Triple-negative Breast Cancer Xenograft Models Research Articles

Peposertib, a DNA-PK Inhibitor, Enhances the Anti-Tumor Efficacy of Topoisomerase II Inhibitors in Triple-Negative Breast Cancer Models.

Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients.

Abstract PO3-25-10: Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics

Abstract Background: Microtubule-targeting agents (MTAs) have been widely utilized in cancer treatment, but the development of novel MTAs has faced limitations. CMPD1, a highly selective inhibitor of MAP kinase-activated protein kinase 2 (MK2) with dual functions as a microtubule inhibitor, shows promise as a targeted therapy for triple-negative breast cancer (TNBC). This study aimed to investigate the effects of CMPD1, targeting MK2 and acting as a microtubule inhibitor, on cell cycle progression, microtubule dynamics, and TNBC-related processes. Methods: CMPD1 was evaluated in non-transformed retinal pigment epithelial cells (RPE1) and TNBC cell lines, including MDA-MB-231 and MDA-MB-468 cells. Time-lapse microscopy and immunofluorescence staining were employed to observe the effects of CMPD1 on mitotic progression, cellular morphology, activation of the spindle assembly checkpoint (SAC), and microtubule dynamics. Anchorage-independent growth assays and in vivo studies using a TNBC xenograft model were performed to assess the impact of CMPD1 on colony formation, tumor growth, and metastasis. Results: CMPD1 treatment induced prometaphase arrest and mitotic slippage in both non-transformed and TNBC cells. This arrest was associated with SAC activation, evidenced by increased levels of Mad1 at kinetochores. CMPD1 exhibited robust microtubule depolymerization, primarily targeting microtubule plus-ends and inhibiting microtubule polymerization in vitro. Furthermore, CMPD1 disrupted the organization of the microtubule network in interphase cells and perturbed the mitotic spindle during mitosis. Additionally, CMPD1 treatment resulted in the formation of cytoplasmic protrusions, membrane deformations, and dynamic movement of clustered mitotic chromosomes. These morphological alterations were indicative of the impact of CMPD1 on cellular structures and processes during mitosis. Notably, CMPD1 demonstrated significant efficacy in inhibiting anchorage-independent growth in TNBC cells, highlighting its potential as an anti-TNBC agent. In the TNBC xenograft model, CMPD1 exhibited superior anti-tumor activity compared to the standard chemotherapeutic agent Taxol. Tumor growth was markedly suppressed, and the formation of metastatic lesions was significantly reduced upon CMPD1 treatment. Furthermore, CMPD1 treatment effectively inhibited TNBC cell migration and invasion, likely through its modulation of microtubule dynamics. The disruption of microtubule dynamics by CMPD1 impaired the ability of TNBC cells to migrate and invade surrounding tissues, which are crucial processes in cancer metastasis. Importantly, CMPD1 showed high selectivity for TNBC cells, exerting minimal cytotoxic effects on normal cells, and could be efficiently washed out. This selectivity and washout ability enhance its potential as a targeted therapeutic agent for TNBC treatment. Conclusions: CMPD1, a highly selective MK2 inhibitor and microtubule inhibitor, exerts profound effects on cell cycle progression, microtubule dynamics, and TNBC-related processes. It induces prometaphase arrest, mitotic slippage, and microtubule depolymerization, resulting in altered cellular morphology. CMPD1 displays high selectivity for TNBC cells, while sparing normal cells and allowing efficient washout, making it a promising therapeutic agent for TNBC treatment. Its inhibition of anchorage-independent growth, suppression of tumor growth in vivo, and ability to impair TNBC cell migration and invasion further underscore its potential as a targeted anti-TNBC therapy. Citation Format: Mamoru Takada, Yu-Chia Chen, Yu Muhan, Hideyuki Yamada, Junta Sakakibara, Hiroshi Fujimoto, Takeshi Nagashima, Masayuki Otsuka, Motoki Takaku, Aussie Suzuki. Selective and Effective Targeting of Triple-Negative Breast Cancer by CMPD1: Inhibition of MAP kinase-activated protein kinase 2 and Microtubule Dynamics [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 PO3-25-10.

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

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

Abstract 5962: Novel xCT-targeted biologics significantly suppress metastasis, improve survival, and sensitize primary tumors to chemotherapy in TNBC xenograft models

