SUM149 Cells Research Articles

Altered ribosomal profile in acquired resistance and reversal associates with pathological response to chemotherapy in inflammatory breast cancer

Therapeutic resistance presents a significant hurdle in combating inflammatory breast cancer (IBC), adding to the complexity of its management. To investigate these mechanisms, we conducted a comprehensive analysis using transcriptomic and proteomic profiling in a preclinical model alone with correlates of treatment response in IBC patients. This included SUM149 cell lines derived from treatment-naïve patients, along with acquired drug resistance (rSUM149) and others in a state of resistance reversal (rrSUM149), aiming to uncover drug resistance networks. We identified specific ribosomal proteins associated with acquiring resistance. These correlated with elevated levels of molecular markers such as pERK, CDK1, XIAP, and SOD2. While resistance reversal in rrSUM149 cells largely normalized the expression profile, VIPER analysis revealed persistent alterations in ribosomal process-related proteins (AGO2, Exportin 1, RPL5), suggesting their continued involvement in drug resistance. Moreover, genes linked to ribosomal processes were significantly enriched (P < 0.001) among overexpressed genes in IBC patients (n = 87) who exhibited a pathological complete response (pCR) to neoadjuvant chemotherapy. Given the common hyperactivation of MAPK in IBC tumors, including rSUM149, we evaluated Merestinib, a multikinase inhibitor in clinical trials. It effectively targeted pERK and peIF4E pathways, suppressed downstream targets, induced cell death in drug-resistant rSUM149 cells, and showed synergistic effects with another tyrosine kinase inhibitor (Lapatinib) in parental cells. This underscores its significant impact on protein synthesis signaling, crucial for combating translational dependence in cancer cells. In summary, our study elucidates adaptive changes in IBC cells in response to therapy and treatment pauses, guiding precision medicine approaches for this challenging cancer type.

Discovery of potent CSK inhibitors through integrated virtual screening and molecular dynamic simulation.

Oncogenic overexpression or activation of C-terminal Src kinase (CSK) has been shown to play an important role in triple-negative breast cancer (TNBC) progression, including tumor initiation, growth, metastasis, drug resistance. This revelation has pivoted the focus toward CSK as a potential target for novel treatments. However, until now, there are few inhibitors designed to target the CSK protein. Responding to this, our research has implemented a comprehensive virtual screening protocol. By integrating energy-based screening methods with AI-driven scoring functions, such as Attentive FP, and employing rigorous rescoring methods like Glide docking and molecular mechanics generalized Born surface area (MM/GBSA), we have systematically sought out inhibitors of CSK. This approach led to the discovery of a compound with a potent CSK inhibitory activity, reflected by an IC50 value of 1.6 nM under a homogeneous time-resolved fluorescence (HTRF) bioassay. Subsequently, molecule 2 exhibits strong growth inhibition of MD anderson - metastatic breast (MDA-MB) -231, Hs578T, and SUM159 cells, showing a level of growth inhibition comparable to that observed with dasatinib. Treatment with molecule 2 also induced significant G1 phase accumulation and cell apoptosis. Furthermore, we have explored the explicit binding interactions of the compound with CSK using molecular dynamics simulations, providing valuable insights into its mechanism of action.

Abstract PO2-24-03: Soluble E-cadherin promotes inflammatory breast cancer tumorigenesis via PDIA4-mediated suppression of ferroptosis

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer known for its rapid progression and high metastatic potential without known distinctive drivers. Currently, there are no FDA-approved targeted therapies specifically for IBC patients, highlighting the urgent need for novel and effective treatments. Through our investigation of metastatic xenograft IBC sublines, we have identified soluble E-cadherin (sEcad), an extracellular proteolytic fragment of full-length E-cadherin, as a protein associated with IBC tumor progression. We hypothesize that sEcad promotes IBC tumorigenesis by suppressing ferroptosis. Methods: MDA-IBC3 (ER–/HER2+) and SUM149 (ER–/HER2–) IBC cell lines were used in this study. Stable overexpression of sEcad in IBC cell lines was achieved using lentiviral vectors. Cell death for ferroptosis was measured by propidium iodide staining using flow cytometry. Mass spectrometry and Bio-ID-based proteomics assays were used to identify sEcad-interacting proteins and potential mechanisms. For in vivo studies, control and sEcad -overexpressing SUM149 and MDA-IBC3 cells were injected into the cleared mammary fat pads of SCID/Beige mice and tumor growth was monitored via caliper measurements. Serum sEcad levels from IBC patients (n=301) were analyzed by ELISA. Results: High serum sEcad levels in IBC patients correlated with poorer OS (p=0.02) and earlier development of metastasis (p=0.006). Overexpression of sEcad in IBC cell lines enhanced cell proliferation and colony formation in vitro. Mice injected with sEcad-overexpressing SUM149 and MDA-IBC3 cells had significantly higher tumor growth rates compared to controls (SUM149: p=0.007; MDA-IBC3: p=0.006). Mechanistically, mass spectrometry and Bio-ID assays identified Protein Disulfide Isomerase Family A Member 4 (PDIA4) as a novel binding partner of sEcad, which was validated through co-immunoprecipitation. Additionally, sEcad increased the expression of PDIA4, an enzyme that plays a role in cellular redox regulation and protein folding. PDIA4 has been shown to regulate key players of ferroptosis such as ATF4, SLC7A11. Our results showed that, compared to controls, sEcad-overexpressing IBC cells displayed significant resistance to ferroptosis inducers, including RSL3, FIN56, and sulfasalazine. Knockdown of PDIA4 in sEcad high-expressing cells significantly increased their sensitivity to ferroptosis-promoting drugs. Moreover, we found that PDIA4 regulates ATF4 to impact ferroptosis-mediated cell death in IBC cells. Conclusions: Our study shows that sEcad affects ferroptosis through PDIA4, impacting tumorigenesis in IBC. These findings uncover a novel and pivotal role for sEcad in IBC tumor growth and progression, providing new insights and potential therapeutic targets for IBC patients. Citation Format: Xiaoding Hu, Yun Xiong, Emilly Villodre, Juhee Song, Debu Tripathy, Wendy Woodward, Savitri Krishnamurthy, Junjie Chen, Bisrat Debeb. Soluble E-cadherin promotes inflammatory breast cancer tumorigenesis via PDIA4-mediated suppression of ferroptosis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-03.

