Chemoresistance In Triple-negative Breast Cancer Research Articles

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.

Abstract P6-11-10: Investigating the role of mitochondrial protein translation in the metabolic adaptation of chemoresistant triple negative breast cancer

Abstract BACKGROUND: Nearly 50% of patients with triple negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NACT) retain residual tumors resulting in high rates of metastatic relapse and poor overall survival. Residual tumors surviving NACT (Adriamycin plus cyclophosphamide; AC) were found to undergo a metabolic transition to heightened mitochondrial oxidative phosphorylation (oxphos; PMID: 30996079). Pharmacologic inhibition of mitochondrial electron transport chain (ETC) complex I with IACS-010759 (PMID: 29892070) had enhanced efficacy in residual, rather than treatment-naïve, tumors of orthotopic patient-derived xenograft (PDX) models. Our analyses of mitochondrial structure and function in human TNBC cell lines revealed differing adaptations in residual cells surviving treatment with conventional NACT agents. While DNA-damaging chemotherapies (e.g.Adriamycin, carboplatin) induced mitochondrial fusion and oxphos, taxanes (e.g.paclitaxel, docetaxel) induced mitochondrial fragmentation and reduced oxphos (Baek et al., Biorxiv Doi 10.1101/2022.02.25.481996). The mechanistic basis of these mitochondrial adaptations is not yet understood. The mitochondrial ETC consists of 92 proteins, 13 of which are encoded in the mitochondrial genome (mtDNA) and translated by the mitoribosome, while the remaining are encoded by the nuclear genome (nDNA), translated by the cytoribosome, and inserted into the inner mitochondrial membrane by accessory proteins, namely Oxidase (Cytochrome C) Assembly 1-Like (OXA1L). Disruption of OXA1L in mammalian cells has been shown to affect the levels and activity of ETC complexes I, III, IV, and V, and thus diminish oxphos. We aim to determine whether mitochondrial translation and OXA1L activity represent therapeutic vulnerabilities to overcome pro-survival metabolic adaptations in chemoresistant TNBC thereby augmenting treatment response. METHODS: Weare evaluating the effects of conventional TNBC chemotherapies singly, and in standard combinations, on mitochondrial translation and ETC formation in human TNBC cells and PDX models(PIM001-P, WHIM14, BCM15116) using metabolomic and proteomic profiling. To perturb these processes genetically, we knocked down (KD) OXA1Lwith siRNA. We are complementing these studies pharmacologically using conventional antibiotics, such as tigecycline, as previous studies showed they inhibit mitochondrial translation in breast and other cancers (PMID: 25625193). These studies will reveal whether OXA1L and mitochondrial translation are required for metabolic adaption and chemotherapy resistance of residual TNBC cells. PDX preclinical trials based on our published residual tumor testing schema (PMID: 30996079), will reveal whether the sequential combination of NACT followed by tigecycline can effectively perturb residual tumor relapse. RESULTS: Proteomic profiling of longitudinally harvested PDX tumors demonstrates substantial disruption of mitochondria-and nuclear-encoded ETC components in residual vs. treatment-naïve tumors. Interestingly, these patterns are distinct between different chemotherapy treatments, with an increase of ETC components in carboplatin-treated residual tumors compared to a decrease in docetaxel-treated residual tumors. Western blot analyses of human cell lines show OXA1LKD perturbs levels of both nuclear-and mitochondria-encoded ETC components. Preliminary findings suggest OXA1LKD increases sensitivity to chemotherapies in human TNBC cell lines. Finally, tigecycline effectively inhibits TNBC cell growth. We next will evaluate whether residual cells not killed by conventional chemotherapies have enhanced tigecycline susceptibility. CONCLUSION: These data suggest targeting mitochondrial translation may be a promising approach to overcome pro-survival metabolic adaptations in residual TNBC cells not killed by conventional chemotherapies. Citation Format: Mariah J. Berner, Lily Baek, Junegoo Lee, Philip L. Lorenzi, Mei Leng, Alexander B. Saltzman, Anna Malovannaya, Lacey E. Dobrolecki, Christina Sallas, Michael T. Lewis, Gloria V. Echeverria. Investigating the role of mitochondrial protein translation in the metabolic adaptation of chemoresistant triple negative breast cancer [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 P6-11-10.

Abstract P6-10-18: Developing novel lysyl oxidase (LOX) inhibitors to overcome chemotherapy resistance in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. It accounts for ~15% of all breast cancer patients yet is responsible for 30% of breast cancer deaths. TNBC is treated primarily with conventional chemotherapy; however, resistance to therapy is common, leading to high mortality rates. Importantly, the benefit of current therapeutic strategies used in chemoresistant TNBC, i.e., immunotherapy and antibody-drug conjugates, is confined to only a fraction of patients, and survival benefit is limited. Therefore, there is an urgent need to identify novel and effective treatment strategies to overcome chemoresistance. Recently, we identified hypoxia-induced ECM re-modeler, lysyl oxidase (LOX), a member of LOX family, as a key mediator of chemoresistance in TNBC. However, currently available LOX inhibitors are either non-selective and/or show toxicity. Here, we performed a high-throughput cell-based LOX activity screen (HTS) with more than 5,000 molecules selected from a diversified compound library and identified the bi-thiazole derivatives as novel potent LOX inhibitors. Our structure activity relationship (SAR) analysis resulted in two lead compounds 6403, a relatively LOX-specific inhibitor, and 6415, a more LOX/LOXL2 inhibitor. Both compounds reduced collagen cross-linking (canonical function of LOX) and led to chemosensitization in TNBC cell lines and in chemoresistant TNBC PDX organoids. In addition, 6403 and 6415 reduced the TGF-beta induced fibrosis and inhibited migration capacity of the breast cancer cell lines. Importantly, 6403 showed excellent pharmacokinetic profile and did not lead to any observable toxicity in mice. Notably, 6403 overcame doxorubicin resistance in LOX-expressing 4T1 syngeneic model with no apparent toxicity. Furthermore, our novel LOX and LOX/LOXL2 dual inhibitors show superior inhibition of LOX activity compared to the recently developed and clinically tested LOX family or LOXL2/LOXL3 inhibitors. Overall, we identified novel potent LOX inhibitors with no observable toxicity for further preclinical development and future clinical testing to overcome chemoresistance in TNBC. Citation Format: Metin Cetin, Ozge Saatci, Ozge Akbulut, Chad Beneker, Abdol-Hossein Rezaeian, Mikhail Chernov, Campbell McInnes, Ozgur Sahin. Developing novel lysyl oxidase (LOX) inhibitors to overcome chemotherapy resistance in triple negative breast cancer [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 P6-10-18.