Abstract Background: Triple negative breast cancer (TNBC) is a highly heterogeneous subset of breast cancer, which has the worst prognosis and lacks specifically targeted therapeutic options. The TNBC subtype termed M/MSL is enriched with cells of high mesenchymal state and increased stemness, and is particularly resistant to current therapies1,2. But this cancer cell phenotype is susceptible to ferroptosis, hence targeting these aggressive tumors with ferroptosis inducers such as xCT inhibitors (xCTi) is an attractive alternative. The cystine/glutamate exchanger xCT/SLC7A11 plays a key role in maintaining redox homeostasis in cancer cells. xCT is expressed on subsets of most solid tumors, including stem/progenitor and metastatic tumor cells. xCT inhibition yields a variety of anti-proliferative phenotypes including death by ferroptosis3. xCT knockout mice exhibit normal development and lifespan. Altogether, targeting xCT outside of CNS may potentially have widespread clinical utility in cancer treatment. Results: We present preclinical data demonstrating development of novel, first-in-class xCT-targeted biotherapeutics (AX68c, AX76c) using studies in TNBC cell lines in vitro and in xenograft. We find that these agents have highly favorable pharmacokinetics, with FcRn-dependent serum half-lives greatly exceeding those of known small molecule xCTi. In vitro, AX68c or AX76c reduces glutathione levels in TNBC cells in accord with inhibition of xCT function. Analysis of anti-proliferative effects in a panel of TNBC cell lines revealed particular potency against those of M/MSL phenotype (IC50s 0.3-1uM). We further tested the susceptibility of the mesenchymal phenotype to AX68c and found that EMT induction in epithelial cancer cells significantly increased sensitivity in a ferroptosis-dependent manner. In wildtype and immunodeficient mice, AX68c is well tolerated at steady-state serum concentrations that are 15-300 fold of its in vitro IC50s in tested TNBC cells. In orthotopic MDA-MB-231 xenografts, AX68c treatment significantly reduced tumor metastasis and sensitized primary tumors to cisplatin and paclitaxel. Bulk RNAseq and GSEA analysis on primary tumors revealed treatment associated alterations in key cancer signaling pathways such as TGFβ, INFγ and TNFα/NFκB. In MDA-MB-436 orthotopic xenografts, AX68c alone led to significantly improved survival primarily through abrogating metastasis. Combining AX68c and cisplatin further prolonged survival through concurrent suppression of metastasis and primary tumor growth. Conclusions: We conclude that our anti-xCT biologics may provide novel targeted therapies for M/MSL-subtype TNBC and metastatic tumors with a large mesenchymal component. 1. Viswanathan, et al., Nature, 2017; 2. Wang, et al., npj Precision Oncology, 2021; 3. Koppula, et al., Protein and Cell, 2021 (review). Citation Format: He Gong, Jun Li, Cyrus Becker, Luika Timmerman, Shaheen Sikandar, Yong Huang. Novel xCT-targeted biologics significantly suppress metastasis, improve survival, and sensitize primary tumors to chemotherapy in TNBC xenograft models [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 5962.

Abstract 6620: PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease

Abstract Chemotherapy, along with the PD1 inhibitor pembrolizumab, is the recommended standard of care for patients with primary triple negative breast cancer (TNBC). Nearly half of TNBC patients treated with standard neoadjuvant chemotherapy (NACT) have excellent responses. However, those patients with significant residual cancer burden (RCB) after NACT are at high risk of recurrence or metastatic relapse within two years. Due to the challenges posed by the inter- and intratumoral heterogeneity of TNBC, it is critical to develop appropriate models for predicting and overcoming therapy resistance. We established a collection of 92 orthotopic patient-derived xenograft (PDX) models of TNBC from 84 patients before, during, and after NACT while they were enrolled in the ARTEMIS trial (NCT02276443) at MD Anderson Cancer Center. Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), after a 3-month course of additional chemotherapy and/or different experimental therapies in cases of AC resistance (post-NACT), and from the metastatic lesions of two patients. The collection includes 12 longitudinal sets. Models were established from chemo-sensitive and -resistant tumors, but engraftment success was higher from those cancers that proved to be treatment resistant (RCB II/III). The PDX collection includes models encompassing a broad heterogeneity of chemotherapy responses, histologic features, and molecular TNBC subtypes. In addition, the majority of models develop spontaneous lung metastases. Whole exome sequencing demonstrated conservation of mutations between patient tumors and corresponding PDX models, with TP53 being the most commonly mutated gene. A similar comparison of patient and PDX transcriptomes revealed conservation of signaling pathway signatures, with those related to immune and stromal interactions being the most variable, as anticipated when adapting human tumors to growth in mice. The subclonal architecture of the tumors exhibited little overall change throughout NACT, suggesting that selection for resistant subclones is not likely to be a significant contributor to resistance. Tumor cells from the longitudinal PDX collections were subjected to high throughput drug susceptibility profiling to identify targetable vulnerabilities associated with resistance to NACT. We found that post-NACT tumors exhibited enhanced sensitivity to drugs targeting protein homeostasis pathways. Preclinical studies with pevonedistat validated the neddylation pathway as an effective target in chemoresistant TNBC. Citation Format: Amanda L. Rinkenbaugh, Yuan Qi, Reid T. Powell, Shirong Cai, Jiansu Shao, Faiza Baameur Hancock, Lei Guo, Xiaomei Zhang, Sabrina Jeter-Jones, Chunxiao Fu, Rebekah Gould, Jason B. White, Clifford Stephan, Gloria V. Echeverria, Peter J. Davies, Stacy Moulder, W. Fraser Symmans, Jeffrey T. Chang, Helen Piwnica-Worms. PDX models of TNBC established from pre- and post-therapy tumors identify vulnerabilities of resistant disease [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 6620.

CD24 negativity reprograms mitochondrial metabolism to PPARα and NF-κB-driven fatty acid β-oxidation in triple-negative breast cancer

CD24 is a well-characterized breast cancer (BC) stem cell (BCSC) marker. Primary breast tumor cells having CD24-negativity together with CD44-positivity is known to maintain high metastatic potential. However, the functional role of CD24 gene in triple-negative BC (TNBC), an aggressive subtype of BC, is not well understood. While the significance of CD24 in regulating immune pathways is well recognized in previous studies, the significance of CD24 low expression in onco-signaling and metabolic rewiring is largely unknown. Using CD24 knock-down and over-expression TNBC models, our in vitro and in vivo analysis suggest that CD24 is a tumor suppressor in metastatic TNBC. Comprehensive in silico gene expression analysis of breast tumors followed by lipidomic and metabolomic analyses of CD24-modulated cells revealed that CD24 negativity induces mitochondrial oxidative phosphorylation and reprograms TNBC metabolism toward the fatty acid beta-oxidation (FAO) pathway. CD24 silencing activates PPARα-mediated regulation of FAO in TNBC cells. Further analysis using reverse-phase protein array and its validation using CD24-modulated TNBC cells and xenograft models nominated CD24-NF-κB-CPT1A signaling pathway as the central regulatory mechanism of CD24-mediated FAO activity. Overall, our study proposes a novel role of CD24 in metabolic reprogramming that can open new avenues for the treatment strategies for patients with metastatic TNBC.