Abstract PO2-24-06: Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1

Abstract Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) have the lowest 5-year survival rates among breast cancers: 40% for IBC and 77% for TNBC. Effective treatments for these aggressive tumors are urgently needed. To develop better treatments, it is crucial to understand the underlying mechanisms driving their aggressive biology. Our recent research has identified N-myc downstream regulated gene 1 (NDRG1) as a crucial driver of tumor progression and metastasis in aggressive breast cancers. We have also found that NDRG1 expression in tumors is correlated with poorer outcomes in IBC patients. Additionally, we have observed that cancer-associated fibroblasts (CAFs), derived from breast cancer biopsies, stimulate NDRG1 expression and phosphorylation as well as tumor stemness. Based on these findings, we hypothesize that NDRG1 is a key regulator of tumor stemness and progression, and secreted stromal factors regulate tumor stemness through NDRG1. Methods: To assess tumor stemness, we conducted in vitro experiments by quantifying the CD44+/CD24- subpopulation and performing mammosphere assays in NDRG1 control and depleted cells (SUM149, BCX010, MDA-IBC3). In vivo, we employed a limiting dilution transplantation assay, where NDRG1 control and knockdown SUM149 cells were transplanted into the mammary fat pad at different dilutions. We treated NDRG1 control and knockdown breast cancer cells with different patient-derived CAF-conditioned media (CAFs-CM, 50%, 24h) or control medium. The expression of NDRG1/pNDRG1 and CD44+/CD24- cells was evaluated by immunoblotting and flow cytometry. qPCR array was performed to evaluate tumor stemness markers. Results: NDRG1 depletion significantly reduced the CD44+/CD24- subpopulation (p&amp;lt; 0.001) and the efficiency of mammosphere formation (p=0.001). In vivo limiting dilution experiments demonstrated a substantial reduction in the frequency of breast cancer stem cells in NDRG1 knockdown cells (p= 1 x 10-12). Treatment of IBC cells with CAF-CM derived from breast cancer patients stimulated the cancer stem cell subpopulation [CD44+/CD24-: Control medium (43.1 ± 0.1) vs CAF-CM (55.1 ± 0.7); p=0.004] and increased NDRG1 and phospho-NDRG1 expression. However, the stimulation of NDRG1 knockdown cells with CAF-CM did not significantly alter the cancer stem cell population (0.2 ± 0.2 in Control medium versus 0.4 ± 0.3 in CAF-CM) and the expression of NDRG1/phospho-NDRG1. Additionally, qPCR array analysis revealed the upregulation of several cancer stem cell and self-renewal markers in CAF-CM stimulated cells compared to control-medium treated cells. Conclusions: Our data strongly support the role of NDRG1 as a critical regulator of tumor stemness. Moreover, we have discovered that conditioned media from patient-derived CAFs induces tumor stemness in breast cancer cells, and this effect is dependent on the expression NDRG1. Citation Format: Emilly Villodre, Jenny M Hogstrom, Xiaoding Hu, Nina Kozlova, Debu Tripathy, Taru Muranen, Bisrat Debeb. Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-06.

Abstract PO4-28-10: Single-Cell Transcriptomic Assessment of Stromal and Immune Crosstalk on Breast Cancer Invasion Using 3D Tumor-On-Chip Model

Abstract Introduction: Our understanding of cancer has evolved over the last decades, with treatments increasingly targeting stromal cells in the tumor microenvironment (TME). The TME comprises transformed cancer cells and various non-cancerous cell types such as fibroblasts, macrophages, and endothelial cells. Despite this understanding, disrupting the tumor-stromal interactions are not targeted mainstream, as the stromal cells in the TME are genetically stable and can induce both beneficial and adverse effect on tumorigenesis. Thus, ex vivo tumor models that can faithfully recapitulate these critical tumor-stroma interactions are required to mechanistically understand the multifaceted reaction occurring within the TME. In this study, we established an innovative and multi-cellular TME on-a-chip model and assessed the synergistic effect of patient-derived Cancer Associated Fibroblast (CAF’s) and macrophages (M𝟇) on breast cancer progression. Materials and Methods: The influence of stromal components on cancer progression was assessed using the microfluidic model that allows spatial organization of various cell types. Tri-culture invasion model was established using Sum159 breast cancer cells, patient-derived CAFs, and THP-1 derived naïve (M𝟇) macrophages. Sum159 mixed with collagen and Matrigel forms the tumor region, while CAF’s and M𝟇 embedded in collagen form the stromal region. Functional and real-time assessments such as migration, morphometric analysis, and proliferation studies were conducted to understand the stromal influence on cancer cells. Gene expression analysis on an array of macrophage activation genes was performed to identify the polarized state of the macrophages after cellular interactions. Finally, Single Cell-RNA sequencing was carried out to unveil the drivers of stromal-immune crosstalk on invasion. Results and Discussion: Using the TME on-a-chip model, we compared cancer cell invasion across 4 different culture conditions namely monoculture (Sum only), co-culture (Sum+CAF, Sum+ M𝟇) and tri-culture (Sum+CAF+ M𝟇). Cancer cell migration was significantly increased in the triculture condition compared to others and stromal components also enhanced cancer cell proliferation. Hierarchical clustering of migration and morphological changes demonstrated that the co-culture conditions grouped together while CAF+ M𝟇 exerted the highest influence on cell migration and clustered separately. To understand the bi-directional communication between tumor and stroma, we assessed the polarized states of naïve macrophages using qPCR. Gene expression analysis showed that tumor-educated macrophages showed characteristics of M1 and M2 phenotypes and communicated reciprocally with tumor cells. Finally, to investigate the molecular mechanics of complex cellular interactions, we performed sc-RNA sequencing on cells extracted from the device and 2D cells (controls). Unsupervised clustering identified 11 different clusters, categorizing cells as Sum159 (tumor cells), CAFs (Stromal), and M𝟇 (Immune) based on cell-specific markers. Notably, we observed a noticeable shift in the clusters of Sum159 cells between 2D and 3D samples, emphasizing the difference between these two cultures. Conclusion: Using the TME on-a-chip, we confirmed the critical influence of microenvironmental components on tumor progression through functional assessment. Gene-expression analysis revealed bi-directional communication between noncancerous stromal components and tumor cells highlighting the importance of microenvironmental crosstalk. Furthermore, sc-RNA sequencing revealed the differences between 2D and 3D cultures. Ongoing work aims to perform transcriptomic and bioinformatic analysis on the sc-RNA sequencing data to gain a deeper understanding of the multifaceted communication within our platform. Citation Format: Kalpana Ravi, Lydia Sakala, Jin Park, Joshua LaBaer, Mehdi Nikkhah. Single-Cell Transcriptomic Assessment of Stromal and Immune Crosstalk on Breast Cancer Invasion Using 3D Tumor-On-Chip Model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-10.