Abstract P2-22-02: The role of circular RNAs in triple-negative breast cancer and chemotherapy resistance

Abstract Background: Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death among women worldwide. Triple-negative breast cancer (TNBC) is an aggressive subtype representing 15-20% of all breast cancers. TNBC often ends with a poor clinical outcome due to high histological grade and recurrence rates with few treatment options. Chemotherapy remains the standard of care for TNBC treatment, but unfortunately, patients frequently develop resistance, and alternative treatment strategies for the chemoresistant disease remain a major unmet need. circRNAs are newly identified non-coding RNA molecules with covalently closed circular structures. Recently, an increasing number of studies have indicated that circRNAs play crucial roles in regulating tumor development and chemoresistance. However, the role of circRNAs in the process of TNBC chemotherapy resistance is not yet fully clear. Materials and methods: To study the molecular functions of circRNAs in TNBC chemoresistance, identify alternative biomarkers for chemotherapy-resistant disease, and elucidate novel therapeutic targets, doxorubicin-resistant (Doxo-R) and paclitaxel-resistant (Taxol-R) cell lines were generated using TNBC models. Results: Doxo-R and Taxol-R cells exhibited a 3- and 12-fold decrease in sensitivity to their respective chemotherapeutic agents. We identified a large spectrum of gene expression changes that were acquired in Doxo-R and Taxol-R cells. The expression of circRNAs was profiled using circRNA microarrays, and top hits were validated using RT-PCR. We identified 429 and 310 circRNAs that were differentially expressed in Doxo-R and Taxol-R resistant cells respectively compared to the parental chemosensitive cell line (|FC| ≥ 1.5; p value < 0.05). Of these, 66 were commonly differentially expressed between the two chemotherapy resistant models. Conclusions: These results revealed that Doxo-R and Taxol-R TNBC cell lines were successfully established and serve as good models for studying the mechanisms of chemotherapy resistance and the regulatory roles of circRNAs in the development of chemoresistance in TNBC. Increasing knowledge of the important functions of circRNAs underlying drug resistance will provide new opportunities for developing efficacious therapeutic strategies and prognostic/predictive biomarkers for TNBC. Citation Format: Xiyin Wang, Michael Emch, John Hawse. The role of circular RNAs in triple-negative breast cancer and chemotherapy resistance [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 P2-22-02.

Abstract P6-01-15: Breast cancer patients with different hormone receptor subtypes receiving neoadjuvant chemotherapy (NAC) experience differential overall survival according to their resistance to NAC

Abstract Background: Response to neoadjuvant chemotherapy (NAC) is an indicator of outcomes and can be quantified as achievement of pathologic complete response (pCR) (absence of residual invasive disease in breast and lymph nodes) and residual percent cellularity. However, the significance of residual tumor cellularity post-NAC is not well understood. We assessed the impact of NAC-induced reduction in tumor cellularity among hormone receptor subtypes and the effect of these reductions on overall survival (OS). Methods: An IRB-approved retrospective review identified demographics, disease presentation, response to treatment, and outcomes. ER and PR status were categorized as low positive (1-9%), positive (≥ 10%) and negative. Treatment response was noted as percent residual cellularity (complete 0%, almost complete < 10%, good 10-30%, moderate >30-80% and poor >80%) in the surgical specimen and pathologic stage. We examined measures of response to NAC within breast tumor subtypes and the effect of these responses on associations with OS among hormone receptor subtypes. Results: The clinical series comprised 384 patients who received curative intent NAC. This series was diverse in presentation, displayed considerable variability in response to NAC (Table 1). 88 (23.6%) patients did not experience tumor down staging, and of those presenting with clinical nodal Stage 1 or higher (n=197), 94 (47.7%) did not experience nodal down staging. Although Triple Negative Breast Cancer (TNBC) status was not significantly associated with post-NAC residual tumor cellularity (p=0.74), ER, PR, and HER2 status were individually associated with this measure of response to NAC (p=0.04, 0.01, and 0.01, respectively). However, none of these associations explained more than 2.5% of the variability in this marker of treatment response. Median non-censored follow-up time was 4.26 years. Accounting for censoring, median survival was 13.65 years (lower 95% confidence limit was 11.79 years). Differential associations with OS were observed for three hormone receptor subtypes (ER: p< 0.001, HER2: p=0.04, and TNBC: p< 0.001) according to residual tumor cellularity, classified by percent residual cellularity categories of complete or almost complete response versus all others. Importantly, although ER negative patients with poor residual cellularity response had worse OS than ER positive patients with good response (Hazard Ratio [HR], 95% Confidence Interval [CI] = 4.74, 2.2-10.2), ER negative patients with good response did not have significantly worse OS than ER positive patients with good response (HR, 95% CI = 1.37, 0.57-3.26). Similar patterns were seen for patients with HER2 negative breast cancer or TNBC. Chemo resistant TNBC patients had higher risks for mortality than if they were not chemo resistant (HR, 95% CI: 2.41, 1.02-5.71). Conclusions: Our data suggest that OS associated with different hormone subtypes differs according to response to NAC. Differential OS according to response to NAC is greatest for patients classified according to ER status influences, with good response eliminating much of the differential mortality risk between ER negative and positive patients. There is a similar difference according to TNBC, although the difference appears to be less complete. Further studies should focus on understanding the chemo resistance of ER negative tumors, and perhaps TNBC, to identify mechanisms that may ultimately drive treatment response and survival. Table 1. Demographic and other characteristics Citation Format: Sangeetha Prabhakaran, V. Shane Pankratz, Christopher McNicoll, Nadja Falk, Jacklyn Nemunaitis, Jain Zhou, Payton Sandoval-Belt. Breast cancer patients with different hormone receptor subtypes receiving neoadjuvant chemotherapy (NAC) experience differential overall survival according to their resistance to NAC [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 P6-01-15.