Development of a HPLC-MS/MS Method to Assess the Pharmacokinetics and Tumour Distribution of the Dimethylarginine Dimethylaminohydrolase 1 Inhibitors ZST316 and L-257 in a Xenograft Model of Triple-Negative Breast Cancer in Mice.

We describe the development and validation of an HPLC-MS/MS method to assess the pharmacokinetics and tumour distribution of ZST316, an arginine analogue with inhibitory activity towards dimethylarginine dimethylaminohydrolase 1 (DDAH1) and vasculogenic mimicry, and its active metabolite L-257 in a xenograft model of triple-negative breast cancer (TNBC). The method proved to be reproducible, precise, and highly accurate for the measurement of both compounds in plasma and tumour tissue following acute and chronic (five days) intraperitoneal administration of ZST316 (30 mg/Kg daily) in six-week-old severe combined immunodeficiency disease (SCID) mice inoculated with MDA-MB-231 TNBC cells. ZST316 was detected in tumour tissue and plasma after 1 h (6.47 and 9.01 μM, respectively) and 24 h (0.13 and 0.16 μM, respectively) following acute administration, without accumulation during chronic treatment. Similarly, the metabolite L-257 was found in tumour tissue and plasma after 1 h (15.06 and 8.72 μM, respectively) and 24 h (0.17 and 0.17 μM, respectively) following acute administration of ZST316, without accumulation during chronic treatment. The half-life after acute and chronic treatment ranged between 4.4-7.1 h (plasma) and 4.5-5.0 h (tumour) for ZST316, and 4.2-5.3 h (plasma) and 3.6-4.9 h (tumour) for L-257. The results of our study demonstrate the (a) capacity to accurately measure ZST316 and L-257 concentrations in plasma and tumour tissue in mice using the newly developed HPLC-MS/MS method, (b) rapid conversion of ZST316 into L-257, (c) good intra-tumour penetration of both compounds, and (d) lack of accumulation of both ZST316 and L-257 in plasma and tumour tissue during chronic administration. Compared to a previous method developed by our group to investigate ZST316 in plasma, the main advantages of the new method include a wider range of linearity which reduces the need for dilutions and the combined assessment of ZST316 and L-257 in plasma and tumour tissue which limits the required amount of matrix. The new HPLC-MS/MS method is useful to investigate the in vivo effects of ZST316 and L-257 on vasculogenic mimicry, tumour mass, and metastatic burden in xenograft models of TNBC.

Abstract C146: VIC-1911, a selective Aurora kinase A inhibitor, synergizes with sacituzumab govitecan in triple-negative breast cancer

Abstract BACKGROUND: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases and 30-40% of U.S. breast cancer deaths. Although current precision treatments improve TNBC patient outcomes, TNBC heterogeneity has led to therapeutic targeting challenges. Also, the resistance of TNBC to conventional treatment remains a significant clinical problem. Therefore, informed evaluation of new targets can improve the treatment of TNBC, representing an unmet need. Dysregulation of the cell cycle is a hallmark of cancer for unlimited proliferation. Abnormal mitosis and cell-cycle checkpoints are crucial in leading to aneuploidy and genetic instability. Aurora kinase A (AURKA), a serine/threonine kinase, regulates mitosis in the early stages (G2/M phase) by regulating centrosome maturation and disjunction, thereby establishing a bipolar mitotic spindle. VIC-1911 (previously known as TAS-119), a novel, orally active, highly selective inhibitor of AURKA with a low toxicity profile, suppresses the growth of various cancer cell lines in vitro and in vivo. As AURKA can modulate DNA damage response, we hypothesized that VIC-1911 combined with sacituzumab govitecan (SG), which has a topoisomerase 1 inhibitor as a payload, has a synergistic antitumor effect on TNBC. MATERIALS AND METHODS: To evaluate the short-term in vitro efficacy of VIC-1911 alone and combination with SG, a sulforhodamine B cell proliferation assay was performed using 10 TNBC cell lines. For long-term treatment conditions, a colony formation assay was conducted. Western blot and immunofluorescent analyses were conducted to confirm treatment-mediated DNA damage and apoptosis. Human TNBC cell line–derived mammary fat pad xenograft models were used to evaluate the antitumor effect of VIC-1911 and SG. RESULTS: In vitro proliferation, data demonstrated that single-agent VIC-1911 had a nanomolar to micromolar range of half-maximal inhibitory concentrations (2.69 nM to 10.6 mM) in the tested TNBC cell lines. The half-maximal inhibitory concentration was correlated with c-Myc expression (R2=0.4876, p=0.0247). Exposure to the combination of VIC-1911 and SG led to significant death of VIC-1911–sensitive TNBC cell lines (p<0.05). Western blot and immunofluorescent analysis revealed that VIC-1911 and SG combination caused TNBC cell death by inducing expression of phosphorylated histone H2AX (DNA damage marker) and cleaved poly (ADP-ribose) polymerase (apoptosis marker). Furthermore, the combination of VIC-1911 and SG had significantly greater antitumor efficacy than did either agent alone in SUM149 TNBC xenograft models (tumor growth inhibition rate (TGI): VIC-1911, 61.4%; SG, 68.3%; combination, 82.9%; p<0.01) and HCC1806 TNBC xenograft models (TGI: VIC-1911, 49.3%; SG, 66.3%; combination, 85.9%; p<0.01). CONCLUSION: The AURKA inhibitor VIC-1911 has a synergistic antitumor effect with SG by inducing DNA damage in TNBC cells. PDX and additional mechanistic studies are planned to further evaluate VIC-1911 and SG as novel combination therapy for TNBC. Citation Format: Jangsoon Lee, YoungJin Gi, Thomas Myers, Linda Paradiso, Debu Tripathy, Naoto T. Ueno. VIC-1911, a selective Aurora kinase A inhibitor, synergizes with sacituzumab govitecan in triple-negative breast cancer [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 C146.

