Triple-negative Breast Cancer Patient-derived Xenografts Research Articles

Evaluation of copanlisib in combination with eribulin in triple negative breast cancer patient-derived xenograft models.

The phosphoinositide 3-kinase (PI3K) pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the anti-tumor effect in TNBC. A panel of 8 TNBC patient-derived xenograft (PDX) models were tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment induced signaling changes were examined by reverse phase protein array, immunohistochemistry, and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET). Compared to each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin induced mitotic arrest. The combination enhanced induction of apoptosis compared to each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan of PDX tumor and AKT phosphorylation by immunohistochemistry. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin. These data led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC.

MerTK Drives Proliferation and Metastatic Potential in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by the absence of the estrogen receptor, progesterone receptor, and receptor tyrosine kinase HER2 expression. Due to the limited number of FDA-approved targeted therapies for TNBC, there is an ongoing need to understand the molecular underpinnings of TNBC for the development of novel combinatorial treatment strategies. This study evaluated the role of the MerTK receptor tyrosine kinase on proliferation and invasion/metastatic potential in TNBC. Immunohistochemical analysis demonstrated MerTK expression in 58% of patient-derived TNBC xenografts. The stable overexpression of MerTK in human TNBC cell lines induced an increase in proliferation rates, robust in vivo tumor growth, heightened migration/invasion potential, and enhanced lung metastases. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, which ultimately drive cell cycle progression, reduce apoptosis, and enhance cell survival. Proteomic profiling indicated increased endoglin (ENG) production in SUM102-MerTK clones, suggesting that MerTK creates a conducive environment for increased proliferative and metastatic activity via elevated ENG expression. To determine ENG's role in increasing proliferation and/or metastatic potential, we knocked out ENG in a SUM102-MerTK clone with CRISPR technology. Although this ENG knockout clone exhibited similar in vivo growth to the parental SUM102-MerTK clone, lung metastasis numbers were significantly decreased ~4-fold, indicating that MerTK enhances invasion and metastasis through ENG. Our data suggest that MerTK regulates a unique proliferative signature in TNBC, promoting robust tumor growth and increased metastatic potential through ENG upregulation. Targeting MerTK and ENG simultaneously may provide a novel therapeutic approach for TNBC patients.

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 PO2-28-08: A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy

Abstract TNBC is one of the most aggressive subtypes of breast cancer. Combating chemotherapy resistance is critical to improving quality of care and reducing fatality among TNBC patients. In order to understand the mechanisms of chemoresistance in TNBC tumors, it has been proposed that the spatial interactions between cell types in the tumor microenvironment (TME) could offer insights into the differential response to therapy among tumors, and metastatic potential of certain tumors over others. Thus, this study utilizes spatially resolved transcriptomic (SRT) technology to profile the spatial interactions in the TNBC TME. With the goal of better understanding chemoresistance in TNBC and providing a proof-of-concept for new technology, we have launched a longitudinal SRT study of a TNBC PDX of residual disease before, during, and after adriamycin and cyclophosphamide (AC) treatment (Tx). SRT by 10X Genomics Visium was performed on vehicle, AC-treated residual tumor (21 days post-Tx), and AC-treated regrown tumor (50 days post-Tx when tumors regrew to starting tumor volume). This study is divided into two parts: 1) development of a computational pipeline; 2) spatial colocalization analysis of residual and regrown tumors to understand chemoresistance. PART 1: Due to the lack of specialized tools for processing and sorting xenograft reads from SRT data, we developed the Xenomake pipeline, which combines a xenograft sorting algorithm (Xengsort) and spatial barcode demultiplexing pipeline to assign reads into the host and graft organisms for each spatial spot. RESULTS: Xenomake permits clustering the spatial spots into stroma-rich (enriched for mouse mRNAs), and epithelial-rich (enriched for human mRNAs) regions. We show that Xenomake can find differential cytokine production in the stroma and epithelium. Since PDX data separate the tumor into stroma and epithelium by organism, the pipeline enables fine-tuned downstream analysis such as stromal-stromal and stromal-epithelial interactions. Xenomake is thus generally applicable for SRT involving PDX samples. PART 2: Previously we detailed patterns of tumor regression into a residual tumor state, followed by uncontrolled regrowth in the absence of treatment in multiple PDX models of TNBC. Although using PDX models necessitates immune-compromised mice, several types of stromal populations are present and analyzable in these models. Thus, using organism-assigned reads processed by Xenomake, we computationally inferred the localization pattern of stromal cell types in Visium spots including cancer associated fibroblasts (CAF), macrophages (MP), endothelial cells (ENDO), monocytes, and perivascular-like cells. We compared the spatial localization profiles (SLP) of stromal cell types across samples. With human reads, we computed the spatial pathway activity map (SPAM) for the ~30 HALLMARK pathways across samples. We correlated the patterns of SPAM with stroma cell-type SLP to survey the stroma-epithelial interactions across samples. RESULTS: Stroma cells are generally distributed in the tumor periphery in vehicle and AC50, while in AC21 there is a notable increase in stroma abundance and stroma infiltration within tumor mass. SLP of MP (Cd68+ and Csf1r+) is correlated with ENDO (Pecam1+), and with CAF (Acta2+ and Pdgfrb+). Additionally, all 3 cell types are correlated with Vim and Cd44 expression. Although all samples show enrichment of MP, ENDO, and CAF, they interact differently with pathway activities of adjacent epithelial cells. In vehicle and AC50, the 3 cell types are colocalized with OXPHOS, MYC targets, E2F pathway, while in AC21, a switch to colocalization with EMT is observed. Furthermore, hypoxia response and glycolysis display anti-correlation with stroma cell types, meaning that these pathway activities are further away from stroma and occupy distinct territories. The results suggest stroma-tumor metabolic crosstalk and ways of targeting residual disease. Citation Format: Benjamin Strope, Katherine Pendleton, William Bowie, Gloria Echeverria, Qian Zhu. A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-28-08.