Abstract P6-11-04: Novel roles of Inosine Monophosphate Dehydrogenase 2 (IMPDH2) in TNBC doxorubicin resistance

Abstract Triple negative breast cancer (TNBC) is one of the major subtypes of breast cancer, being associated with the lowest survival rate after metastasis occurs. TNBC patients face limited therapeutic options, relying mostly on a combination of chemotherapies (anthracycline, taxanes and cyclophosphamide). However, around 40% of patients treated with chemotherapy relapse due to onset of chemoresistance, contributing to the dismal survival of this aggressive subtype. Therefore, understanding the molecular mechanisms underlying chemoresistance is critical to identify better therapeutic target options. Guanosine triphosphate (GTP) is important for several biological processes, including cell proliferation. We and others have shown that inosine 5’-monophosphate dehydrogenase 2 (IMPDH2), the rate limiting enzyme in the de novo synthesis of GTP, is important in migration and invasion of cancer, including TNBC, however the role it plays in chemoresistance remains to be elucidated. Our preliminary data revealed that IMPDH2 expression levels modulate doxorubicin sensitivity in different TNBC cell lines. Moreover, we have generated MDA-MB-231 cells that are resistant to doxorubicin (referred to as DoxoR), and revealed IMPDH2 to be upregulated in DoxoR cells, at both protein and mRNA levels as well as having higher activity. Consistently, DoxoR cells have ~50% more GTP than their naïve counterpart and, interestingly, are twice more sensitive to treatment with Ribavirin, a well-tolerated FDA approved IMPDH inhibitor, suggesting that Ribavirin could be repurposed for the treatment of chemoresistant TNBC. As expected, RNA sequencing of DoxoR cells revealed increased stemness properties when compared to naïve cells, which we confirmed experimentally in tumor sphere formation assays. Importantly, knockdown of IMPDH2 reverted the ability of DoxoR cells to form bigger tumor spheres, as well as reduced the IC50 of doxorubicin to that of naïve cells. Therefore, our data suggests that increased IMPDH2 and GTP levels in resistant setting could be a potential new vulnerability to be leveraged therapeutically to suppress and/or prevent the growth of chemoresistant lesions. Citation Format: Tatiane da Silva Fernandes, Anna Bianchi-Smiraglia. Novel roles of Inosine Monophosphate Dehydrogenase 2 (IMPDH2) in TNBC doxorubicin resistance [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 P6-11-04.

Abstract P6-01-01: Validation of an Optical Imaging Platform to Identify Metabolic Vulnerabilities in Chemo-Resistant and Sensitive Tumors

Abstract Less than 20% of Triple Negative Breast Cancer (TNBC) patients experience long-term responses to mainstay chemotherapy, as tumors develop chemo-resistance. While combinations of chemotherapies and targeted therapies show potential improvements in TNBC clinical outcomes, patient stratification and prediction of treatment response is critical. Spatio-temporal metabolic reprogramming holds promise as a biomarker of therapy response as resistant tumor subpopulations utilize alternate metabolic pathways to escape therapy, enter minimum residual disease (MRD) and recur. Currently, there are limited tools to temporally evaluate heterogeneous changes along distinct metabolic axes in vivo at a spatial resolution capable of resolving vulnerabilities of residual tumor subpopulations. Here, we utilized an optical imaging-based platform to identify in vivo, longitudinal differences in metabolic reprogramming between a resistant and sensitive tumor model at high resolutions along three metabolic axes of TNBC chemoresistance (oxidative phosphorylation, glycolysis, and fatty acid oxidation). Xenografts were established by orthotopic cell injection and mice were treated with Paclitaxel (PTX), a commonly used chemotherapeutic drug in TNBC treatment, under a conventional maximum dose density regimen once the tumor reached a volume ~150mm3. MDA-MB-231 xenografts were resistant to PTX, defined as an initial response to PTX, a period of minimal residual disease, and a resurgence in tumor volume at ~60 days post drug withdrawal (n=3). HCC-1806 xenografts were sensitive to PTX, defined as an initial response to PTX in all mice and a complete cure in 7/10 mice. A separate cohort of mice for each tumor line was implanted with window chambers and imaged longitudinally at distinct stages of the tumor’s lifecycle with previously validated fluorophores 2-NBDG, TMRE, and Bodipy to directly report on glucose uptake, mitochondrial membrane potential, or fatty acid uptake, respectively. Wide field fluorescence imaging of MDA-MB-231 mice showed a significant increase in TMRE as early as two days after the 3rd PTX dose (n=5, p< 0.05), a significant decrease in 2-NBDG as early as two days after the 5th PTX dose (n=5, p< 0.05) and no significant changes in bodipy uptake. This increase in non-glucose-driven mitochondrial respiration was sustained during MRD. An increase in heterogeneity of TMRE uptake was seen during disease regression, MRD, and recurrence (n=5, p< 0.05). HCC-1806 tumors showed increased glucose uptake, decreased fatty acid uptake, and no significant changes in mitochondrial membrane potential during acute treatment. Metabolic changes were transient, with no significant changes in probe uptakes after drug withdrawal and during MRD. Unlike the MDA-MB-231 tumors, no significant changes in the heterogeneity of TMRE uptake were seen following paclitaxel withdrawal in HCC-1806 tumors (n=5, p>0.05). Our results point towards a metabolic switch from glycolysis to non-glucose, non-fat-driven mitochondrial respiration in MDA-MB-231 mice, possibly suggesting amino acid catabolism as a fuel during MRD. In the sensitive HCC-1806 line, we observed changes in glucose and fatty acid uptake during acute treatment, but no significant changes in metabolism during MRD. Consistent with the literature, our results point toward an increase in metabolic plasticity and heterogeneity in a chemo-resistant model compared to a chemo-sensitive one. Together, our results show the potential of using metabolic changes following therapy as a biomarker of therapy response, while highlighting the importance of tracking the change (or lack of change) in metabolism longitudinally. We aim to use this system to visualize and exploit early, in vivo metabolic vulnerabilities of disease regression that accompany local and distal recurrences. Citation Format: Enakshi D. Sunassee, Elizabeth Maydew, Brian Crouch, Megan Madonna, Gregory M Palmer, Nimmi Ramanujam. Validation of an Optical Imaging Platform to Identify Metabolic Vulnerabilities in Chemo-Resistant and Sensitive Tumors [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 P6-01-01.

Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment.

Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in ~45% of patients. Unfortunately, TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. We previously demonstrated mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual TNBC cells surviving NACT. We sought to investigate the mechanism underlying this enhanced reliance on mitochondrial metabolism. Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. The functional impact of mitochondrial structure on metabolic output is highly context dependent. Several chemotherapy agents are conventionally used for neoadjuvant treatment of TNBC patients. Upon comparing mitochondrial effects of conventional chemotherapies, we found that DNA-damaging agents increased mitochondrial elongation, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes instead decreased mitochondrial elongation and OXPHOS. The mitochondrial effects of DNA-damaging chemotherapies were dependent on the mitochondrial inner membrane fusion protein optic atrophy 1 (OPA1). Further, we observed heightened OXPHOS, OPA1 protein levels, and mitochondrial elongation in an orthotopic patient-derived xenograft (PDX) model of residual TNBC. Pharmacologic or genetic disruption of mitochondrial fusion and fission resulted in decreased or increased OXPHOS, respectively, revealing longer mitochondria favor oxphos in TNBC cells. Using TNBC cell lines and an in vivo PDX model of residual TNBC, we found that sequential treatment with DNA-damaging chemotherapy, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22, a specific inhibitor of OPA1, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibit regrowth of residual tumor cells. Our data suggest that TNBC mitochondria can optimize OXPHOS through OPA1-mediated mitochondrial fusion. These findings may provide an opportunity to overcome mitochondrial adaptations of chemoresistant TNBC.

CENPF knockdown inhibits adriamycin chemoresistance in triple-negative breast cancer via the Rb-E2F1 axis

Drug resistance occurs frequently in triple-negative breast cancer (TNBC) and leads to early relapse and short survival. Targeting the DNA damage response (DDR) has become an effective strategy for overcoming TNBC chemoresistance. CENPF (centromere protein) is a key regulator of cell cycle progression, but its role in TNBC chemotherapy resistance remains unclear. Here, we found that CENPF, which is highly expressed in TNBC, is associated with a poor prognosis in patients receiving chemotherapy. In addition, in vitro CENPF knockdown significantly increased adriamycin (ADR)-induced cytotoxicity in MDA-MB-231 cells and ADR-resistant cells (MDA-MB-231/ADR). Then, we demonstrated that CENPF targets Chk1-mediated G2/M phase arrest and binds to Rb to compete with E2F1 in TNBC. Considering the crucial role of E2F1 in the DNA damage response and DNA repair, a novel mechanism by which CENPF regulates the Rb-E2F1 axis will provide new horizons to overcome chemotherapy resistance in TNBC.

Pseudo-temporal dynamics of chemoresistant triple negative breast cancer cells reveal EGFR/HER2 inhibition as synthetic lethal during mid-neoadjuvant chemotherapy

In the absence of targetable hormonal axes, chemoresistance for triple-negative breast cancer (TNBC) often compromises patient outcomes. To investigate the underlying tumor dynamics, we performed trajectory analysis on the single-nuclei RNA-seq (snRNA-seq) of chemoresistant tumor clones during neoadjuvant chemotherapy (NAC). It revealed a common tumor trajectory across multiple patients with HER2-like expansions during NAC. Genome-wide CRISPR-Cas9 knock-out on mammary epithelial cells revealed chemosensitivity-promoting knock-outs were up-regulated along the tumor trajectory. Furthermore, we derived a consensus gene signature of TNBC chemoresistance by comparing the trajectory transcriptome with chemoresistant transcriptomes from TNBC cell lines and poor prognosis patient samples to predict FDA-approved drugs, including afatinib (pan-HER inhibitor), targeting the consensus signature. We validated the synergistic efficacy of afatinib and paclitaxel in chemoresistant TNBC cells and confirmed pharmacological suppression of the consensus signature. The study provides a dynamic model of chemoresistant tumor transcriptome, and computational framework for pharmacological intervention.