Thrombin induces ferroptosis in triple-negative breast cancer through the cPLA2α/ACSL4 signaling pathway

Ferroptosis is a recently identified form of regulated cell death that plays a crucial role in tumor suppression. In this study, we found that F2 (the gene encoding thrombin) was strongly upregulated in breast cancer (BRCA, TCGA Study Abbreviations) compared with normal samples and that lower F2 levels were associated with poorer prognosis in breast cancer patients. Thrombin induces ferroptosis in triple-negative breast cancer (TNBC) cells by activation of cytosolic phospholipase A2α (cPLA2α) activity to increase the release of arachidonic acid (AA). TNBC in all breast cancer subtypes exhibited the highest levels of PLA2G4A (the gene encoding cPLA2α) and Acsl4, and inhibition of cPLA2α and its downstream enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) reversed thrombin toxicity. In a mouse xenograft model of TNBC, thrombin treatment suppressed breast cancer growth which can be inhibited by ferroptosis inhibitor Liproxstatin-1 (Lip-1). Our study underscores the potential of the thrombin-ACSL4 axis as a promising therapeutic target for the treatment of TNBC.

Targeting UBR5 inhibits postsurgical breast cancer lung metastases by inducing CDC73 and p53 mediated apoptosis.

UBR5 is a HECT domain E3 ubiquitin ligase that is frequently amplified in breast, ovarian and prostate cancers. Heightened UBR5 expression plays a profound role in tumor growth through immune-dependent mechanisms; however, its mode of action in driving tumor metastasis has not been definitively delineated. Herein, we used a tetracycline (Tet)-inducible RNAi-mediated expression silencing cell system to investigate how UBR5 enables postsurgical mammary tumor metastatic growth in mouse lungs without the continuous influence of the primary lesion. In vitro, Ubr5 knockdown induces morphological and molecular changes characteristic of epithelial-mesenchymal transition (EMT). In vivo, UBR5 promotes lung metastasis in an E3 ubiquitin ligase-dependent manner. Moreover, doxycycline-induced UBR5 expression knockdown in metastatic cells in the lungs, following removing the primary tumors, resulted in increased apoptosis, decreased proliferation and prolonged survival, whereas silencing the expression of cell division cycle 73 (CDC73), a tumor suppressor and E3 ligase substrate of UBR5, reversed these effects. Transcriptome analyses revealed a prominent role of the p53 pathway in dovitinib-induced apoptosis of tumor cells differentially regulated by UBR5 and CDC73. In human triple-negative breast cancer (TNBC) patient specimens, a strong inverse correlation was observed between UBR5 and CDC73 protein levels, with reduced CDC73 expression at metastatic sites compared to primary lesions. Furthermore, a xenograft model of human TNBC recapitulated the metastatic properties and characteristics of the unique UBR5-CDC73 functional antagonism. This study reveals the novel and critical roles and intricate relationships of UBR5, CDC73 and p53 in postsurgical breast cancer metastasis and indicates the potential of targeting this pathway in cancer therapy.

VNLG-152R and its deuterated analogs potently inhibit/repress triple/quadruple negative breast cancer of diverse racial origins in vitro and in vivo by upregulating E3 Ligase Synoviolin 1 (SYVN1) and inducing proteasomal degradation of MNK1/2.

Triple-negative breast cancer (TNBC) and its recently identified subtype, quadruple negative breast cancer (QNBC), collectively account for approximately 13% of reported breast cancer cases in the United States. These aggressive forms of breast cancer are associated with poor prognoses, limited treatment options, and lower overall survival rates. In previous studies, our research demonstrated that VNLG-152R exhibits inhibitory effects on TNBC cells both in vitro and in vivo and the deuterated analogs were more potent inhibitors of TNBC cells in vitro. Building upon these findings, our current study delves into the molecular mechanisms underlying this inhibitory action. Through transcriptome and proteome analyses, we discovered that VNLG-152R upregulates the expression of E3 ligase Synoviolin 1 (SYVN1), also called 3-hydroxy-3-methylglutaryl reductase degradation (HRD1) in TNBC cells. Moreover, we provide genetic and pharmacological evidence to demonstrate that SYVN1 mediates the ubiquitination and subsequent proteasomal degradation of MNK1/2, the only known kinases responsible for phosphorylating eIF4E. Phosphorylation of eIF4E being a rate-limiting step in the formation of the eIF4F translation initiation complex, the degradation of MNK1/2 by VNLG-152R and its analogs impedes dysregulated translation in TNBC cells, resulting in the inhibition of tumor growth. Importantly, our findings were validated in vivo using TNBC xenograft models derived from MDA-MB-231, MDA-MB-468, and MDA-MB-453 cell lines, representing different racial origins and genetic backgrounds. These xenograft models, which encompass TNBCs with varying androgen receptor (AR) expression levels, were effectively inhibited by oral administration of VNLG-152R and its deuterated analogs in NRG mice. Importantly, in direct comparison, our compounds are more effective than enzalutamide and docetaxel in achieving tumor growth inhibition/repression in the AR+ MDA-MD-453 xenograft model in mice. Collectively, our study sheds light on the involvement of SYVN1 E3 ligase in the VNLG-152R-induced degradation of MNK1/2 and the therapeutic potential of VNLG-152R and its more potent deuterated analogs as promising agents for the treatment of TNBC across diverse patient populations.