Abstract LB188: PEA-15 phosphorylation mediates pro-tumor effects by promoting β-catenin activity in TNBC

Abstract Triple negative breast cancer (TNBC) is a subtype of breast cancer that is defined by a lack of estrogen receptor, progesterone receptor, and HER2-neu gene product expression. Previously, we found that TNBC cells with phosphorylated PEA-15 adaptor protein exhibited decreased clonogenic and anchorage-independent growth in vitro, and decreased growth and lung metastasis in vivo. Further, we found lower levels of phosphorylated β-catenin at S675 in PEA-15-AA (non-phosphorylatable) mutants than in PEA-15-DD (phosphomimetic) mutants. We therefore hypothesized that phosphorylated PEA-15 enhances the aggressiveness of TNBC by regulating the phosphorylation of β-catenin at S675. We first screened a panel of TNBC cells for β-catenin levels via western blot. Next to assess the biological role of β-catenin we knocked down its expression in TNBC cells (SUM149, BT549). In the β-catenin knockouts, proliferation decreased by 20%(P&amp;lt;0.0001) mammosphere formation by 69% (P&amp;lt;0.001), and clonogenic growth by 65% (P&amp;lt;0.0001), when compared to the vector control. To determine if PEA-15 mediates pro-tumor effects by phosphorylating β-catenin at S675, we created a nonphosphorylatable (S675A) and a phosphomimetic (S675D) form of β-catenin. We transfected BT549 cells with S675A and observed a decrease of 26% (P=0.011) in proliferation, 65% (P&amp;lt;0.001) in mammospheres, and 54% (P=0.0001) in clonogenic formation when compared with WT β-catenin transfected cells. Next, we transfected SUM149 cells with S675D and found an increase of 58% (P&amp;lt;0.001) in proliferation, 79% (P=0.001) in mammospheres, and 23% (P=0.0303) in clonogenic formation when compared with WT β-catenin transfected cells. To test if PEA-15 phosphorylation mediates β-catenin activity, we transfected the S675A and S675D form of β-catenin into MDA-MB-468 cell lines which stably express PEA-15-AA and PEA-15-DD. When we performed a rescue experiment with PEA-15-AA cells transfected with S675D we found a 78% (P=0.0008) increase in mammospheres when compared to vector transfected cells. Conversely, when PEA-15-DD cells were transfected with S675A we found a 53% (P=0.0004) decrease in mammospheres when compared to vector transfected cells. Next, we did a multiplex staining of a tissuemicroarray (n= 102 human TNBC tumors). We observed that in patients with grade 3 tumors, the percentages of cells expressing total PEA-15(P=0.005), P-PEA-15-S116(P=0.038), and p-β-catenin (P=0.0005) were significantly higher than those in patients with grade 2 tumors. Together, these results show that PEA-15 phosphorylation mediates tumor aggressiveness in TNBC by promoting β-catenin activity. Future studies will evaluate the clinical significance of PEA-15 and β-catenin and their phosphorylated forms in TNBC patients. These results will establish β-catenin and PEA-15 phosphorylation as potential markers of, and targets against TNBC progression. Citation Format: Alex W. Tan, Mohd Mughees, Wendy A. Woodward, Debu Tripathy, Natalie Fowlkes, Chandra Bartholomeusz. PEA-15 phosphorylation mediates pro-tumor effects by promoting β-catenin activity in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB188.

Abstract 4482: Development of small molecule inhibitors of lipocalin 2 for the treatment of aggressive cancers

Abstract Lipocalin-2 (LCN2), a secreted protein pivotal in iron homeostasis, immune responses, siderophore transport, and epithelial cell differentiation. LCN2 is highly abundant in aggressive forms of many cancers including breast, pancreatic, ovarian, and esophageal cancer. Elevated LCN2 levels in aggressive tumors correlate with heightened cancer cell motility, proliferation, angiogenesis, invasion, and metastasis. After the protein folds it makes a barrel structure which encloses a three-calyx pocket that can bind and transport molecules. A first generation of small molecule inhibitors (SMIs) targeting LCN2 demonstrated efficacy in attenuating the proliferative capacity of Inflammatory breast cancer (IBC) cells. IBC stands as a rare and highly aggressive variant, accounting for 1 to 5% of all BC cases, with 20-30% presenting as de novo metastatic disease. In the previous studies the concentration of LCN2 inhibitors to reduce cell proliferation in 50% was around 10 µM. In pursuit of better LCN2 SMIs, we conducted in-silico analysis using a library of 369,000 ligands. Through the High-Performance Computer Facility at UPR Río Piedras, we employed AutoDock Vina for virtual screening, targeting the LCN2-calyx with Lys134 as the central coordinate. Docking protocols, predicting binding affinities, identified 8,162 out of 327,638 ligands that have been docked already with values below -9.6 kcal/mol for further consideration. The next phase involves subjecting these potential inhibitors to the SwissADME tool for a comprehensive evaluation of absorption, distribution, metabolism, and excretion parameters, as well as pharmacokinetics, drug-likeness, and medicinal chemistry considerations. The parameters to be evaluated are the Lipinski rules, which states that an orally active drug may have a maximum of one violation and the pann-assay interference compounds (PAINS) alerts tool, a source of false positives for biologically active compounds. The top candidates, exhibit binding affinities within the range of -11.5 to -12.2. After SwissADME filtering, we will re-rank the ligand list, identifying 30 commercially available candidates for subsequent cell viability assays in SUM149 (TNBC) and SUM190 (HER2+) cell lines. This multifaceted approach integrates computational methodologies with rigorous experimental validation, paving the way for potential therapeutic interventions in IBC treatment. Citation Format: Marienid Flores-Colón, Cristal A. Peña-Vázquez, Eliud Hernández-O'Farril, Luis E. Vázquez-Quiñones, Pablo E. Vivas-Mejía. Development of small molecule inhibitors of lipocalin 2 for the treatment of aggressive cancers [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 4482.