DANCR Induces Cisplatin Resistance of Triple-Negative Breast Cancer by KLF5/p27 Signaling

An increasing body of evidence suggests that long noncoding RNAs play critical roles in human cancer. Breast cancer is a heterogeneous disease and the potential involvement of long noncoding RNAs in breast cancer remains poorly understood. Herein, the study identified a long noncoding RNA, DANCR, which promotes cisplatin chemoresistance in triple-negative breast cancer (TNBC) cells. Mechanistically, binding of DANCR to Krüppel-like factor 5 (KLF5) induced acetylation of KLF5 at lysine 369 (K369), and DANCR knockdown resulted in down-regulation of KLF5 protein levels. Furthermore, DANCR/KLF5 signaling pathway induced hypersensitivity to cisplatin in chemoresistant patients by inhibiting p27 transcription. In summary, this study reinforced the potential presence of a growth regulatory network in TNBC cells, and documented a DANCR/KLF5/p27 signaling pathway mediating cisplatin chemoresistance in TNBC.

DAB2IP attenuates chemoresistance of triple-negative breast cancer through sequestration of RAC1 to prevent β-catenin nuclear accumulation.

Although chemotherapy, the most widely used systemic treatment in triple-negative breast cancer (TNBC), markedly improved the patients' outcome, chemoresistance always occurs. This study purposed to explore new therapeutic strategies for the treatment of chemoresistance. The expression and prognostic value of DAB2IP were investigated in TNBC tissues and cell lines. Low DAB2IP expression predicted high mortality risk in TNBC. Inhibition of DAB2IP expression conferred cancer stem cell capacity and chemoresistance in TNBC cell lines. Using murine breast cancer (BC) xenograft models, we evaluated the association with DAB2IP and chemoresistance. DAB2IP inhibited TNBC tumourigenesis and chemoresistance in vivo. Further, we revealed that DAB2IP inhibited β-catenin nuclear transport through competitive interaction with RAC1 and decreased β-catenin accumulation in the cell nucleus. Finally, we found that the DNA methylation level was negatively associated with DAB2IP expression in TNBC. Inhibition of DNA methylation restored the DAB2IP expression and attenuated chemoresistance in TNBC. We revealed that DAB2IP attenuates chemoresistance of TNBC via inhibition of RAC1-mediated β-catenin nuclear accumulation. Decitabine treatment results in re-expression of DAB2IP by inhibiting DNA methylation and could be a potential therapeutic strategy for chemoresistance in TNBC.

USP7 Induces Chemoresistance in Triple-Negative Breast Cancer via Deubiquitination and Stabilization of ABCB1.

Triple-negative breast cancer (TNBC) accounts for 15–20% of all breast cancer. TNBC does not express the estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2. Cytotoxic chemotherapy and surgery are the current therapeutic strategies for TNBC patients, but the chemoresistance of TNBC limits the efficiency of this strategy and shortens the lifespan of patients. The exploration of targeted therapy is ongoing in TNBC research. The aim of the present study was to identify the mechanism underlying acquired resistance in TNBC through the exploration of the relationship between the expression of USP7 and of ABCB1. We found that ubiquitin specific protease 7 (USP7) is a potential therapeutic target for overcoming the chemoresistance of TNBC. USP7 overexpression increased the chemoresistance of TNBC, while the knockdown of USP7 effectively increased the chemosensitivity of chemoresistant TNBC. A USP7 inhibitor effectively induced apoptosis and suppressed metastasis in chemoresistant TNBC. We further clarified that USP7 is a specific deubiquitinating enzyme for ABCB1 that plays an essential role in drug resistance. USP7 directly interacted with ABCB1 and regulated its stability. We concluded that USP7 promotes the chemoresistance of TNBC by stabilizing the ABCB1 protein.

Omics Analysis of Chemoresistant Triple Negative Breast Cancer Cells Reveals Novel Metabolic Vulnerabilities

The emergence of drug resistance in cancer poses the greatest hurdle for successful therapeutic results and is associated with most cancer deaths. In triple negative breast cancer (TNBC), due to the lack of specific therapeutic targets, systemic chemotherapy is at the forefront of treatments, but it only benefits a fraction of patients because of the development of resistance. Cancer cells may possess an innate resistance to chemotherapeutic agents or develop new mechanisms of acquired resistance after long-term drug exposure. Such mechanisms involve an interplay between genetic, epigenetic and metabolic alterations that enable cancer cells to evade therapy. In this work, we generated and characterized a chemoresistant TNBC cell line to be used for the investigation of mechanisms that drive resistance to paclitaxel. Transcriptomic analysis highlighted the important role of metabolic-associated pathways in the resistant cells, prompting us to employ 1H-NMR to explore the metabolome and lipidome of these cells. We identified and described herein numerous metabolites and lipids that were significantly altered in the resistant cells. Integrated analysis of our omics data revealed MSMO1, an intermediate enzyme of cholesterol biosynthesis, as a novel mediator of chemoresistance in TNBC. Overall, our data provide a critical insight into the metabolic adaptations that accompany acquired resistance in TNBC and pinpoint potential new targets.