Abstract PO1-16-04: Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression

Abstract Purpose: Low molecular weight isoforms of cyclin E (LMW-E) have been implicated in various human cancers, including triple negative breast cancer (TNBC), and are associated with a poor prognosis. However, targeted therapies for TNBC based on biomarkers are currently lacking. This study aims to investigate LMW-E as a potential therapeutic target in TNBC and evaluate the efficacy of RP-6306, a selective inhibitor of the Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1), in LMW-E-positive breast tumors. Experimental Design: Immunohistochemical (IHC) analysis was performed on pre-treatment tumor specimens from TNBC patients (n=36) to assess the correlation between LMW-E expression, CDK1 phosphorylation at Threonine 14 (pT14), and pathologic complete response to neoadjuvant chemotherapy. LMW-E inducible human mammary epithelial cells (hMEC) and breast cancer cell lines were used to investigate the regulatory effect of LMW-E on PKMYT1, the kinase responsible for CDK1 phosphorylation at T14, and the response to RP-6306, a first in-class and selective inhibitor of PKMYT1. Patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models of TNBC expressing human LMW-E (hLMW-E) were also utilized to assess LMW-E as a biomarker for predicting response to RP-6306. Results. Analysis of TNBC tumor biopsies revealed a significant positive correlation between LMW-E expression and CDK1 pT14, and both biomarkers were associated with a lack of pathological complete response to neoadjuvant chemotherapy. In vitro results using LMW-E inducible hMECs and breast cancer cell lines demonstrated that LMW-E up-regulates PKMYT1 and CDK1 pT14, acting as a PKMYT1 binding protein and enhancing PKMYT1 protein stability. High LMW-E protein levels predicted a favorable response to RP-6306, resulting in the accumulation of sub-G1 and polyploid cells, decreased tolerance to replication stress, increased DNA damage, chromosomal breakage, and apoptosis. In vivo treatment of TNBC PDX models and hLMW-E transgenic tumors with RP-6306 resulted in a significant reduction in tumor volume only in mice harboring high LMW-E tumors, while low cyclin E models showed no response. Immunohistochemical analysis confirmed increased γ-H2AX and decreased CDK1-pT14 and Ki67 levels, indicating the efficacy of RP-6306 in both PDX and transgenic models. Conclusion: This study highlights the regulatory axis from LMW-E to PKMYT1 and its predictive value for pathological complete response in TNBC patients receiving neoadjuvant chemotherapy. The selective PKMYT1 kinase inhibitor RP-6306 consistently induced DNA damage and inhibited tumor growth in in vitro and in vivo pre-clinical breast tumor models. Co-expression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306, emphasizing its therapeutic significance. Citation Format: Mi Li, Amriti Lulla, Cansu Karakas, Spiridon Tsavaschidis, Yan Wang, Tuyen Nguyun, Tuyen Bui, Gary Marshall, Kelly Hunt, Khandan Keyomarsi. Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression [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 PO1-16-04.

Abstract PO1-06-14: Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (TNBC)