Synthesis and mechanism of action of new purine derivatives against triple negative breast cancer

Triple negative breast cancer (TNBC) is considered to be the most difficult subtype of breast cancer to treat because of its extremely prone to metastasis and the lack of targeted therapy drugs. New purine derivatives were synthesized and evaluated in a series of kinases and cell lines. The most active compounds 3g and 3j were selected based on their antiproliferative activities, then their pharmaceutical activity and mechanism in MDA-MB-231 cells were analyzed. The results in vitro indicated that compounds 3g and 3j can induce MDA-MB-231 cells apoptosis, and inhibit its migration and angiogenesis through influencing protein expression such as Bcl-2, Bax, Bcl-xl, P38, MMP2, MMP9, AKT and EGFR. In vivo results indicate that compounds 3g and 3j can inhibit tumor growth and metastasis and reduce the expression of Ki67 and CD31 protein in TNBC xenograft models. These findings not only broaden our understanding of the anti-TNBC effects and mechanisms of compounds 3g and 3j, but also provide new ideas and reference directions for the treatment of TNBC.

Targeting FAK/PYK2 with SJP1602 for Anti-Tumor Activity in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) presents significant challenges due to its aggressive nature and limited treatment options. Focal adhesion kinase (FAK) has emerged as a critical factor promoting tumor growth and metastasis in TNBC. Despite encouraging results from preclinical and early clinical trials with various FAK inhibitors, none have yet achieved clinical success in TNBC treatment. This study investigates the therapeutic potential of a novel dual inhibitor of FAK and PYK2, named SJP1602, for TNBC. In vitro experiments demonstrate that SJP1602 effectively inhibits FAK and PYK2 activities, showing potent effects on both kinases. SJP1602 shows concentration-dependent inhibition of cell growth, migration, invasion, and 3D spheroid formation in TNBC cell lines, surpassing the efficacy of other FAK inhibitors. Pharmacokinetic studies in rats indicate favorable bioavailability and sustained plasma concentrations of SJP1602, supporting its potential as a therapeutic agent. Furthermore, in TNBC xenograft models, SJP1602 exhibits significant dose-dependent inhibition of tumor growth. These promising results emphasize the potential of SJP1602 as a potent dual inhibitor of FAK and PYK2, deserving further investigation in clinical trials for TNBC treatment.

An autophagy-inducing stapled peptide induces mitochondria dysfunction and triggers autotic cell death in triple-negative breast cancer

Autophagy is a lysosome-dependent bulk degradation process essential for cell viability but excessive autophagy leads to a unique form of cell death termed autosis. Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with notable defect in its autophagy process. In previous studies, we developed stapled peptides that specifically targeted the essential autophagy protein Beclin 1 to induce autophagy and promote endolysosomal trafficking. Here we show that one lead peptide Tat-SP4 induced mild increase of autophagy in TNBC cells but showed potent anti-proliferative effect that could not be rescued by inhibitors of programmed cell death pathways. The cell death induced by Tat-SP4 showed typical features of autosis including sustained adherence to the substrate surface, rupture of plasma membrane and effective rescue by digoxin, a cardioglycoside that blocks the Na+/K+ ATPase. Tat-SP4 also induced prominent mitochondria dysfunction including loss of mitochondria membrane potential, elevated mitochondria reactive oxygen species and reduced oxidative phosphorylation. The anti-proliferative effect of Tat-SP4 was confirmed in a TNBC xenograft model. Our study uncovers three notable aspects of autosis. Firstly, autosis can be triggered by moderate increase in autophagy if such increase exceeds the endogenous capacity of the host cells. Secondly, mitochondria may play an essential role in autosis with dysregulated autophagy leading to mitochondria dysfunction to trigger autosis. Lastly, intrinsic autophagy deficiency and quiescent mitochondria bioenergetic profile likely render TNBC cells particularly susceptible to autosis. Our designed peptides like Tat-SP4 may serve as potential therapeutic candidates against TNBC by targeting this vulnerability.

Targeting of microRNA-22 Suppresses Tumor Spread in a Mouse Model of Triple-Negative Breast Cancer.

microRNA-22 (miR-22) is an oncogenic miRNA whose up-regulation promotes epithelial-mesenchymal transition (EMT), tumor invasion, and metastasis in hormone-responsive breast cancer. Here we show that miR-22 plays a key role in triple negative breast cancer (TNBC) by promoting EMT and aggressiveness in 2D and 3D cell models and a mouse xenograft model of human TNBC, respectively. Furthermore, we report that miR-22 inhibition using an LNA-modified antimiR-22 compound is effective in reducing EMT both in vitro and in vivo. Importantly, pharmacologic inhibition of miR-22 suppressed metastatic spread and markedly prolonged survival in mouse xenograft models of metastatic TNBC highlighting the potential of miR-22 silencing as a new therapeutic strategy for the treatment of TNBC.

Abstract 6215: Peposertib, a DNA-PK inhibitor, enhances the antitumor efficacy of anthracyclines in triple-negative breast cancer models in vitro and in vivo