Abstract 6203: An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients

Abstract Background: Drug resistance is a major challenge in inflammatory breast cancer (IBC), a hyperproliferative subtype characterized by the presence of diffuse tumor cell clusters that show high level of heterogeneity. As the degree of tumor heterogeneity during cancer progression can cause evolution of cell states that can either compete or co-operate resulting in therapeutic failure, the study objective was to identify drug resistance networks that can serve as biomarkers or therapeutic targets. Methods: Integrated transcriptomic and proteomic profiling approach was conducted in a progression model of acquired drug resistance and its reversal comprised of a treatment-naïve patient-derived SUM149 cell line, along with two isogenic derivatives and representing drug-resistant (rSUM149) and resistance-reversal states (rrSUM149). Global gene expression analysis in IBC patients (n=87) recorded responses to neoadjuvant chemotherapy. Based on the data, targeted multikinase inhibitors were evaluated in viability and signaling assays. Results: We identified specific ribosomal proteins associated with resistance acquisition, which correlated with high levels of pERK, CDK1, XIAP, and SOD2. Conversely, the resistance reversal in rrSUM149 showed almost complete normalization to the profile of SUM149. However, VIPER analysis revealed proteins related to ribosomal processes (AGO2, Exportin 1, RPL5) that did not revert in rrSUM149, suggesting this pathway may have pleiotropic effects in governing drug resistance. Notably, genes linked to ribosomal processes were significantly enriched (P&amp;lt;0.001) among the overexpressed genes in IBC patients (n=87) who exhibited pCR to neoadjuvant chemotherapy. This is crucial as IBC tumors, including rSUM149, exhibit MAPK hyperactivation including in the presence of EGFR tyrosine kinase inhibitors. Treatment with Merestinib, a multikinase inhibitor, suppressed pERK, peIF4E, downstream targets like CDK1, and ribosomal protein IMP3 underscoring the impact on protein synthesis signaling crucial for countering translational dependence in cancer cells. Additionally, Merestinib synergized with EGFR-TKI (lapatinib) in increasing cell death in all three states. Conclusion: Overall, our study describes adaptive changes in response to both therapeutic stress and therapy break that have the potential to serve as biomarkers or extended for pharmacological interrogation toward precision medicine for this rare, understudied cancer. Furthermore, the ability of Merestinib, in clinical trials, to target translational dependence is attractive as cancer cells often appropriate these signaling pathways to gain tolerance to various stress stimuli. Support: NIH-R01CA264529; DoD Breast Cancer Breakthrough level 2 Award W81XWH2010153 Citation Format: Gayathri R. Devi, Pritha PAI, Matthew W. Foster, Dorababu S. Sannareddy, Seayoung Lee, Steven V. Laere. An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients [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 6203.

UCHL1 contributes to insensitivity to endocrine therapy in triple-negative breast cancer by deubiquitinating and stabilizing KLF5

BackgroundUbiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that regulates ERα expression in triple-negative cancer (TNBC). This study aimed to explore the deubiquitination substrates of UCHL1 related to endocrine therapeutic responses and the mechanisms of UCHL1 dysregulation in TNBC.MethodsBioinformatics analysis was conducted using online open databases. TNBC representative MDA-MB-468 and SUM149 cells were used for in vitro and in-vivo studies. Co-immunoprecipitation was used to explore the interaction between UCHL1 and KLF5 and UCHL1-mediated KIF5 deubiquitination. CCK-8, colony formation and animal studies were performed to assess endocrine therapy responses. The regulatory effect of TET1/3 on UCHL1 promoter methylation and transcription was performed by Bisulfite sequencing PCR and ChIP-qPCR.ResultsUCHL1 interacts with KLF5 and stabilizes KLF5 by reducing its polyubiquitination and proteasomal degradation. The UCHL1-KLF5 axis collaboratively upregulates EGFR expression while downregulating ESR1 expression at both mRNA and protein levels in TNBC. UCHL1 knockdown slows the proliferation of TNBC cells and sensitizes the tumor cells to Tamoxifen and Fulvestrant. KLF5 overexpression partially reverses these trends. Both TET1 and TET3 can bind to the UCHL1 promoter region, reducing methylation of associated CpG sites and enhancing UCHL1 transcription in TNBC cell lines. Additionally, TET1 and TET3 elevates KLF5 protein level in a UCHL1-dependent manner.ConclusionUCHL1 plays a pivotal role in TNBC by deubiquitinating and stabilizing KLF5, contributing to endocrine therapy resistance. TET1 and TET3 promote UCHL1 transcription through promoter demethylation and maintain KLF5 protein level in a UCHL1-dependent manner, implying their potential as therapeutic targets in TNBC.

TR-57 Treatment of SUM159 Cells Induces Mitochondrial Dysfunction without Affecting Membrane Potential.

Recent works identified ClpXP, mitochondrial caseinolytic protease, as the only target of imipridones, a new class of antitumor agents. Our study of the mechanism of imipridone derivative TR-57 action in SUM159 human breast cancer cells demonstrated mitochondrial fragmentation, degradation of mitochondrial mtDNA and mitochondrial dysfunction due to inhibition of Complex I and Complex II activity. Complete inhibition of oxidative phosphorylation accompanied 90, 94, 88 and 87% decreases in the content of Complex I, II, III and IV proteins, respectively. The content of the FOF1-ATPase subunits decreased sharply by approximately 35% after 24 h and remained unchanged up to 72 h of incubation with TR-57. At the same time, a disappearance of the ATPIF1, the natural inhibitor of mitochondrial FOF1-ATPase, was observed after 24 h exposure to TR-57. ATPase inhibitor oligomycin did not affect the mitochondrial membrane potential in intact SUM159, whereas it caused a 65% decrease in TR-57-treated cells. SUM159 cells incubated with TR57 up to 72 h retained the level of proteins facilitating the ATP transfer across the mitochondrial membranes: VDAC1 expression was not affected, while expression of ANT-1/2 and APC2 increased by 20% and 40%, respectively. Thus, our results suggest that although TR-57 treatment leads to complete inhibition of respiratory chain activity of SUM159 cells, hydrolysis of cytoplasmic ATP by reversal activity of FOF1-ATPase supports mitochondrial polarization.

