyzed cytokine production that may alter tumor cells and TME. Crosstalk with other immune cells will be evaluated. We are currently evaluating the tumor volume changes using an EGFRvIII-targeted CART cell therapy on TNBC model systems, followed by apoptosis and EGFR analysis using FACS. Conclusion: Our COMPASS 2 model platform enables studies that require recapitulating hurdles to CAR therapy, including antigen escape, T cell dysfunction and trafficking; to reveal an essential role of the endogenous immune response mediating the antitumor effect following CAR therapy. Citation Format: Damieanus O. Ochola, Huckie Dell Mundo, Preethi Samuel, Natalia Korkorina, Bhuvanesh Dave. Indication agnostic evaluation of CAR T cell function using patient-derived immunocompetent tumoroid modelling platform [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 4216.

Abstract 2909: ST101, a clinical CEBPβ antagonist peptide, promotes an immune-active tumor microenvironment by multiple cellular mechanisms

Abstract CCAAT/Enhancer Binding Protein β (C/EBPβ) is a transcription factor that is an established driver of cellular transformation in several cancer types with poor prognosis, including glioblastoma (GBM), triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). These cancers’ growth is often supported by an immunosuppressive (IS) tumor microenvironment (TME), characterized by high expression of TAMs, MDSCs, Tregs, and in the case of GBM, IS microglia. In addition to its cell-autonomous role of promoting survival and proliferation in cancer cells, C/EBPβ is also critical for IS M2 macrophage polarization, while its role in other TME populations is less understood. ST101 is a peptide antagonist of C/EBPβ that is being evaluated in a Phase 2 clinical study in patients with recurrent and newly diagnosed glioblastoma (NCT04478279). Here we explored the impact of ST101 on IS TME cell populations and on activation of cytotoxic T-cells in macrophage co-culture systems. ST101 increased the M1:M2 ratio up to 40-fold in hPBMC-derived macrophages cultured under conditions that typically drive M2 polarization. Further, ST101 promoted M2 repolarization to the M1 cell type, demonstrating that C/EBPβ is required for maintenance of the M2 program. When added to co-cultures of T cells with M2 macrophages, ST101 induced a 3-4-fold increase in intracellular IFN-γ staining indicating enhanced T cell activation. Notably, in M1 cultures ST101 further enhanced expression of the immune activity marker CD80 and T cell activation. Similar to its effect on M2 repolarization, ST101 induced activation of iPs-derived IS human microglia cultures toward an M1-like program. ST101 also impacted IS cell populations in vivo, as demonstrated in an orthotopic TNBC model in which ST101 exposure was associated with a reduction of the TAM fraction, increased intratumoral M1/M2 ratios, reduction of Tregs, increase of CD8:Treg fraction, and enhanced activity in combination with anti-PD-1. Similar observations were observed in clinical samples, where following ST101 exposure a significant decrease in expression of factors involved in M2 polarization, including CD209, SIGLEC5, and IL-24, and T cell suppression, including FoxP3 and inhibitory KIR proteins, were observed. Gene expression analysis suggests that ST101 induced a decrease in the intratumoral Treg:TIL ratio, indicating a shift towards a more immune-active TME. Overall, these results provide novel evidence for the contribution of C/EBPβ to the expression of multiple IS cancer-associated cell types and support the use of ST101 to antagonize C/EBPβ and promote an immune-active TME. Further, these data provide rational for evaluating the clinical impact of ST101 in combinations with immune checkpoint inhibitors. Citation Format: Claudio Scuoppo, Julia Diehl, Ricardo Ramirez, Mark Koester, Erin Gallagher, Siok Leong, Jerel Gonzales, Karen Mendelson, Zach Mattes, Lila Ghamsari, Gene Merutka, Abi Vainstein-Haras, Barry J. Kappel, Jim A. Rotolo. ST101, a clinical CEBPβ antagonist peptide, promotes an immune-active tumor microenvironment by multiple cellular mechanisms [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 2909.