Abstract Background: Aberrant PI3K pathway signaling is frequently observed in TNBC. Increasing evidence indicate that PI3K pathway activation maintains the stemness and chemoresistance of breast cancer stem cells (CSCs). PI3K inhibition sensitizes CSCs to chemotherapy. Eribulin (E), a non-taxane microtubule dynamics inhibitor, showed overall survival benefit in metastatic HER2 negative BC. Preclinically, the addition of copanlisib (C), a potent pan-class I PI3K inhibitor that is highly selective against α and δ PI3K isoforms, improved anti-tumor effect in both E-sensitive and resistant TNBC patient-derived xenograft models, irrespective of PIK3CA/PTEN alteration status. Here we report the preliminary data of the phase I dose escalation trial of E+C in patients with metastatic TNBC. Methods: This trial includes a phase I study to determine the dose limiting toxicity (DLT) and recommended phase 2 dose (RP2D) of E+C, followed by a randomized phase II study of E+C (at RP2D) versus E (stratified by tumor PIK3CA/PTEN mutation status) with progression-free survival (PFS) as the primary endpoint. Key eligibility included patients with: metastatic TNBC who progressed on ≤5 lines of chemotherapy, prior anthracycline/taxane chemotherapy (unless contraindicated), ECOG 0-1, adequate organ function and known tumor PIK3CA/PTEN/AKT mutation status. Key exclusions include: prior receipt of E or any PI3K/mTOR/AKT inhibitor, grade ≥2 neuropathy, presence of tumor AKT mutation, and uncontrolled diabetes, hypertension or congenital QT prolongation. A 3+3 design was used to determine the DLT and RP2D. Dose level 1 (DL1) started with E at 1.1 mg/m2 mg IV and C at 45 mg IV on days (D) 1/8 of 21-D cycle, with the potential to escalate to 2 additional dose levels – DL2: E 1.4 mg/m2 and C 45 mg on D 1/8; DL3: E 1.4 mg/m2 and C 60 mg on D 1/8. Results: Eight patients were enrolled in the phase I study and received at least 1 dose of E+C. The median age was 48.5 (range, 26-67) years. The majority of patients identified as White (additionally 2 Hispanic, 1 Black) and had an ECOG performance status of 1 (n=6, 75.0%). Tumor PIK3CA and PTEN alterations were each observed in 1 patient. Median number of cycles received were 2 (range, 1-5). All 8 patients had at least 1 treatment-related adverse event (TRAE). The most common TRAEs across all dose levels were: lymphopenia (n=5, 62.5%), neutropenia (n=5, 62.5%), hyperglycemia (n=4, 50.0%), anemia (n=4, 50.0%) and fatigue (n=4, 50.0%); most toxicities were grade (G) 1-2. G3 hyperglycemia and hypertension were observed in 1 patient each, were medically managed and did not lead to treatment interruption. 1 patient was diagnosed with pulmonary embolism, possibly attributed to study therapy and breast cancer. DLTs were reported in 2 of 2 patients treated at DL2 - 1 patient developed G3 rash responsive to oral corticosteroids, and 1 patient developed G3 acute kidney injury in the setting of hypovolemia and discontinued study therapy. No additional patients were enrolled in DL2. No DLTs were observed in all 6 patients treated at DL1. 1 patient treated at DL1 had possible G1 non-infectious pneumonitis that resolved spontaneously on short interval imaging. Stable disease was observed in 2 of 6 response-evaluable patients (33.3%). 1 patient achieved regression of cutaneous disease following 1 cycle of therapy as assessed by the treating investigator. DL1, E at 1.1 mg/m2 plus C at 45 mg IV, was determined to be the RP2D. Tumor tissue biomarker analysis is underway. Conclusion: These data indicate that E+C at DL1 is a well-tolerated regimen warranting further investigation. The study is actively enrolling patients to the phase II study. Clinical trial information: NCT04345913. Funding Source: National Cancer Institute. Citation Format: Nusayba Bagegni, Brittney Haas, Leslie Nehring, Jingqin Luo, Laura Kennedy, Meghna Trivedi, Manali Bhave, Rabih Said, Cynthia Ma. Preliminary results of a phase I dose escalation study of eribulin in combination with copanlisib in patients with metastatic triple negative breast cancer (TNBC) [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 PO1-06-14.

Targeting CD73 with flavonoids inhibits cancer stem cells and increases lymphocyte infiltration in a triple-negative breast cancer mouse model.

Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients, improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target, given its elevated expression on tumor, stromal, and specific immune cells, and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation, while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity, further suppressing anti-tumor immunity. While debulking the tumor mass, chemotherapy also enriches heterogenous cancer stem cells (CSC), potentially leading to tumor relapse. Therefore, drugs targeting both CD73, and CSCs hold promise for enhancing chemotherapy efficacy, overcoming treatment resistance, and improving clinical outcomes. However, safe and effective inhibitors of CD73 have not been developed as of now. We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry, RT-qPCR, CD73 activity, cell viability, tumorsphere formation, and other in vitro functional assays. For assessment of clinical translatability, TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses. We identified quercetin and luteolin, currently used as over-the-counter supplements, to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen, we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44highCD24low CSCs, and co-targeting CD73, YAP, and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore, triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors. Conclusively, our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new, safe, and cost-effective therapeutic strategies for TNBC.