A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

Abstract A020: Reversing triple negative breast cancer stem cells by disrupting the Notch signaling pathway via BCL6 targeting

Abstract Triple negative breast cancer (TNBC) is a heterogeneous molecular breast cancer (BC) subtype that still has a very poor prognosis. Genetic, molecular and clinical tumor heterogeneity represent major pathobiological factors involved in poor clinical outcome. The sequence specific transcriptional repressor and proto-oncogene B cell lymphoma protein 6 (BCL6) has emerged as a suitable therapeutic target as it plays broad and critical roles in many hematological and solid cancers. BCL6 exerts control over cellular proliferation and survival by binding to specific responsive elements located within the regulatory regions of target genes and recruiting of chromatin modifying co-repressor complexes such as silencing mediator for retinoid and thyroid receptors (SMRT), BCL6 corepressor (BCoR) and histone deacetylases (HDACs). Moreover, in hematological malignancies, BCL6 transcriptional activity is also associated to the recruitment of the chromatin remodeling factor, Enhancer of Zeste 2 (EZH2). The role of BCL6 in BC is still not well defined; however emerging studies report its involvement in tumorigenesis, disease progression and poor survival. We recently showed that BCL6 is expressed at significantly higher levels in TNBC mammospheres, 3D multicellular structures enriched in cancer stem cells (CSCs), than in their differentiated bulk cell counterparts. Additionally, we found that the functional role of BCL6 is crucial for mammosphere formation, as its loss of function decreased this process. Intriguingly, we uncovered a functional interplay occurring between BCL6 and EZH2, which triggers the BCL6/Notch stemness signaling axis by inhibiting Numb transcription in both SUM149 and SUM159 TNBC cell lines. We exploited the molecular mechanisms responsible for this specific interplay, taking advantage of two molecules, FX1, a small molecule inhibitor able to block BCL6 recruiting activity and EED226, a potent Polycomb Repressive Complex 2 (PRC2) inhibitor. In order to define the role of both BCL6, as epigenetic regulator, and NUMB, as inhibitor of NOTCH stemness pathway in TNBC, SUM149 and SUM159 cell lines were stably transduced with lentiviral shRNA to derive stable NUMB-silenced matched cells. The expression of Notch downstream target genes, such as HES1/2 and HEY1/2, was assessed by qRT PCR analysis in the engineered TNBC cell models and resulted only slightly increased following NUMB downmodulation. Upon FX1 or EED226 administration, a strongest effect was observed, indicating that NUMB is not the only player in regulating the Notch pathway. Further experiments are currently ongoing aiming at identify additional players and molecular pathway(s). Collectively, our preliminary results provide additional preclinical evidences on the role exerted by BCL6 as epigenetic regulator of TNBC and underlie its interplay with EZH2thus paving the way for a novel potential therapeutic strategy targeting BCL6/Notch signaling axis. Citation Format: Francesca De Santis, Francesca Putti, Giorgia Bravin, Carla Portulano, Federica Zonca, Lorenzo Castagnoli, Michele Magni, Filippo de Braud, Serenella M Pupa, Massimo Di Nicola. Reversing triple negative breast cancer stem cells by disrupting the Notch signaling pathway via BCL6 targeting [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A020.

Mechanisms of Intrinsic Radioresistance in Breast Cancer Identify Potential Therapeutic Vulnerabilities

Clinical management of breast cancer (BC) includes radiation therapy (RT) for most women, though the molecular mechanisms that underly RT response and intrinsic radioresistance are poorly understood. Both in vitro and in vivo models aid in our understanding of radiobiology, and we hypothesized that transcriptional changes caused by radiation in vitro in BC cell lines would be recapitulated in an in vivo mouse xenograft model and uncover targetable mechanisms of radioresistance in BC. Radiosensitivity was measured with clonogenic survival assays in 16 cell lines. RNA-seq experiments in vitro and in vivo were performed in an RT resistant (SUM-159) and RT sensitive (ZR-75) cell line 24 hrs after 4 Gy or after 2 Gy x 6 fractions, respectively. Differentially expressed genes (DEGs) were identified from RNA-seq data with DeSeq2 followed by pathway analysis with iPathwayGuide. RT sensitivity was subtype independent in 16 BC cell lines, with SUM-159 radioresistant (SF 0.88) and ZR-75-1 radiosensitive (SF 0.29). There were 75 unique pathways that were significantly altered after RT in SUM-159 cells (53 pathways in vivo only, 36 pathways in vitro only, 14 both conditions; adjusted p-value < 0.05) and 85 unique pathways that were significantly altered after RT in ZR-75-1 cells (16 pathways in vivo only, 72 in vivo only, 3 both conditions; adjusted p-value < 0.05). Pathways that were significantly affected in both cell lines exclusively in the in vitro condition include canonical RT response pathways such as cell cycle, cellular senescence, and DNA replication, though the direction of DEGs were opposite in the two cell lines for each of these pathways. The IL-17 signaling pathway was significantly altered for both cell lines in vivo. Of the pathways that were significantly altered in both conditions for SUM-159 cells, inflammation, including chemokine signaling pathway and cytokine-cytokine receptor interaction, were among the most significant. Significantly more cytokines were upregulated following RT in vivo than in vitro. Cytokines were not upregulated in ZR-75-1 cells in vitro or in vivo. Taken together, the significant changes in the IL-17 pathway and the upregulation of cytokines only in vivo indicate a potential of the tumor microenvironment in the in vivo condition that the in vitro condition lacks. Increased heterogeneity in vivo relative to in vitro may also explain the absence of several canonical RT response pathways in the in vivo conditions for each cell line. Notably, the opposite direction of DEG changes in the canonical RT response pathways between the 2 cell lines with disparate radiosensitivity levels may point to important biologic vulnerabilities that may be targeted in the resistant SUM-159 cells. Future studies are underway using additional BC cell lines and single-cell analysis to better understand RT response heterogeneity.