Abstract 7553: Dual inhibition of chromatin remodeling and DNA methylation as a novel treatment for triple negative breast cancer

Abstract Epigenetics plays a central role in the initiation, establishment, and progression of cancer. In this capacity, epigenetics regulates several abnormal nuclear processes in the cancer cell including DNA replication, DNA damage repair and most prominently transcription. Because of their reversibility, epigenetic modifications have been aggressively pursued pharmacologically to correct abnormal cancer biology in the hopes of reversing cancer phenotypes. To develop novel epigenetic based therapeutic strategies for triple negative breast cancer (TNBC) we combined the DNA methyltransferase inhibitor (DNMTi) guadecitabine (Gua) with a genetic CRISPR/Cas9 mediated knockout (KO) of the chromatin remolding protein Bromodomain PHD-finger Transcription Factor (BPTF). Using the E0771 and 4T1 mouse TNBC models we show that BPTF depleted tumor cells are more susceptible to the antitumor effects of Gua. These antitumor effects are not observed in culture, or in immune compromised NSG mice, suggesting that their effects on cancer cell growth are not cancer cell intrinsic. RNA-Seq, ATAC-Seq and MBD-Seq analysis from FACS purified cancer cells shows that the combination of BPTF inhibition and Gua alters immune regulatory cytokine expression (BMP4 and CSF3) in the cancer cell, and it is theorized that these changes alter the tumor microenvironment to become less immune suppressive and permissive to the antitumor effects of immune cells. This hypothesis is supported by Cytek phenotyping of tumor resident and peripheral immune cells from treated mice showing reduced levels of immune suppressive myeloid derived suppressor cells (MDSC). Inhibiting BPTF pharmacologically using the bromodomain inhibitor AU1 does not recapitulate the effects of the genetic inhibition suggesting that the benefits of BPTF depletion to sensitization to Gua are not captured by inhibition of its bromodomain. To model the effects of pharmacologic BPTF inhibition we created a BPTF mouse KO genetically modified mouse model (GEMM). We show using this GEMM that BPTF KO in the adult mouse (a tamoxifen inducible Cre) is not lethal and has beneficial effects to the control of E0771 tumors, possibly through depleting tumor resident naïve T cells, and that transplanting BPTF KO E0771 tumors into BPTF KO adult mice further improves tumor growth control. These benefits in tumor growth control are prevented with CD8 depletion. These results in total suggests that a combination therapy regimen including BPTF inhibition (a PROTAC degradation approach) with a DNMTi could provide clinical benefits to patients with TNBC through both tumor cell intrinsic and extrinsic mechanisms. Citation Format: Joseph W. Landry, Jubin Kang, Christiane Morecock, Harry Bear, Rebecca Martin. Dual inhibition of chromatin remodeling and DNA methylation as a novel treatment for 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 7553.

Abstract 708: Targeting aurora kinase B (AURKB) as a radiosensitizing strategy in syngeneic models of triple negative breast cancer (TNBC)

Abstract Purpose: Triple negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype with few treatment options. Radiation therapy (RT) is a mainstay therapy for the treatment of BC, but local recurrence following RT therapy is common. Consequently, decreasing local recurrence in patients with TNBC is a critical clinical need. Prior work demonstrated that AURKB is overexpressed in TNBC, and over-expression correlates with poor prognosis. Here, we examined the effects of AURKB inhibition as a novel radiosensitizing strategy in syngeneic TNBC models. Methods: Cell viability assays were used to determine the half-maximal inhibitory concentrations (IC50) of the AURKB inhibitors Barasertib-HQPA and SP-96 72 hours post treatment. Clonogenic survival assays were used to assess the radiosensitivity of TNBC murine cell lines to AURKB inhibition. In these assays, AURKB inhibitors were delivered at sub-IC50 concentrations 24 hours prior to RT, and radiation enhancement ratios (rERs) were calculated. Immunofluorescent microscopy using DAPI stain assessed micronuclei. Propidium iodide staining to assess aneuploidy was completed via flow cytometry. To assess the radiosensitizing effects of AURKB inhibition in vivo, the 4T1 syngeneic TNBC cell line was injected bilaterally into Balb/c mice and treated with Barasertib and RT. Tumor volume was recorded twice weekly throughout the study. Results: Aurora kinase B inhibitors (500-750 nM Barasertib-HQPA, 100-200 nM SP-96) delivered 24 hours prior to radiation therapy induced radiosensitization in the murine TNBC cells 4T1 (rER: 1.24-1.56) and Py8119 (rER: 1.51-1.72). Mechanistically, combined AURKB inhibition and RT significantly increased micronuclei formation in 4T1 cells 24 hours after RT compared to vehicle control (p<0.0001). Furthermore, combined AURKB inhibition and RT induced aneuploidy in murine TNBC cell lines 24 hours after radiation therapy compared to vehicle control. Combined AURKB inhibition (Barasertib 25 mg/kg, IP) and RT (8 Gy RT in 1 fraction) significantly decreased tumor volume compared to mice that had received vehicle control (777 ± 61 mm3 vs 1623 ± 126 mm3; p <0.0001). Conclusion: AURKB inhibition induces radiosensitization in syngeneic models of TNBC and leads to increased micronuclei and aneuploidy, suggesting a mechanism of sensitization. These results suggest that AURKB is a potential radiosensitizing strategy for the treatment of triple negative disease. Ongoing studies are further refining the underlying mechanisms of AURKB inhibition and RT on the antitumoral immune response. Citation Format: Kassidy M. Jungles, Zhuwen Wang, Caroline Bishop, Cydnee Wilson, Meilan Liu, Jadyn James, Michael Green, James M. Rae, Corey W. Speers, Lori J. Pierce. Targeting aurora kinase B (AURKB) as a radiosensitizing strategy in syngeneic models of triple negative breast cancer (TNBC) [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 708.