Abstract 1986: Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts

Abstract Sacituzumab Govitecan (SG) is an antibody drug conjugate that targets the epithelial glycoprotein Trop-2. SG has been approved for the treatment of patients with triple negative breast cancer (TNBC) or estrogen receptor positive (ER+) breast cancer after they have received at least two previous systemic treatments. The objectives of this study were to identify biomarkers of SG response using breast cancer patient-derived xenografts (PDXs) and determine if SG efficacy changes when cells become carboplatin resistant (CR). Analysis of transcriptomic and proteomic levels of Trop2 using bulk RNAseq, scRNA-seq, and tandem mass tag spectrometry found that overall ER+ PDXs had significantly lower RNA (p-value less than 0.001) and protein (p-value less than 0.001) expression of Trop-2 than TNBC PDXs. Immunohistochemical assessment of 42 TNBC and 27 ER+ patient tumor samples identified variation in Trop-2 expression; within each cohort, expression varied from highly positive to completely negative. To test the association of Trop-2 with drug responsiveness, cells from 19 different PDXs were cultured in vitro and administered increasing doses of SG. TNBC cells were more responsive to SG treatments compared to ER+ cells (p-value equaling 0.005). Correlation analysis identified a positive relationship of drug response with protein abundance which was stronger in TNBC samples. In vivo studies with 7 TNBC PDXs found that over 70% were highly susceptible to SG treatments, with some tumors being completely eradicated or exhibiting a total inhibition of tumor growth which resulted in a long-term durable response after 10 weeks of treatment. In vivo across all the PDXs, Trop-2 expression alone did not strongly correlate with SG treatment success. Interestingly, CR sublines derived from 2 carboplatin sensitive PDXs were significantly less responsive to SG than their parental PDXs (p-value less than 0.001). These two CR sublines were also significantly less sensitive to the majority of 555 other drugs identified by the NCI compared to their parental PDXs. Ongoing studies are defining mechanisms of reduced SG efficacy in CR models. Overall, these studies find that SG is highly effective in many TNBC PDX models and should be utilized earlier in treatment protocols, especially in metastatic patients. Citation Format: Carson J. Walker, Julia E. Altman, Emily K. Zboril, Rachel K. Myrick, Nicole S. Hairr, David C. Boyd, Bin Hu, Mikhail G. Dozmorov, J. Chuck Harrell. Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1986.

Abstract 3876: A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy

Abstract Combating chemotherapy resistance in TNBC is critical to improving quality of care and reducing fatality. To understand chemoresistance, it has been proposed that the spatial interactions between cell types in the tumor microenvironment (TME) could offer insights into the differential response to therapy and metastatic potential. Thus, this study utilizes spatially resolved transcriptomics (SRT) to profile the spatial interactions in TNBC TME. With the goal of understanding chemoresistance and providing a proof-of-concept for new technology, we launched a longitudinal SRT study of a TNBC PDX of residual disease before, during, and after adriamycin and cyclophosphamide (AC) treatment (Tx). SRT by 10X Visium was performed on Veh, AC-residual tumor (21 d post-Tx), and AC-regrown tumor (50 d post-Tx when tumors regrew). This study is divided into two parts. 1. Due to the lack of specialized tools for processing xenograft reads from SRT, we developed the Xenomake pipeline, which combines xenograft sorting and spatial barcode demultiplexing to assign reads into the host and graft organisms per spatial spot. It permits clustering the spots into stroma-rich (mouse), and epithelial-rich (human) regions. We show that it can find differential cytokines in the stroma (S) and epithelium (E), and finding SS, EE, and SE interactions. 2. Although using PDX necessitates immune-compromised mice, several types of stromal populations are present and analyzable. Thus, using Xenomake, we inferred the localization of stromal cell types in Visium spots including cancer associated fibroblasts (CAF), macrophages (MP), endothelial (ENDO), monocytes, and perivascular cells. We compared the spatial localization profiles (SLP) of stromal cell types across samples. With human reads, we computed the spatial pathway activity map (SPAM) for HALLMARK pathways. We correlated SPAM with stroma cell type SLP to survey stroma-epithelial interactions. Stroma cells are distributed in the tumor periphery in vehicle and AC50, while in AC21 there is a notable increase in stroma abundance and stroma infiltration within tumor mass. SLP of MP (Cd68+ and Csf1r+) is correlated with ENDO (Pecam1+), and CAF (Acta2+ and Pdgfrb+). Additionally, all 3 cell types are correlated with Vim and Cd44 expression. Although all samples show enrichment of MP, ENDO, and CAF, they interact differently with pathway activities of adjacent epithelial cells. In vehicle and AC50, the 3 cell types are colocalized with OXPHOS, MYC targets, E2F pathway, while in AC21, a switch to colocalization with EMT is observed. Further, hypoxia response and glycolysis display anti-correlation with stroma cell types, meaning that these pathway activities are further away from stroma and occupy distinct territories. The results suggest stroma-tumor metabolic crosstalk and ways of targeting residual disease. Citation Format: Benjamin Strope, Katherine Pendleton, William Bowie, Gloria Echeverria, Qian Zhu. A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy [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 3876.

Abstract 2405: Harnessing liquid biopsy and genetic barcoding in evaluating diversity within triple-negative breast cancer