Abstract Background: Triple-negative breast cancer (TNBC) is the most lethal breast cancer subtype, exhibiting poor response rates toward current chemotherapy regimens and lacking additional effective treatment options. Approximately 30% of patients with TNBC respond well to anthracycline/taxane-based standard-of-care chemotherapy regimens. However, remaining patients demonstrate limited improvements in clinical outcomes, highlighting the critical need for effective strategies to improve anthracycline/taxane-based chemotherapy (1, 2). DNA-dependent protein kinase (DNA-PK), a member of the phosphoinositide 3-kinase-related kinase protein family, promotes nonhomologous end joining (NHEJ) as part of the DNA damage response. Peposertib is a potent, selective, and orally bioavailable DNA-PK inhibitor that strongly potentiates the antitumor effects of ionizing radiation and DNA double-strand break-inducing agents, including anthracyclines, in preclinical models. Here we report the synergistic antitumor effects of peposertib combined with topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC models in vitro and in vivo. Methodology: Combinations of peposertib with TOPO II inhibitors were evaluated in TNBC cell lines by viability assays, immunoblotting, and flow cytometry. Gene expression analysis was performed using the nCounter PanCancer pathways panel and results were confirmed by quantitative polymerase chain reaction (qPCR). In vivo efficacy was assessed in cell-line derived and patient-derived xenograft models. Results: When administered in combination with doxorubicin, epirubicin, and etoposide, peposertib exhibited synergistic antiproliferative activity in TNBC cell lines in vitro. The downstream analysis of pharmacodynamic biomarkers revealed induction of NHEJ mediated repair upon TOPO II inhibitor treatment and provided mechanistic insights into the synergistic antitumor effects of peposertib combined with TOPO II inhibitors. Furthermore, this combination treatment induced ataxia telangiectasia mutated (ATM)-dependent compensatory signaling and inflammatory responses, potentially creating a proinflammatory tumor microenvironment. To evaluate whether peposertib enhanced the antitumor effects of TOPO II inhibitors in vivo, we established a well-tolerated preclinical treatment schedule for the combined use of peposertib and pegylated liposomal doxorubicin, which was capable of achieving tumor regression in patient- and cell line-derived xenograft models of TNBC. Conclusion: Our findings suggest that cotreatment with the DNA-PK inhibitor peposertib can enhance the efficacy of anthracycline/TOPO II-based chemotherapies. This work was supported by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945). 1) Davison C et al. NPJ Breast Cancer. 2021;7(1):38. 2) Bianchini G et al. Nat Rev Clin Oncol. 2016;13(11):674-690. Citation Format: Steffie Revia, Christian Sirrenberg, Antonia Schach, Astrid Zimmermann, Frank T. Zenke, Joachim Albers. Peposertib, a DNA-PK inhibitor, enhances the antitumor efficacy of anthracyclines in triple-negative breast cancer models in vitro and in vivo. [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 6215.

ImmunoPET Imaging Identifies the Optimal Timepoint for Combination Therapy in Xenograft Models of Triple-Negative Breast Cancer.

(1) Purpose: The glycoprotein non-metastatic melanoma B (gpNMB) is a type 1 transmembrane protein that is overexpressed in numerous cancers, including triple-negative breast cancer (TNBC). Its overexpression is associated with lower overall survival of patients with TNBC. Tyrosine kinase inhibitors such as dasatinib can upregulate gpNMB expression, which has the potential to enhance therapeutic targeting with anti-gpNMB antibody drug conjugates such as glembatumumab vedotin (CDX-011). Our primary aim is to quantify the degree and identify the timeframe of gpNMB upregulation in xenograft models of TNBC after treatment with the Src tyrosine kinase inhibitor, dasatinib, by longitudinal positron emission tomography (PET) imaging with the 89Zr-labeled anti-gpNMB antibody ([89Zr]Zr-DFO-CR011). The goal is to identify the timepoint at which to administer CDX-011 after treatment with dasatinib to enhance therapeutic efficacy using noninvasive imaging. (2) Methods: First, TNBC cell lines that either express gpNMB (MDA-MB-468) or do not express gpNMB (MDA-MB-231) were treated with 2 μM of dasatinib in vitro for 48 h, followed by Western blot analysis of cell lysates to determine differences in gpNMB expression. MDA-MB-468 xenografted mice were also treated with 10 mg/kg of dasatinib every other day for 21 days. Subgroups of mice were euthanized at 0-, 7-, 14-, and 21-days post treatment, and tumors were harvested for Western blot analysis of tumor cell lysates for gpNMB expression. In a different cohort of MDA-MB-468 xenograft models, longitudinal PET imaging with [89Zr]Zr-DFO-CR011 was performed before treatment at 0 (baseline) and at 14 and 28 days after treatment with (1) dasatinib alone (2) CDX-011 (10 mg/kg) alone, or (3) sequential treatment of dasatinib for 14 days then CDX-011 to determine changes in gpNMB expression in vivo relative to baseline. As a gpNMB-negative control, MDA-MB-231 xenograft models were imaged 21 days after treatment with dasatinib, combination of CDX-011 and dasatinib, and vehicle control. (3) Results: Western blot analysis of MDA-MB-468 cell and tumor lysates showed that dasatinib increased expression of gpNMB in vitro and in vivo at 14 days post treatment initiation. In PET imaging studies of different cohorts of MDA-MB-468 xenografted mice, [89Zr]Zr-DFO-CR011 uptake in tumors (SUVmean = 3.2 ± 0.3) was greatest at 14 days after treatment initiation with dasatinib (SUVmean = 4.9 ± 0.6) or combination of dasatinib and CDX-011 (SUVmean= 4.6 ± 0.2) compared with that at baseline (SUVmean = 3.2 ± 0.3). The highest tumor regression after treatment was observed in the combination-treated group with a percent change in tumor volume relative to baseline (%CTV) of -54 ± 13 compared with the vehicle control-treated group (%CTV = +102 ± 27), CDX-011 group (%CTV = -25 ± 9.8), and dasatinib group (%CTV = -23 ± 11). In contrast, the PET imaging of MDA-MB-231 xenografted mice indicated no significant difference in the tumor uptake of [89Zr]Zr-DFO-CR011 between treated (dasatinib alone or in combination with CDX-011) and vehicle-control groups. (4) Conclusions: Dasatinib upregulated gpNMB expression in gpNMB-positive MDA-MB-468 xenografted tumors at 14 days post treatment initiation, which can be quantified by PET imaging with [89Zr]Zr-DFO-CR011. Furthermore, combination therapy with dasatinib and CDX-011 appears to be a promising therapeutic strategy for TNBC and warrants further investigation.