CircRNA-CREIT inhibits stress granule assembly and overcomes doxorubicin resistance in TNBC by destabilizing PKR

BackgroundCircular RNAs (circRNAs) represent a novel type of regulatory RNA characterized by high evolutionary conservation and stability. CircRNAs are expected to be potential diagnostic biomarkers and therapeutic targets for a variety of malignancies. However, the regulatory functions and underlying mechanisms of circRNAs in triple-negative breast cancer (TNBC) are largely unknown.MethodsBy using RNA high-throughput sequencing technology, qRT-PCR and in situ hybridization assays, we screened dysregulated circRNAs in breast cancer and TNBC tissues. Then in vitro assays, animal models and patient-derived organoids (PDOs) were utilized to explore the roles of the candidate circRNA in TNBC. To investigate the underlying mechanisms, RNA pull-down, RNA immunoprecipitation (RIP), co immunoprecipitation (co-IP) and Western blotting assays were carried out.ResultsIn this study, we demonstrated that circRNA-CREIT was aberrantly downregulated in doxorubicin resistant triple-negative breast cancer (TNBC) cells and associated with a poor prognosis. The RNA binding protein DHX9 was responsible for the reduction in circRNA-CREIT by interacting with the flanking inverted repeat Alu (IRAlu) sequences and inhibiting back-splicing. By utilizing in vitro assays, animal models and patient-derived organoids, we revealed that circRNA-CREIT overexpression significantly enhanced the doxorubicin sensitivity of TNBC cells. Mechanistically, circRNA-CREIT acted as a scaffold to facilitate the interaction between PKR and the E3 ligase HACE1 and promoted proteasomal degradation of PKR protein via K48-linked polyubiquitylation. A reduced PKR/eIF2α signaling axis was identified as a critical downstream effector of circRNA-CREIT, which attenuated the assembly of stress granules (SGs) to activate the RACK1/MTK1 apoptosis signaling pathway. Further investigations revealed that a combination of the SG inhibitor ISRIB and doxorubicin synergistically inhibited TNBC tumor growth. Besides, circRNA-CREIT could be packaged into exosomes and disseminate doxorubicin sensitivity among TNBC cells.ConclusionsOur study demonstrated that targeting circRNA-CREIT and SGs could serve as promising therapeutic strategies against TNBC chemoresistance.

Abstract 2774: Reference free transcriptomic characterization of chemoresistant triple negative breast cancers provides a promising reservoir of predictive biomarkers of early chemoresistance

Abstract Triple Negative Breast Cancers (TNBCs) are the most aggressive breast cancers and so far, benefited very little from personalized medicine. Unlike in hormone positive breast cancers, coding transcriptomic signatures like Lehmann’s failed to predict which TNBC patients will benefit from perioperative chemotherapy. Here, we analyzed the total transcriptome of a unique cohort of 44 TNBC tumors before neoadjuvant chemotherapy (NAC), including both patients that were chemosensitive (N=26) and chemoresistant (N=18) to following treatment. Differential gene expression analysis (DE-seq) was first performed on annotated genes and de novo Scallop RNA-profiled transcripts. Nearly half of detected differential transcripts were from non-coding genes that were mostly long non-coding (lnc)RNAs (90%). More than half differential lncRNAs were not annotated in gencode. Next, De-kupl, a new reference-free analysis of differential fragments of transcripts, was applied. This approach is based on 31-nucleotide long k-mers that are later merged into contigs without annotation bias. These differential contigs were able to separate correctly, in an unsupervised manner, all TNBC patients regarding their response to chemotherapy, outperforming our previous DE-seq analysis of gencode and scallop transcripts. Strikingly, 80% of the DE-kupl differential contigs were not derived from DE-seq differential genes. Furthermore, as BRCA 1/2 mutated TNBCs are now well known for their chemosensitivity, we excluded them from subsequent analysis and repeated the differential expression analysis. Our results showed that DE-kupl differential contigs once more perfectly classified all patients and outperformed the whole length transcripts from DE-seq. Finally, we applied the LASSO regression analysis machine learning method, on both all misregulated genes and all misregulated contigs in parallel, to generate minimal signatures characterizing pre-chemotherapy TNBC tumors that are going to be chemoresistant. We obtained an 8-contig-signature that overpassed the 11-gene-signature in the accuracy to discriminate chemosensitive and chemoresistant TNBC patients before chemotherapy. These results suggest that a contig based unreferenced differential analysis of TNBCs regarding their response status to NAC provides promising predictive biomarkers of early TNBC chemoresistance and thus need further confirmation in a larger and independent validation cohort. Citation Format: Nouritza Torossian, Dominika Foretek, Marc Gabriel, Linda Larbi Cherif, Charlotte Lecerf, Maud Kamal, Christophe Le Tourneau, Daniel Gautheret, Sergio Roman-Roman, Antonin Morillon. Reference free transcriptomic characterization of chemoresistant triple negative breast cancers provides a promising reservoir of predictive biomarkers of early chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2774.

Abstract 151: Mixed-lineage kinase 4 is involved in the activation of the ATM-NEMO signaling axis in response to DNA damage and confers chemoresistance in triple-negative breast cancer