Abstract Introduction: While liquid biopsy shows promise as a non-invasive precision medicine tool, its ability to comprehensively represent the complexity of a tumor or only a subset of its clones remains uncertain. This issue is particularly crucial in the context of triple-negative breast cancer (TNBC), a type of cancer known for its significant heterogeneity. The goal of this study is to examine the clonal diversity within Circulating Biomaterials (CBMs): namely circulating tumor cells (CTCs), extracellular vesicles RNA (evRNA), and circulating tumor DNA (ctDNA), employing genetic barcoding and patient-derived TNBC xenografts. Methodology: Cells from TNBC patients' resected tumors were tagged with a unique barcode sequence through vector transduction. These barcoded cells were subsequently introduced into immunodeficient mice to form xenografts. When these xenografts grew to a certain size (15mm in diameter), the mice were humanely euthanized, and the tumor tissues as well as CBMs from their blood and plasma were collected. CTCs were isolated through negative enrichment and DEPArray technology; evRNA was extracted using ultracentrifugation and TRIzol™ Plus RNA Purification method; ctDNA was retrieved using the QIAamp DNA Micro Kit. Tumor tissues were processed for RNA and DNA using RNeasy micro kit and Qiamp DNA micro kit, respectively. RNA sequencing of the tumor tissues was carried out using the Lexogen - CORALL Total RNA-Seq Kit and Illumina NextSeq high output reagents. Results: Analysis of xenografts from four patients showed that ctDNA was the predominant genetic material detected through liquid biopsy. Crucially, clonal analysis using Next Generation Sequencing (NGS) demonstrated that ctDNA provides an accurate reflection of the primary tumor's clonal heterogeneity. The method efficiently identified both main tumor clones and subclones, even those constituting as little as 1% of the tumor, with a 50% detection probability. Transcriptomic analysis of the tumor tissues uncovered a distinct mesenchymal and pro-metastatic signature, more pronounced in tumors with higher ctDNA release. Clonal analysis of CTCs and evRNA is still ongoing. Conclusions: This investigation affirms the effectiveness of ctDNA analysis in liquid biopsy for identifying subclonal diversity in TNBC. Our results suggest that liquid biopsy via ctDNA is capable of managing TNBC's extensive intra-tumoral heterogeneity, including the identification of smaller subclones. Furthermore, a link was identified between the release of ctDNA and a mesenchymal, prometastatic gene expression pattern in the tumors. Citation Format: Davide Ceresa, Barbara Cardinali, Antonio Daga, Roberta Tasso, Andrea Sciutto, Irene Appolloni, Daniela Marubbi, Franca Carli, Piero Fregatti, Paolo Malatesta, Lucia Del Mastro. Harnessing liquid biopsy and genetic barcoding in evaluating diversity within triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2405.

Abstract 6429: Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer

Abstract Objective: In the cancer research, a highly promising in vivo platform for personalized treatment involves the adoption of an advanced approach—specifically, the grafting of patient-derived xenografts (PDX) onto humanized mouse models. These models capture distinctive aspects of interaction with the human immune system and the tumor microenvironment (TME). In this study, we have engineered humanized triple-negative breast cancer (TNBC) PDX models to observe alterations in differentially expressed genes, thereby enhancing our understanding of the intricacies of personalized treatment in this context. Method: We utilized two TNBC PDX models established for the humanized models, where CD34+ hematopoietic stem cells were transplanted into immunodeficient mice. RNA sequencing was performed to identify altered gene expression in humanized PDX models compared to NSG PDXs. Additionally, we generated stable cell lines overexpressing TMEM176B in MDAMB231 and Hs578T cells, and we knocked down TMEM176B in 4T1 cells using recombinant techniques Results: In the humanized model, the growth of PDX tumors did not show a significantly faster or markedly different rate compared to previous studies. However, upon conducting RNA sequencing in the PDX model, the expression of the TMEM176B gene was consistently downregulated. Consequently, we transfected and observed that the increased expression of TMEM176B in MDAMB231 and Hs578T cell lines resulted in enhanced cell proliferation in vitro. Transplanting MDAMB231 cells overexpressing TMEM176B into athymic nude mice resulted in accelerated tumor growth. The study further investigates the expression of TMEM176B in an immune-competent environment by knocking down the gene in 4T1 cells or transplanting the control group into a balb/c mouse model. Tumors with TMEM176B knockdown exhibited inhibited growth, increased CD3+ T cell infiltration, and significant inhibition when treated with anti-PD-L1 compared to the control group. Conclusion: This research demonstrates the utility of PDX models, particularly humanized PDX models, in studying the complexities of cancer biology and the tumor microenvironment. The findings regarding TMEM176B and its role in tumorigenesis and immune responses in triple-negative breast cancer provide valuable insights for further research and potential therapeutic interventions. Citation Format: Yujeong Her, Jihui Yun, Woohang Heo, Jong-Il Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6429.