Forkhead Box Q1 is a novel regulator of autophagy in breast cancer cells.

Forkhead Box Q1 (FoxQ1) transcription factor is overexpressed in luminal-type and basal-type human breast cancers when compared to normal mammary tissue. This transcription factor is best known for its role in promotion of breast cancer stem-like cells and epithelial to mesenchymal transition. The present study documents a novel function of FoxQ1 in breast cancer cells. Overexpression of FoxQ1 in basal-like SUM159 cells and luminal-type MCF-7 cells resulted in increased conversion of microtubule-associated protein light chain 3 beta-I (LC3B-I) to LC3B-II, which is a hallmark of autophagy. Autophagy induction by FoxQ1 overexpression was confirmed by visualization of LC3B puncta as well as by transmission electron microscopy. Expression profiling for genes implicated in autophagy regulation revealed upregulation of many genes, including ATG4B, ATG16L1, CTSS, CXCR4 and so forth but downregulation of BCL2L1, DRAM1, TNF, ULK2 and so forth by FoxQ1 overexpression in SUM159 cells. Western blot analysis confirmed upregulation of ATG4B and CXCR4 proteins by FoxQ1 overexpression in both SUM159 and MCF-7 cells. Chromatin immunoprecipitation assay revealed recruitment of FoxQ1 at the promoter of ATG4B. Pharmacological inhibition of ATG4B using S130 significantly increased apoptosis induction by DOX in empty vector transfected as well as FoxQ1 overexpressing SUM159 and MCF-7 cells but this effect was statistically significantly lowered by FoxQ1 overexpression indicating the protective role of FoxQ1 on apoptosis. Treatment of SUM159 cells with S130 and DOX enhanced LC3B-II level in both empty vector transfected cells and FoxQ1 overexpressing SUM159 cells but not in FoxQ1 overexpressing MCF-7 cells. In conclusion, FoxQ1 is a novel regulator of autophagy.

Natural Compounds, Optimal Combination of Brusatol and Polydatin Promote Anti-Tumor Effect in Breast Cancer by Targeting Nrf2 Signaling Pathway.

Triple-negative breast cancer (TNBC) has been clearly recognized as a heterogeneous tumor with the worst prognosis among the subtypes of breast cancer (BC). The advent and application of current small-molecule drugs for treating TNBC, as well as other novel inhibitors, among others, have made treatment options for TNBC more selective. However, there are still problems, such as poor patient tolerance, large administration doses, high dosing frequency, and toxic side effects, necessitating the development of more efficient and less toxic treatment strategies. High expression of Nrf2, a vital antioxidant transcription factor, often promotes tumor progression, and it is also one of the most effective targets in BC therapy. We found that in MDA-MB-231 cells and SUM159 cells, brusatol (BRU) combined with polydatin (PD) could significantly inhibit cell proliferation in vitro, significantly downregulate the expression of Nrf2 protein as well as the expression of downstream related target genes Heme Oxygenase-1 (HO-1) and NAD(P)H dehydrogenase, quinone 1 (NQO1), and promote reactive oxygen species (ROS) levels to further strengthen the anti-tumor effect. Furthermore, we discovered in our in vivo experiments that by reducing the drug dosage three times, we could significantly reduce tumor cell growth while avoiding toxic side effects, providing a treatment method with greater clinical application value for TNBC treatment.

Abstract 3635: TBK1 inhibition potentiates the efficacy of AXL-targeted therapy by modulating tumor microenvironment in aggressive breast cancers

Abstract Introduction: Triple-negative and inflammatory breast cancer (TNBC and IBC) are the most aggressive breast cancer subtypes. Novel actionable targets and complementary therapies are critically needed. AXL, a receptor tyrosine kinase, drives pleiotropic phenotypes of TNBC and IBC aggressiveness. Targeting AXL reduces IBC tumor growth in vivo and inhibits polarization of M2 macrophages. To identify novel tumor microenvironment (TME) targets to enhance the efficacy of AXL-targeted therapy, we conducted a synthetic lethal kinome siRNA screening. We identified TBK1 as a candidate to synergize with AXL growth inhibition. TBK1 is a serine/threonine protein kinase that regulates innate, adaptive responses and antitumor immune responses. We hypothesized that inhibition of TBK1 enhances the antitumor effect of AXL-targeted therapy in aggressive breast cancers. Methods: We used knockdown, knockout (KO) and inhibitors to suppress AXL and TBK1 pathways and tested the synergistic effect of targeting AXL and TBK1 on the growth of human SUM149 and BCX010 IBC cells, and HS578T TNBC cells in vitro. To determine the synergistic effect of targeting both pathways in vivo, we inoculated control or TBK1 KO murine TNBC 4T1.2 cells into BALB/c mice. We assessed the activity of AXL inhibitor TP-0903 in reducing tumor growth. Using multicolor flow cytometry, we studied the effects of targeting AXL and TBK1 on the TME. We tested the activity of TP-0903 combined with a TBK1 inhibitor in another TNBC mouse model, E0771. We used RNA-sequencing and real-time PCR/Western blot to determine the molecular mechanisms of how TBK1 inhibition synergizes AXL-targeted therapy in these cancers. Results: Compared with AXL or TBK1 suppression alone, genomic or pharmacologic suppression of AXL and TBK1 significantly reduced the growth of SUM149, BCX010, and HST578T cells in vitro. In both 4T1.2 and E0771 syngeneic mouse models, TP-0903 was more active in reducing tumor growth in TBK1-inhibited tumors than in control tumors. Tumors with TBK1 inhibition and treated with TP-0903 had a significantly higher population of cytotoxic T cells than control tumors. Depletion of CD8+ T cells blocked the synergistic effect of targeting AXL and TBK1 pathways on reducing tumor growth, suggesting that cytotoxic T cells contributed to the anti-tumor synergy of targeting AXL and TBK1. Mechanistically, TBK1 induced M2 macrophage migration via IRF3-regulated CCL5 secretion in SUM149 and HS578T cells, and AXL KO attenuated the polarization and migration of M2 macrophages by inhibiting the CCR5/CCL5 axis. Conclusions: Targeting TBK1 enhances the efficacy of AXL-targeted therapy in aggressive breast cancer by suppressing the paracrine effect of CCR5/CCL5 axis. This combination represents a novel and effective therapy modulating the TME of aggressive breast cancer, which warrants further investigation in the clinical setting. Citation Format: Lan Phi, Takashi Semba, Ngu V. Trinh, Fang Zou, Jason M. Foulks, Steven L. Warner, Savitri Krishnamurthy, James P. Long, James M. Reuben, Debu Tripathy, Naoto T. Ueno, Xiaoping Wang. TBK1 inhibition potentiates the efficacy of AXL-targeted therapy by modulating tumor microenvironment in aggressive breast cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3635.