Abstract Resistance to chemotherapy leads to high rates of relapse and poor outcomes in patients with triple-negative breast cancer (TNBC). Therefore, there is a need to identify novel molecular targets that could be exploited to overcome TNBC chemoresistance. Mixed-lineage kinase 4 (MLK4) is a member of MLK family of serine/threonine kinases. Large-scale genomic and transcriptomic data indicated that MLK4 gene (MAP3K21/KIAA1804) is amplified and overexpressed in approximately 50% of triple-negative tumors. We have recently demonstrated that MLK4 promotes the aggressive growth and metastatic potential of TNBC cells. However, the functional role of MLK4 in resistance to chemotherapy has not been investigated so far. In our recent study, we have shown that MLK4 contributes to TNBC chemoresistance by regulating the pro-survival response to DNA-damaging therapies. MLK4 knock-down or inhibition sensitized TNBC cell lines to doxorubicin and etoposide in vitro. Moreover, loss of MLK4 enhanced sensitivity of HCC1806 TNBC cell line towards doxorubicin in a xenograft mouse model. Our data indicated that MLK4 silencing induced persistent DNA damage accumulation and enhanced apoptosis in TNBC cells treated with chemotherapeutics. To further determine cellular mechanisms responsible for the identified MLK4-dependent chemoresistance in TNBC, we performed a quantitative phosphoproteomic analysis of control and MLK4-depleted HCC1806 cells, either untreated or treated with doxorubicin. We found that the doxorubicin-induced phosphorylation of several core DNA damage response components, including ATM, TRIM28 (KAP-1), MDC1 and TP53BP1, was impaired in cells with MLK4 knock-down. These findings were further confirmed using HCC1806 cells with CRISPR/Cas9-mediated deletion of MLK4, which showed markedly reduced phosphorylation of ATM kinase at Ser1981 - a site critical for ATM monomerization and activation. ATM was shown to activate NF-κB transcription in response to DNA damage through the well-established nuclear to cytoplasmic signaling pathway involving NEMO. Interestingly, we observed diminished phosphorylation of NEMO and reduced expression of several NF-кB-associated pro-survival cytokines (IL6, IL8, CXCL1, CXCL6) in MLK4-depleted TNBC cells treated with doxorubicin. These data, along with the additional immunoprecipitation and kinase assay experiments, indicate that MLK4 is involved in the regulation of DNA damage response signaling and ATM-NEMO pathway activation. Moreover, our recent results suggest that MLK4 controls autocrine/paracrine signaling loops in cancer cells and tumor microenvironment (TME) upon treatment with chemotherapy. We currently aim to further determine how MLK4-dependent crosstalk between TNBC cells and TME contributes to chemoresistance and tumor progression. Citation Format: Dawid Mehlich, Michal Lomiak, Aleksandra Sobiborowicz, Alicja Mazan, Dagmara Dymerska, Lukasz M. Szewczyk, Anna Mehlich, Agnieszka Borowiec, Monika K. Prelowska, Dominika Nowis, Anna A. Marusiak. Mixed-lineage kinase 4 is involved in the activation of the ATM-NEMO signaling axis in response to DNA damage and confers chemoresistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 151.

Abstract 1595: Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry

Abstract Shifts in tumor cell phenotype in response to selective pressures (i.e. changing microenvironments, drug treatments) pose one of the biggest obstacles to successful breast cancer therapies. Phenotypically diverse breast tumor and stroma subpopulations, and interactions between them that alter tumor cell biology, represent unique and spatially distinct niches. We hypothesize that localized neighborhoods of breast tumor cells possess specialized phenotypes that mediate chemoresistance and represent novel therapeutic vulnerabilities. In order to assess these potential phenotypes, we utilized imaging mass cytometry (IMC), a highly multiplexed imaging modality that allows simultaneous measurement of 30-40 antigens while retaining the spatial architecture of the cancer tissue. We constructed an IMC antibody panel that combines markers for tissue architecture, tumor and stromal cell phenotyping, and signaling pathway activation. IMC was applied to patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC).Our TNBC PDX collection was established from tumors obtained before and after neoadjuvant Adriamycin and cyclophosphamide (AC). IMC analysis of 18 PDX models representing eight patients revealed that stromal cell phenotypes were generally shared between all models, but tumor cell phenotypes were largely patient-specific. While every model was comprised primarily of a few major tumor cell phenotypes, we noted that each case also harbored several minor, unique populations, suggesting that specialized neighborhoods may exist within the tumor mass. Comparison of paired PDX models showed a wide range of phenotypic responses to chemotherapy, ranging from stable tumor composition to widespread changes in tumor phenotypes. These phenotypic changes arose despite relatively consistent genomic architecture. Vimentinhi fibroblasts were present more often in post-AC models, while SMAhi fibroblasts were unchanged after treatment. Comparison of pre-/post-AC PDX pairs revealed spatially constrained MAPK activation emerged after treatment. To capture acute changes in tumor phenotype, we treated treatment-naïve PDX models with AC and evaluated tumors by IMC. As tumors regressed and then regrew, we identified novel phenotypic shifts, again including increased MAPK signaling localized to discrete neighborhoods, suggesting this property may be a common feature of chemoresistant TNBC. Analysis of adjacent cells revealed seven distinct neighborhoods, and ongoing work is aimed at determining whether these neighborhoods are altered in response to chemotherapy treatment. Taken together, our findings suggest that distinct tumor phenotypes arise following treatment. Our goal is to determine whether these unique phenotypic niches functionally contribute to chemoresistance and if disruption of these niches enhances chemosensitivity. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Pankaj Singh, Yuan Qi, Jiansu Shao, Xiaomei Zhang, Gloria V. Echeverria, W. Fraser Symmans, Stacy L. Moulder, Helen Piwnica-Worms. Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1595.

Abstract 6384: Morphological and functional plasticity of mitochondria promotes chemotherapy resistance in triple negative breast cancer