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

Abstract Introduction: Eliminating triple-negative breast cancer (TNBC) resistance to neoadjuvant chemotherapy is a critical unmet clinical need. TNBC accounts for 20% of breast cancer and is more aggressive due to increased metastasis, high recurrence rate, and significant resistance to chemotherapy (e.g., Doxorubicin, DOX). Napabucasin (NAPA, a small organic molecule) is recognized to kill cancer stem cells by targeting the transcription factor STAT3 pathway and is currently in clinical trials. Our previous in vitro results showed significant effects with hypoxia-responsive targeted polymersomes compared to control in MDA-MB-231 and Patient-Derived Xenograft (PDX) TNBC cells. Further, we observed increased NRP-1 expression in TNBC patient-derived xenograft (PDX) cells under hypoxic (0.2% oxygen) conditions compared to normoxia (21% oxygen). Hence, we hypothesize that targeted hypoxia-responsive polymersome drug therapy would reduce the tumor growth of the PDX mouse model of triple-negative breast cancer in vivo. Methods: We have established the TNBC PDX mouse model with two major phases, initial propagation (F1-F3) and final study phase (F3-F6), for in vivo study (TNBC tumor samples received from Sanford Broadway Clinic, Fargo, IRB approved). We utilized 3-6 months old female adult NSG mice (Jackson Lab) weighing 18-25 g. PDX tumor-bearing female NSG mice were administered polymersome-encapsulated drug (doxorubicin and other groups) by intravenous injection twice a week for 4 weeks and then observed the animals for an additional week. The percentage of tumor volume growth was calculated on these treatment groups and compared to vehicle. The tumors from all the groups were excised and dissected after the treatment and fixed in 10% formalin for histological evaluation. Tumors from all the groups after treatment were stained using hematoxylin and eosin to analyze the tumor core necrosis, and the average area of necrosis was compared among all the groups. Results: The TNBC PDX mouse model treated with DOX alone and a combination of encapsulated DOX with NAPA showed significant weight loss compared to the control (over 4 weeks). DOX-alone treated mice have shown reduced tumor growth and exhibited a significant difference compared to the control group. Notably, the targeted DOX-encapsulated polymersome demonstrated a much lower tumor volume and displayed a marked difference compared to non-targeted vehicles, free-DOX, and saline. In the initial analysis, we observed a higher necrosis in the tumor of the targeted DOX-encapsulated polymersome compared to the control. Summary and Conclusion: Overall, the hypoxia-responsive targeted polymersomes showed potent antitumor activity in the novel TNBC PDX animal model. With further developments, the targeted polymersomes might have translational potential as drug carriers for treating TNBC. Citation Format: Yogaraj S Ramakrishnan, Shubhashri Ambhore, Connor Edvall, Jiyan Mohammed, Sanku Mallik, Daniel M Tuvin, Venkatachalem Sathish. Development of hypoxia responsive targeted polymersomes drug therapy validated using patient derived xenograft of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3390.

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

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

Potential Perturbations of Critical Cancer-regulatory Genes in Triple-Negative Breast Cancer Cells Within the Humanized Microenvironment of Patient-derived Xenograft Models.

In this study, we aimed to establish humanized patient-derived xenograft (PDX) models for triple-negative breast cancer (TNBC) using cord blood (CB) hematopoietic stem cells (HSCs). Additionally, we attempted to characterize the immune microenvironment of the humanized PDX model to understand the potential implications of altered tumor-immune interactions in the humanized PDX model on the behavior of TNBC cells. To establish a humanized mouse model, high-purity CD34+ HSCs from CB were transplanted into immunodeficient NOD scid γ mice. Peripheral and intratumoral immune cell compositions of humanized and non-humanized mice were compared. Additionally, RNA sequencing of the tumor tissues was performed to characterize the gene expression features associated with humanization. After transplanting the CD34+ HSCs, CD45+ human immune cells appeared within five weeks. A humanized mouse model showed viable human immune cells in the peripheral blood, lymphoid organs, and in the tumor microenvironment. Humanized TNBC PDX models showed varying rates of tumor growth compared to that of non-humanized mice. RNA sequencing of the tumor tissue showed significant alterations in tumor tissues from the humanized models. tumor necrosis factor receptor superfamily member 11B (TNFRSF11B) is a shared downregulated gene in tumor tissues from humanized models. Silencing of TNFRSF11B in TNBC cell lines significantly reduced cell proliferation, migration, and invasion in vitro. Additionally, TNFRSF11B silenced cells showed decreased tumorigenicity and metastatic capacity in vivo. Humanized PDX models successfully recreated tumor-immune interactions in TNBC. TNFRSF11B, a commonly downregulated gene in humanized PDX models, may play a key role in tumor growth and metastasis. Differential tumor growth rates and gene expression patterns highlighted the complexities of the immune response in the tumor microenvironment of humanized PDX models.

Abstract LB_C04: Novel orally bioavailable macrocycles that target cyclin A and B elicit antitumor activity in breast cancer patient-derived xenograft models