Multi-omics analyses reveal ClpP activators disrupt essential mitochondrial pathways in triple-negative breast cancer.

ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in vitro and in vivo studies, including clinical trials for refractory solid tumors. Though progress has been made in identifying specific phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP activation leads to broad anti-cancer activity has yet to be fully elucidated. In this study, we utilized a multi-omics approach to identify the ClpP-dependent proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the TR compound TR-57 in triple-negative breast cancer cells. Applying mass spectrometry-based methods of proteomics and metabolomics, we identified ∼8,000 proteins and 588 metabolites, respectively. From proteomics data, 113 (ONC201) and 191 (TR-57) proteins significantly increased and 572 (ONC201) and 686 (TR-57) proteins significantly decreased in this study. Gene ontological (GO) analysis revealed strong similarities between proteins up- or downregulated by ONC201 or TR-57 treatment. Notably, this included the downregulation of many mitochondrial processes and proteins, including mitochondrial translation and mitochondrial matrix proteins. We performed a large-scale transcriptomic analysis of WT SUM159 cells, identifying ∼7,700 transcripts (746 and 1,100 significantly increasing, 795 and 1,013 significantly decreasing in ONC201 and TR-57 treated cells, respectively). Less than 21% of these genes were affected by these compounds in ClpP null cells. GO analysis of these data demonstrated additional similarity of response to ONC201 and TR-57, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, cell cycle, and nucleus, and increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparison of response between both compounds demonstrated a highly similar response in all -omics datasets. Analysis of metabolites also revealed significant similarities between ONC201 and TR-57 with increases in α-ketoglutarate and 2-hydroxyglutaric acid and decreased ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ClpP activation in TNBC cells. Further analysis identified multiple pathways that were specifically impacted by ClpP activation, including ATF4 activation, heme biosynthesis, and the citrulline/urea cycle. In summary the results of our studies demonstrate that ONC201 and TR-57 induce highly similar and broad effects against multiple mitochondrial processes required for cell proliferation.

Abstract P6-12-04: An Inducible Lineage Tracing System for the Identification of Tumor-Initiating Cells

Abstract Background: Metastasis and therapeutic resistance are a major clinical challenge, responsible for the vast majority of cancer deaths. A subpopulation of tumor cells known to have intrinsic resistance to standard therapies and contribute to metastasis function as “cancer stem”, or tumor-initiating, cells (TICs). Enriched populations of TICs are typically identified by markers such as aldehyde dehydrogenase activity, the cell surface marker combination of CD44+/CD24-, or fluorescent reporters for signaling pathways that regulate TIC function such as STAT3. Despite their utility, TIC markers and reporters have limitations. Marker expression can be unstable and there is no established method to lineage trace long-lived TICs or to follow them as they undergo cell state changes. Methods: To augment existing TIC reporters, we developed a two component TAM-inducible, Cre recombinase-dependent, STAT3 signaling-specific lentiviral LT system. The first component is a vector that labels cells with active STAT3 signaling (EGFP+), followed by a self-cleaving peptide and TAM-inducible Cre-recombinase (4M67-EGFP-P2A-CreERT2). The second component is a constitutively expressed dual-color switching Cre-dependent reporter vector (EFS-loxPdsRedloxP-mNeptune2). Addition of TAM drives color switching from dsRed to mNeptune2 via CreERT2 recombination in STAT3 signaling cells. Both lentiviral vectors were constructed using Gateway® Cloning. Sum159 cells were transduced with the LT system and reporter activity was validated both in vitro and in vivo using confocal microscopy and flow cytometry. Four LT cell populations (EGFP+/mNeptune2+, EGFP+/dsRed+, EGFP-/mNeptune2+, and EGFP-/dsRed+) were enriched using fluorescence activated cell sorting, then analyzed by single cell RNA sequencing (scRNA seq). Results: Our results confirm the STAT3 LT reporter identifies STAT3 signaling cells (EGFP+), which can be labelled (mNeptune2+) upon addition of TAM. We conducted a TAM dose response curve to identify the optimal TAM dose for complete and partial labeling of STAT3 signaling cells. scRNA seq uncovered gene expression patterns within the TIC compartment and revealed similarities and differences in gene expression between the TIC compartment and the remaining LT reporter populations. Conclusion: These data demonstrate our LT system is a powerful tool that can be applied to uncover the role of TICs in metastasis and therapeutic resistance, as well as identify genetic vulnerabilities to specifically target TICs. Citation Format: Eric P. Souto, Michael T. Lewis. An Inducible Lineage Tracing System for the Identification of Tumor-Initiating Cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-04.