Abstract 3327: L-pampo™, a TLR2/3 agonist, enhances anti-tumor immune responses and synergistically improves the anti-tumor efficacy of immune checkpoint inhibitors in cold tumors

Abstract Introduction: Immunotherapy is a promising therapeutic approach for a wide range of cancers. However, some tumors called "cold tumors" lack infiltrating T-cells and are not responsive to immune checkpoint inhibitors (ICIs), leading to worse clinical outcomes. This study demonstrates that L-pampo, a novel TLR2/3 agonist, promotes an anti-tumor immune response in cold tumors. Also, combining L-pampo with ICIs can improve the anti-tumor efficacy more safely than standard-of-care (SoC) chemotherapies. Method: We utilized syngeneic tumor models using CT26 colon cancer and 4T1 triple-negative breast cancer (TNBC). L-pampo was administered alone or in combination with ICIs such as anti-PD-1 (aPD-1) and anti-CTLA-4 (aCTLA-4) antibodies to tumor-bearing mice (n=8/group). Tumor size was measured every three days, and the survival rate was monitored until day 60. On day 21 or 27 after transplantation, we analyzed the tumor weight and tumor-infiltrating immune cells. Furthermore, we assessed the respective anti-tumor efficacies and safety of L-pampo and SoC chemotherapies (FOLFOX and paclitaxel). Result: In the CT26 colon cancer model, L-pampo effectively reduced the tumor size compared to the vehicle group (83.7%). The combination of L-pampo and ICI reduced tumor size by 86.1% (with aCTLA4) and 90.5% (with aPD-1), while the ICI alone did not effectively inhibit tumor growth (aPD-1; 17.3%, aCTLA-4; 23.6%). Notably, the triple concurrent treatment with L-pampo, aPD-1 and aCTLA-4 reduced tumor size by 96.6% and induced completed tumor regression in 1 out of 8. In the 4T1 TNBC model, L-pampo reduced tumor size by 64.2% compared to the vehicle treatment, and combined treatment with L-pampo and a-PD-1 suppressed tumor growth compared to aPD-1 alone (9.6% vs. 61.5%). Additionally, L-pampo improved median overall survival in each tumor model (CT26; 25.5 vs. 35.5, 4T1; 29.5 vs. 53.5 days). To compare the anti-tumor efficacy of L-pampo and conventional cytotoxic chemotherapy, we treated tumor-bearing mice with FOLFOX or paclitaxel as SoC regimen, respectively. Compared with each chemotherapy, L-pampo revealed comparable antitumor efficacies (Tumor growth inhibition CT26; 75.6% vs. 70.6%, 4T1; 65.5% vs. 58.8%). Interestingly, L-pampo inhibited tumor growth without significant adverse effects. In contrast, conventional chemotherapies induced acute hematological side effects such as leukopenia/neutropenia/thrombocytopenia and rapid weight loss. Furthermore, L-pampo increased the active cytotoxic T-cells infiltration through antigen-presenting cells stimulation within the tumors. Conclusion: This study suggests that L-pampo, a novel TLR2/3 agonist, can be a potent anti-tumor agent, improving the anti-tumor efficacy of ICIs in cold tumors with enhanced anti-tumor immunity and minimal toxicities than conventional chemotherapies. Citation Format: Guentae Kim, Yoonki Heo, Hye Jung Kim, Sejung Park, Byung Cheol Ahn, Won Suk Lee, Chan Kim, Hong Jae Chon, Eunyoung Chun, Jung Sun Yum. L-pampo™, a TLR2/3 agonist, enhances anti-tumor immune responses and synergistically improves the anti-tumor efficacy of immune checkpoint inhibitors in cold tumors [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 3327.