Abstract Background: Cyclin/cyclin-dependent kinase (CDK) complexes regulate the activity of RB1 and E2F to drive cell cycle progression while ensuring fidelity of DNA replication. The interaction between cyclins and a critical subset of their substrates, including RB1 and E2F, is mediated by a conserved short linear RxL motif. RxL peptides have been shown to inhibit substrate phosphorylation by CDK2/cyclin A (AACR 2022 poster #5379). Previously, we have shown that macrocycles that selectively inhibit RxL-mediated binding to cyclins A and B (RxL inhibitors) lead to growth inhibitory activity in multiple cancer cell lines, and result in tumor regression in small cell lung cancer (SCLC) and ovarian cancer xenograft models (AACR 2023 poster #1560). Here, we test the antitumor activity of CID-016 in patient-derived xenograft (PDX) models from breast cancer. Methods: Bioinformatic analyses using RNA sequencing (RNAseq) and whole exome sequencing (WES) data from 39 breast cancer cell lines was employed to identify biomarker profiles that associate with sensitivity to CID-016. The antiproliferative activity of CID-016 was assessed by GI50 after 4-8 days of treatment. Hallmark pathway scores were calculated by Gene Set Variation Analysis (GSVA) method using MSigDb Hallmark collection from cell lines and existing PDX models. Based on the calculated hallmark pathway score, triple negative breast cancer (TNBC) PDX models were selected to test the anti-tumor activity of CID-016. In vivo, NMRI-Foxn1nu/nu mice were implanted with tumor fragments in the lower flank and treated with vehicle or CID-016 oral formulation of 50-100 mg/kg BID or 100 mg/kg QD, respectively for at least 28 days. Tumor volume and body weight were measured biweekly. Pharmacodynamic (PD) analyses will be conducted after treatment with CID-016. Results: Treatment of the breast cancer cell line panel with CID-016 demonstrated greater potency by GI50 in TNBC compared to other breast cancer cell lines (p=0.009). Gene expression levels of CCNB1, CCNA2, CCNE1 as well as target hallmark scores for “E2F targets”, “G2M checkpoint” and “Mitotic spindle” were significantly higher in TNBC vs other breast cancer cell lines. Oral treatment with CID-016 resulted in tumor regression in at least one triple-negative breast cancer PDX model and was well tolerated with no significant body weight changes. In vivo PD analyses will be assessed after 5 days of treatment with CID-016. Conclusions: The selective cyclin A/B RxL inhibitor CID-016 exhibits potent antitumor activity in TNBC preclinical models, consistent with the proposed mechanism of RB1/E2F pathway dysregulation. Given their compelling characteristics, orally bioavailable RxL inhibitors are advancing as the only first-in-class orally bioavailable dual inhibitors of cyclins A and B into clinical development. Citation Format: Mariana Paes Dias, Li-Fen Liu, Bernard Levin, Cristina Molina, Marta Guzmán, Olga Rodríguez, Evelyn W. Wang, Violeta Serra. Novel orally bioavailable macrocycles that target cyclin A and B elicit antitumor activity in breast cancer patient-derived xenograft models [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 LB_C04.

Discovery of VIP236, an αvβ3-Targeted Small-Molecule-Drug Conjugate with Neutrophil Elastase-Mediated Activation of 7-Ethyl Camptothecin Payload for Treatment of Solid Tumors.

The emerging field of small-molecule-drug conjugates (SMDCs) using small-molecule biomarker-targeted compounds for tumor homing may provide new perspectives for targeted delivery. Here, for the first time, we disclose the structure and the synthesis of VIP236, an SMDC designed for the treatment of metastatic solid tumors by targeting αvβ3 integrins and extracellular cleavage of the 7-ethyl camptothecin payload by neutrophil elastase in the tumor microenvironment. Imaging studies in the Lewis lung mouse model using an elastase cleavable quenched substrate showed pronounced elastase activity in the tumor. Pharmacokinetics studies of VIP236 in tumor-bearing mice demonstrated high stability of the SMDC in plasma and high tumor accumulation of the cleaved payload. Studies in bile-duct-cannulated rats showed that biliary excretion of the unmodified conjugate is the primary route of elimination. Treatment- and time-dependent phosphorylation of H2AX, a marker of DNA damage downstream of topoisomerase 1 inhibition, verified the on-target activity of the payload cleaved from VIP236 in vivo. Treatment with VIP236 resulted in long-lasting tumor regression in subcutaneous patient-derived xenograft (PDX) models from patients with non-small-cell lung, colon, and renal cancer as well as in two orthotopic metastatic triple-negative breast cancer PDX models. In these models, a significant reduction of brain and lung metastases also was observed.

Tumor targeting and therapeutic assessments of RNA nanoparticles carrying α9-nAChR aptamer and anti-miR-21 in triple-negative breast cancers

Triple-negative breast cancer (TNBC) is highly aggressive with a poor prognosis because of a lack of cell markers as drug targets. α9-Nicotinic acetylcholine receptor (nAChR) is expressed abundantly in TNBC; thus, it is a valuable biomarker for TNBC detection and treatment. In this study, we utilized thermodynamically stable three-way junction (3WJ) packaging RNA (pRNA) as the core to construct RNA nanoparticles with an α9-nAChR RNA aptamer as a targeting ligand and an anti-microRNA-21 (miR-21) as a therapeutic module. We compared the configuration of the two RNA nanoparticles and found that 3WJ-B-α9-nAChR-aptamer fluorescent RNA nanoparticles (3WJ-B-α9-apt-Alexa) exhibited better specificity for α9-nAChR in TNBC cells compared with 3WJ-C-α9-nAChR. Furthermore, 3WJ-B-α9-apt-Alexa bound more efficiently to TNBC patient-derived xenograft (PDX) tumors than 3WJ fluorescent RNA nanoparticles (3WJ-Alexa) with little or no accumulation in healthy organs after systemic injection in mice. Moreover, 3WJ-B-α9-nAChR-aptamer RNA nanoparticles carrying anti-miR-21 (3WJ-B-α9-apt-anti-miR-21) significantly suppressed TNBC-PDX tumor growth and induced cell apoptosis because of reduced miR-21 gene expression and upregulated the phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4) proteins. In addition, no pathological changes were detected upon toxicity examination of treated mice. In conclusion, the 3WJ-B-α9-nAChR-aptamer RNA nanoparticles established in this study efficiently deliver therapeutic anti-miR-21, indicating their potential as a novel TNBC therapy.

FRA-1 as a Regulator of EMT and Metastasis in Breast Cancer.