Abstract P6-10-14: Lysosomal acid lipase (LIPA) as a novel therapeutic vulnerability for treating TNBC

Abstract Background: TNBCs have the highest mortality rate among all BC subtypes. There is thus an urgent and unmet need for effective targeted therapies in TNBC. Recently we, identified a novel agent ERX-41 that showed good efficacy in treating TNBC in preclinical mouse models, however, its molecular action remain unknown. In this study, we identified LIPA as novel molecular target of ERX-41. Methods: We have used CRISPR knockout pooled library and multiple TNBC models for identifying molecular target of ERX-41. Mechanistic studies were performed using LIPA mutants, RNA-seq, Turbo-ID mapping, Mass spectrometry, Immunoprecipitation, and Western blotting. The in vivo efficacy of ERX-41 was examined using four different patient-derived xenograft (PDX) models. We evaluated LIPA protein expression in TNBC using tissue microarray (TMA). Results: To identify the molecular target of ERX-41, we performed an unbiased CRISPR–Cas9 knockout (KO) screen in MDA-MB-231 cells and the results identified LIPA as a top hit. KO of LIPA alone (which encodes lysosomal acid lipase (LAL) abrogated cytotoxic response to ERX-41. Cellular thermal shift assays confirmed that ERX-41 binds to LAL. In silico modelling and mutational studies confirmed that ERX-41 interacts with LAL through residues in its LXXLL domain and that ERX-41 ability to induce ER stress and cell death in TNBC is independent of the lipase activity of LAL. Unbiased RNA-seq studies with and without ERX-41 in parental and LIPA KO SUM-159 cells revealed induction of genes involved in ER stress and UPR response by ERX-41 in parental SUM-159 cells but not in cells with LIPA KO. Ultrastructural studies using live-cell confocal microscopy show that LIPA KO abrogated ER morphological changes at 2 and 4 h after ERX-41 treatment. Further, subcellular localization studies showed LIPA localizes to endoplasmic reticulum (ER). Unbiased proteomic approaches (TurboID and DIA mass spec) identified a core set of proteins that were both LAL binders and affected by ERX-41 treatment. GO analyses of LAL binding proteins confirmed their involvement in protein folding. Tumor micro array (TMA) analyses confirmed that &amp;gt;80% of primary TNBC tumors had significant and detectable LAL protein expression in contrast, normal breast tissue had lower LAL expression. ERX-41 (10 mg/kg body weight) decreased growth of four distinct TNBC patient-derived xenografts (PDXs) in vivo. Conclusions: Our results identified a new molecular target (LAL) for ERX-41 and novel mechanism of action (disruption of protein folding and induction of ER stress) that may have utility in treating patients with TNBC. Citation Format: Suryavathi Viswanadhapalli, Xihui Liu, Uday Pratap, Gangadhara R. Sareddy, Susan T. Weintraub, Ganesh V. Raj, Jung-Mo Ahn, Ratna K. Vadlamudi. Lysosomal acid lipase (LIPA) as a novel therapeutic vulnerability for treating TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-14.

Abstract P4-07-50: mRNA expression and delivery efficacy of lipid nanoparticles in the cells breast tumor microenvironment: in vitro and in vivo evaluation

Abstract During the COVID-19 pandemics we have all witnessed the clinical importance of mRNA as current vaccines and future therapeutics. mRNA therapies have a potential to revolutionize cancer treatment. Delivery of mRNA requires lipid nanoparticles (LNP) to protect the cargo from degradation. mRNA has a negative charge and depends on positively charged lipids to be encapsulated in LNP. These lipids can be either ionizable at certain pH or constantly cationic. Even though previous studies had evaluated the formulation properties of ionizable and cationic LNP systems, there is the need to understand their specificity in terms of mRNA delivery and protein expression in breast cancer tumor microenvironment. The objective of this work was to assess the kinetics of LNP cellular uptake and mRNA expression inv breast cancer (BC) cells and fibroblasts, the most frequent cell type in the tumor microenvironment cells, while studying the mechanisms involved in differential behaviors of LNP formulated with cationic and ionizable lipids. To achieve this goal mRNA-LNP containing ionizable lipids (LNP-A) and cationic lipids (LNP-B) were designed and formulated using Nanoassemblr® Benchtop microfluidics mixer (Precision NanoSystems). mRNA-LNP were characterized for size, zeta potential using dynamic light scattering (DLS) and mRNA encapsulation efficiency using RiboGreen assay. LNP were tagged with rhodamine lipid to investigate the uptake kinetic and a reporter GFP mRNA to evaluate mRNA expression in murine 4T1 and human MCF7, MDA-231, SUM-159 and T-47D breast cancer cells and BJ fibroblasts. Live fluorescence microscopy imaging, IncuCyte S3®, was used to determine the LNP uptake and GFP mRNA expression. In vitro biocompatibility was assessed with WST-1 assay. Additionally, expression of mRNA delivered from LNP in tumor microenvironment was evaluated in vivo in a syngeneic 4T1 breast cancer model using mRNA luciferase and IVIS imaging. mRNA-LNPs possessed an average diameter of 77 - 107 nm, narrow size distribution, neutral zeta potential and high mRNA encapsulation efficiency (&amp;gt;94%). Our results demonstrated that mRNA expression was higher in breast cancer cells when delivered from LNP-A formulation and in BJ fibroblasts when delivered from LNP-B. LNP-A, the ionizable LNP, was tested in the breast cancer cells to confirm the efficacy of the delivery. The highest transfection efficacy, from high to low, T-47D, MCF7, SUM-159, 4T1 and MDA-231.We have further investigated the cellular uptake mechanisms of LNP using uptake pathway inhibitors for caveolae endocytosis, clathrin endocytosis, and phagocytosis. Our data confirm that there are differences in mechanisms that govern the uptake of mRNA LNP in breast cancer cells and fibroblasts. Clathrin-mediated endocytosis was active in 4T1 breast cancer cells for ionizable and cationic LNP. Interestingly, despite in vitro differences in uptake and mRNA expression, in vivo results show that both formulations efficiently delivered luciferase-mRNA in the tumor microenvironment. Histology results demonstrated similar luciferase expression for both LNP in tumors. Additionally, we were able to confirm the prominent presence of fibroblast and similar distribution in the 4T1 subcutaneous model which could explain the similar efficacy of cationic and ionizable LNP. Understanding uptake and mRNA expression of different LNP formulations in the tumor microenvironment can help in achieving the necessary protein expression for breast cancer therapies. Furthermore, determining the most efficient carrier in early stages may reduce the time required for clinical translation. Acknowledgement: This research was supported in part by CPRIT Core for RNA Therapeutics and Research. Citation Format: Karem A. Court, Anjana Tiwari, Eric Chau, John Cooke, Biana Godin. mRNA expression and delivery efficacy of lipid nanoparticles in the cells breast tumor microenvironment: in vitro and in vivo evaluation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-50.