Abstract P4-08-18: Engineered Toxin Bodies Specific for TROP2 Positive Cancers

Abstract Engineered Toxin Bodies Specific for TROP2 Positive Cancers Authors: Garrett L. Cornelison, Ileana Pedraza, Kendra Garrison, Elizabeth M. Kapeel, Channing Pletka, Abdul Khan, Jessica Momb, Rebecca Martin, Adam Bartos, Joseph D. Dekker, Jay Zhao, John Majercak, Garrett L. Robinson Molecular Templates, Austin, TX Lanier Biotherapeutics, Bogart, GA Molecular Templates produces next generation immunotoxins called Engineered toxin bodies (ETBs). ETBs are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forced internalization, undergoing retrograde translocation to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes resulting in the inhibition of protein synthesis and induction of apoptosis. In addition, Molecular Templates has expanded the ETB platform to include Antigen Seeding Technology (AST) to generate ETBs with the ability to deliver foreign protein antigen to targeted populations of tumor cells. This mechanism of action allows for the intracellular processing of antigen and subsequent surface MHC-I presentation required for activation of a re-directed T lymphocyte response and the capacity to restore a functional immune clearance program against the tumor. Three ETBs are in clinical development (MT-5111 targeting HER2, MT-0169 targeting CD38, and AST enabled MT-6402 targeting PD-L1). The novel mechanisms of action have potential benefit in different indications including in the relapsed setting, when disease has progressed after chemotherapies and other targeted therapies, and additionally may be able to combine with standard of care. ETBs are being developed that target other cell surface receptors expressed on solid tumors including tumor-associated calcium signal transducer 2 (TROP2). TROP2 is a clinically validated target in metastatic triple-negative breast cancer (mTNBC) and other cancers such as metastatic urothelial carcinoma (mUC) using antibody drug conjugate (ADC) therapies such as sacituzumab govitecan (Trodelvy®). In vitro, ETBs targeting TROP2 specifically and directly kill tumor cells expressing TROP2 with picomolar activity. ETBs can bind to TROP2 in the presence of the Trodelvy parent monoclonal antibody, sacituzumab, and ETBs retain potency on TROP2 positive cell lines in the presence of clinically relevant concentrations of sacituzumab. AST enabled Trop2 targeted ETBs retain direct cell killing activity and can deliver multiple viral antigens to induce cytokine secretion and T-cell mediated killing in a co-culture assay of TROP2 target cells with antigen matched HLA type and antigen specific T-cells. In vivo, TROP2 targeted ETBs demonstrate good tolerability in a murine HCC1806 triple-negative breast cancer xenograft model and significantly reduce tumor burden relative to vehicle control. These pre-clinical in vitro and in vivo data suggest AST enabled Trop2 targeted ETBs have the potential to deplete Trop2 positive malignancies through multiple unique mechanisms of action. Citation Format: Garrett L. Cornelison, Ileana Pedraza, Kendra Garrison, Elizabeth M. Kapeel, Channing Pletka, Abdul Khan, Jessica Momb, Rebecca Martin, Adam Bartos, Joseph D. Dekker, Jay Zhao, John Majercak, Garrett L. Robinson. Engineered Toxin Bodies Specific for TROP2 Positive Cancers [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 P4-08-18.

Abstract P6-10-08: Evaluation of potent novel second-generation MELK-selective inhibitor in aggressive breast cancers

Abstract Background Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are aggressive breast cancer subtypes with poor clinical outcomes due to the lack of well-validated and actionable targets and the onset of chemo-resistant metastasis. TNBC and IBC account for 15-20% and 2-4% of all breast cancer diagnoses but result in 30% and 8%–10% of breast cancer-related deaths, respectively. MELK (maternal embryonic leucine zipper kinase) is a potential therapeutic target in both TNBC and triple-negative IBC (TN-IBC). We have shown that 1) MELK expression is higher in basal tumors (mostly TNBC) and in IBC than in non-IBC, 2) high MELK expression is an independent prognostic factor for poor overall survival (P=0.0002) and poor distant metastasis-free survival (P=0.008) in breast cancer, and 3) MELK knockout leads to significantly lower metastatic burden and prolonged survival in a xenograft model of TNBC (unpublished data). MELK inhibitor (MELKi) OTS167 is in clinical trials but it lacks specificity and cross-reacts with other essential kinases. It is critical to discover novel, selective inhibitors with good bioavailability that target MELK and minimize adverse effects. We have developed a second-generation small-molecule MELK inhibitor 30e that is a highly potent and slow-binding ATP-competitive inhibitor of MELK. 30e exhibits potent cellular inhibition of MELK (IC50< 10 nM), as assessed using a live cell NanoBRET assay, and is highly selective as assessed by live-cell KiNativ profiling experiments in MDA-MB-231 TNBC cells. Methods We screened a panel of TNBC and IBC cell lines for MELK expression by western blot and qPCR analysis. We assessed the effects of MELK inhibition with the 30e on migration and invasion. The role of MELK in regulating cancer stem cell phenotypes was assessed using the mammosphere assay and flow cytometry (CD44+/CD24-/low surface marker). MELK’s protumorigenic role was determined in vitro using a soft agar assay, and the anti-tumor activity of 30e was assessed in vivo using a TN-IBC orthotopic xenograft mouse model. Results We found that TNBC had high mRNA levels of MELK (62%) compared to non-TNBC cell lines (25%), which correlated with MELK protein levels. 30e inhibited cell viability in TNBC and IBC cells in a dose-dependent manner and with IC50s ranging from 0.45 to 1.76 μM (P≤0.05). However, no significant effect was observed when normal MCF-10A breast cells were treated with 30e (IC50>20 μM). 30e also significantly reduced colony formation ability. Further, we observed that 30e reduced mammosphere formation efficiency and the CD44+/CD24-/low subpopulation in both TNBC and IBC cells in a dose-dependent manner (P≤0.05), suggesting the potential of 30e to inhibit the stem cell population in vitro. The soft agar assay also showed a significant decrease in colony formation in the TNBC and IBC cells after treatment with 30e, an indirect indicator of in vivo tumorigenicity. To assess the efficacy of 30e treatment on tumor growth, we evaluated this inhibitor as a single agent in a SUM-149 (TN-IBC) orthotopic mouse model. The mice were treated with intraperitoneal injections daily with 3 doses (2.5, 5, and 10 mg/kg) of 30e. Our data show that 30e suppressed tumor growth in a dose-dependent manner (P< 0.007). Conclusions and Future Directions Our data demonstrate that a novel, potent and specific MELK inhibitor inhibits tumor growth by suppressing the cancer stem cell phenotype. MELK is a promising target for aggressive cancers such as TNBC and IBC. We are evaluating how MELK inhibition modulates the tumor microenvironment, which is a critical component of breast cancer response to treatment. We performed a cytokine antibody array and are validating the top targets. Further, we will evaluate 30e in combination with standard-of-care treatment for toxicity and inhibition of metastasis. Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Hector Gonzalez, Emilly S. Villodre, Bisrat Debeb, Debu Tripathy, Geoffrey Bartholomeusz, Juhyeon Lee, Kevin Dalby, Chandra Bartholomeusz. Evaluation of potent novel second-generation MELK-selective inhibitor in aggressive breast cancers [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 P6-10-08.