Abstract Mitochondrial metabolism plays a key role in triple negative breast cancer (TNBC) aggressiveness. As TNBC has limited targeted therapy options, chemotherapies remain the mainstay treatment. Nearly 50% of TNBC patients harbor substantial residual cancer following chemotherapy, leading to high rates of recurrence. Using longitudinal biopsies from orthotopic patient-derived xenograft (PDX) models and TNBC patients, we found residual tumors following chemotherapy transitioned to a unique metabolic state characterized by high mitochondrial oxidative phosphorylation (oxphos). This state was transient, with tumors reverting to their baseline glycolysis-high phenotype when they were allowed to regrow in the absence of treatment. Using genomic sequencing and cellular barcode-mediated clonal tracking, we found this mechanism of chemoresistance arose in the absence of clonal selection, suggesting chemotherapy induced plastic (i.e., non-genomic) programs enabling cell survival following treatment. Blocking oxphos with an inhibitor of electron transport chain Complex I (IACS010759; PMID:29892070) was significantly more efficacious against residual than pre-treated tumors (PMID:30996079), providing evidence that dynamic metabolic phenotypes represent targetable therapeutic vulnerabilities for TNBC. Using longitudinal samples collected from PDX models undergoing treatments with anthracyclines, platinums, and/or taxanes, we visualized and quantified mitochondrial structure in two- and three-dimensions by electron microscopy. These studies revealed extensive alteration of mitochondrial structure and number in residual tumor cells, and these changes reverted when residual tumors were allowed to regrow in the absence of treatment. We then administered chemotherapeutics to human TNBC cells, revealing that DNA-damaging chemotherapeutics increased mitochondrial elongation, but microtubule poisons increased mitochondrial fragmentation. These findings suggested chemotherapeutics may alter the dynamics of mitochondrial fission and fusion in TNBC cells. These structural changes were accompanied by increased or decreased oxphos rates, glucose-driven TCA cycle flux, and mitochondrial content, respectively. Driving mitochondrial fusion by genetic or pharmacologic inhibition of the mitochondrial fission factor Drp1 increased oxphos and chemoresistance, whereas driving mitochondrial fission by genetic or pharmacologic inhibition of the mitochondrial fusion protein Opa1 decreased oxphos and chemoresistance. These findings provide evidence that modulating mitochondrial fission and fusion may be a promising strategy to overcome metabolic states contributing to chemoresistance in TNBC. Our ongoing investigations are aimed at rational targeted therapies and scheduling approaches to overcome chemoresistance in in vivo models of TNBC. Citation Format: Lily Baek, Junegoo Lee, Mariah J. Berner, Katherine E. Pendleton, Emily B. Goff, Karen Wang, James P. Barrish, Bora Lim, Philip J. Lorenzi, Weston Porter, Michael T. Lewis, Gloria V. Echeverria. Morphological and functional plasticity of mitochondria promotes chemotherapy resistance in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6384.

Abstract LB085: ATYR2810, a fully humanized monoclonal antibody targeting the VEGF-NRP2 pathway sensitizes highly aggressive and chemoresistant TNBC subtypes to chemotherapy

Abstract Introduction: Triple negative breast cancer (TNBC) represents 15% of all breast cancers, and is a notably aggressive subtype that is prone to early recurrence following initial diagnosis with a median survival of only 9 months following recurrence. One of the few treatment options for patients with advanced and metastatic TNBC is platinum-based chemotherapy, however not all tumors respond, and those that do often become refractory during the course of treatment. The cell surface receptor Neuropilin-2 (NRP2), which acts as a co-receptor for vascular endothelial growth factors (VEGFs), has been shown to be both highly expressed and associated with therapeutic resistance in TNBC. NRP2 also serves as a co-receptor for semaphorins, which may play tumor-suppressor functions in breast cancer. We have previously demonstrated that ATYR2810, a highly specific humanized monoclonal antibody which effectively blocks NRP2/VEGF signaling without disrupting NRP2/Semaphorin 3F signaling, down-regulated epithelial-mesenchymal transition (EMT) genes such as ZEB1 and sensitizes TNBC to chemotherapy both in vitro and in vivo. Results: In this study we have further characterized the breast cancer subtypes that are most responsive to ATYR2810 treatment. Using in vitro 3D colony formation assays (CFAs), we have interrogated ~15 breast cancer cell lines covering luminal, HER2+ and various TNBC subtypes, for responsiveness to ATYR2810 treatment in combination with chemotherapy. All responsive cell lines were associated with the more aggressive subtypes of TNBC such as BL2 (basal-like 2), ML (mesenchymal-like) and MSL (mesenchymal-stem like) subtypes. Additionally, we screened five patient derived xenografts (PDXs) and again found that the responsive PDXs were associated with the more aggressive and chemoresistant subtypes of TNBC. To gain insight into the potential mechanism for enhanced chemosensitivity by ATYR2810 treatment, we performed RNAseq on the PDX tumors. Interestingly, the chemokine receptor CXCR4, which is known to promote drug resistance and tumorigenic potential, was found to be significantly downregulated in responder PDX tumors treated with ATYR2810 in combination with cisplatin as compared to those treated with cisplatin alone. Further, gene set enrichment analysis (GSEA) showed that CXCR4 was found in numerous downregulated pathways enriched in the PDX responder tumors treated with ATYR2810/cisplatin, suggesting that CXCR4 may be driving the responder phenotype of the PDXs. Flow cytometry analysis of TNBC cells treated with ATYR2810 monotherapy in vitro resulted in a reduced frequency of cells expressing CXCR4. Furthermore, ATYR2810 monotherapy inhibited spontaneous lung metastasis in experimental models of mesenchymal TNBC. Conclusion: Taken together, our data suggest that ATYR2810 enhances chemosensitivity in highly aggressive TNBC subtypes, and that this response may be mediated through the downregulation of genes known to be associated with aggressive cancer states such as ZEB1 and CXCR4. ATYR2810 may therefore serve as a novel therapeutic agent for the treatment of advanced and metastatic TNBC and potentially other aggressive cancer types. Zhiwen Xu and Alison Barber are co-first authors of this abstract. Citation Format: Zhiwen Xu, Alison Barber, Christoph Burkart, Hira Lal Goel, Justin Rahman, Kristina Hamel, Zachary Fogassy, Lisa Eide, Clara Polizzi, Jasmine Stamps, Sofia Klopp Savino, Luke Burman, Esther Chong, Suzanne Paz, Arthur M. Mercurio, Leslie A. Nangle. ATYR2810, a fully humanized monoclonal antibody targeting the VEGF-NRP2 pathway sensitizes highly aggressive and chemoresistant TNBC subtypes to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB085.