Among FOS-related components of the dimeric AP-1 transcription factor, the oncoprotein FRA-1 (encoded by FOSL1) is a key regulator of invasion and metastasis. The well-established FRA-1 pro-invasive activity in breast cancer, in which FOSL1 is overexpressed in the TNBC (Triple Negative Breast Cancer)/basal subtypes, correlates with the FRA-1-dependent transcriptional regulation of EMT (Epithelial-to-Mesenchymal Transition). After summarizing the major findings on FRA-1 in breast cancer invasiveness, we discuss the FRA-1 mechanistic links with EMT and cancer cell stemness, mediated by transcriptional and posttranscriptional interactions between FOSL1/FRA-1 and EMT-regulating transcription factors, miRNAs, RNA binding proteins and cytokines, along with other target genes involved in EMT. In addition to the FRA-1/AP-1 effects on the architecture of target promoters, we discuss the diagnostic and prognostic significance of the EMT-related FRA-1 transcriptome, along with therapeutic implications. Finally, we consider several novel perspectives regarding the less explored roles of FRA-1 in the tumor microenvironment and in control of the recently characterized hybrid EMT correlated with cancer cell plasticity, stemness, and metastatic potential. We will also examine the application of emerging technologies, such as single-cell analyses, along with animal models of TNBC and tumor-derived CTCs and PDXs (Circulating Tumor Cells and Patient-Derived Xenografts) for studying the FRA-1-mediated mechanisms in in vivo systems of EMT and metastasis.

Homologous recombination deficiency derived from whole-genome sequencing predicts platinum response in triple-negative breast cancers

The high frequency of homologous recombination deficiency (HRD) is the main rationale of testing platinum-based chemotherapy in triple-negative breast cancer (TNBC), however, the existing methods to identify HRD are controversial and there is a medical need for predictive biomarkers. We assess the in vivo response to platinum agents in 55 patient-derived xenografts (PDX) of TNBC to identify determinants of response. The HRD status, determined from whole genome sequencing, is highly predictive of platinum response. BRCA1 promoter methylation is not associated with response, in part due to residual BRCA1 gene expression and homologous recombination proficiency in different tumours showing mono-allelic methylation. Finally, in 2 cisplatin sensitive tumours we identify mutations in XRCC3 and ORC1 genes that are functionally validated in vitro. In conclusion, our results demonstrate that the genomic HRD is predictive of platinum response in a large cohort of TNBC PDX and identify alterations in XRCC3 and ORC1 genes driving cisplatin response.

Abstract 6553: The development of homologous recombination repair deficiency (HRD) score methodology using WES data and its application in patient-derived xenograft models

Abstract Homologous recombination repair deficiency (HRD) and mutations in genes involved with homologous recombination repair (HRR) are associated with sensitivity to platinum-based chemotherapy, topoisomerase 1 inhibitors and inhibitors targeting DNA repair pathway. Use of the Myriad HRD test for HRD determination in preclinical models is not feasible globally. To address this challenge, we developed an approach to generate HRD scores from whole exome sequencing (WES) data and applied it to assess HRD status in patient-derived xenograft (PDX) models. We used python package CNVkit for segmentation and gene-level/allele-specific copy number calling with customized settings according to CNVkit documentation. The reference was constructed using the WES BED file and hg38 reference genome when there are no paired control samples available. Next R package scarHRD was used to determine the HRD score that is the sum of loss of heterozygosity (LOH), telomere allele imbalances (TAI), and large fragment migrations (LST). The method was applied to the WES of 25 Xentech triple negative breast cancer (TNBC) PDX models to derive HRD scores. The samples were also separately profiled using Myriad’s MyChoice CDx assay. The two scores showed moderate correlation with Pearson r = 0.56. However, when n = 6 PDX models with low coverages (average depth <100x) were excluded, the Pearson r improved to r = 0.76. We also found that the absolute value of HRD score calculated by CNVkit/scarHRD is lower than that of Myriad HRD score. Furthermore, we evaluated the genomic landscape in this cohort of TNBC PDX models for the selected genes of HRR pathway (BRCA1, BRCA2, ATM, BRIP1, PALB2, RAD51C, BARD1, CDK12, CHEK1/2, FANCL, RAD51B, RAD51D, RAD54L). Sixteen models found to carry HRR mutations, with ATM and BRCA1 mutations being the most common ones, being found in 9 and 5 of 25 models, respectively. Although HRRm positive (HRRm+) models have higher HRD score for both Myriad HRD score and HRD score generated with CNVkit/scarHRD approach, the association between HRR (HRRm+/HRRm-) and Myriad HRD (HRD+/HRD- defined by HRD threshold of 42) is not statistically significant (Fisher’s exact test p value = 0.1998, odds ratio=3.5), likely due to small cohort sizes. These results suggest that combination of CNVkit and scarHRD for HRD score calculation may be an alternative approach for HRD identification in preclinical PDX models. Citation Format: Jixin Wang, Krista Kinneer, Wenyan Zhong, Zhongwu Lai. The development of homologous recombination repair deficiency (HRD) score methodology using WES data and its application in patient-derived xenograft models [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 6553.