Abstract P6-11-15: Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which limited targeted therapies are available. Therefore, conventional chemotherapy remains the backbone of standard neoadjuvant treatment (NACT) for TNBC patients. Unfortunately, ~45% of patients will have substantial residual tumor burden post neoadjuvant chemotherapy, leading to poor prognoses (PMID: 28135148). Recently, it has been demonstrated that mitochondrial oxidative phosphorylation (oxphos) is upregulated and is a therapeutic vulnerability in chemoresistant TNBC (PMID: 30996079; Baek et al., BioRxiv doi.org/10.1101/2022.02.25.481996). However, mechanisms driving increased oxphos in chemoresistant TNBC are not understood. Upregulated fatty acid (FA) metabolism is a common adaptation in tumors, providing an energy source through fatty acid β-oxidation (FAO), and promoting lipid accumulation after fatty acid synthesis (FAS) when energy needs are met. Chemotherapy can induce oxidative stress through the generation of reactive oxygen species. Cancer cells adapt to these damaging molecules by increasing de novo lipogenesis, resulting in the accumulation of lipid droplets (LDs) in the cytosol (PMID: 32782526, 20876798). We hypothesize that TNBC cells metabolically adapt to the stress of NACT by upregulating lipid metabolic pathways, providing highly energetic molecules that can be utilized to drive oxphos in chemoresistant TNBC. Methods: Using orthotopic patient-derived xenograft (PDX) models of TNBC (PIM001-P, PMID: 30996079, HCI-010, PMID: 22019887; WHIM14, PMID:24055055), we are measuring protein levels of fatty acid synthase (FASN) in vehicle tumors vs residual tumors surviving treatment with the standard front-line neoadjuvant chemotherapy regimens (Adriamycin plus cyclophosphamide (AC), docetaxel, carboplatin, or docetaxel+carboplatin) using immunohistochemistry (IHC). Vectra 3 microscopy (Akoya) is being used to quantify tumor cell-specific staining. We complemented our IHC analysis with reverse-phase protein array (RPPA). To assess LD accumulation in residual PDX tumors, we conducted transmission electron microscopy (TEM). To complement these PDX studies, we modeled the residual tumor metabolic state in cultured human TNBC cells. Following treatment with the IC50 of standard chemotherapeutic agents (AC, carboplatin, paclitaxel, docetaxel), we assessed oxphos by measuring oxygen consumption rate (OCR) using a Seahorse Bioanalyzer (Agilent). Further, we tested LD accumulation using LipidTOX staining. In ongoing studies, we are measuring incorporation of 13C palmitate into the tricarboxylic acid cycle (TCA) prior to and following chemotherapy treatments to assess if lipids fuel mitochondrial metabolism in residual TNBC cells. Results/Discussion: IHC in the PIM001-P PDX model after in vivo AC treatment revealed increased levels of FASN in post-AC residual tumors compared to the treatment-naive tumors. Further, key proteins involved in fatty acid synthesis, FASN and Acetyl-CoA carboxylase, were significantly increased in residual PIM001-P cells that survived AC compared to vehicle by RPPA. TEM analysis of the HCI-010 PDX revealed significantly more LDs in carboplatin-treated tumors compared to vehicle. This finding was supported by increased LDs observed in TNBC cell lines treated with NACT compared to vehicle in our LipidTOX analyses. Taken together, these data indicate that NACT induces increased expression of key lipid metabolism proteins and accumulation of cytosolic LDs. Our future experiments will reveal if chemoresistant TNBC cells preferentially utilize and incorporate lipids into the tricarboxylic acid cycle, in turn driving oxphos. These data have the potential to provide rationale for the incorporation of FAO/LD inhibitors in sequential combinations with conventional chemotherapies to more effectively kill TNBC cells that are chemo-refractory. Citation Format: Katherine E. Pendleton, Mokryun L. Baek, Junegoo Lee, Lin Tan, Hannah L. Johnson, Lacey E. Dobrolecki, James P. Barrish, Michael T. Lewis, Philip L. Lorenzi, Fabio Stossi, Gloria V. Echeverria. Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment [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 P6-11-15.