Breast Cancer Patient-derived Xenograft Models Research Articles

PLK1 Inhibitor Onvansertib Enhances the Efficacy of Alpelisib in PIK3CA-Mutated HR-Positive Breast Cancer Resistant to Palbociclib and Endocrine Therapy: Preclinical Insights.

Endocrine therapy (ET) combined with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) is the preferred first-line treatment for hormone receptor-positive (HR+)/HER2- metastatic breast cancer. Although this is beneficial, acquired resistance leads to disease progression, and patients harboring PIK3CA mutations are treated with targeted therapies such as the PI3Kα inhibitor, alpelisib, alongside ET. Drug-associated resistance mechanisms limit the efficacy of alpelisib, highlighting the need for better combination therapies. This study aimed to evaluate the efficacy of combining alpelisib with a highly specific PLK1 inhibitor, onvansertib, in PIK3CA-mutant HR+ breast cancer preclinical models. We assessed the effect of the alpelisib and onvansertib combination on cell viability, PI3K signaling pathway, cell cycle phase distribution and apoptosis in PI3K-activated HR+ breast cancer cell lines. The antitumor activity of the combination was evaluated in three PIK3CA-mutant HR+ breast cancer patient-derived xenograft (PDX) models, resistant to ET and CDK4/6 inhibitor palbociclib. Pharmacodynamics studies were performed using immunohistochemistry and Simple Western analyses in tumor tissues. The combination synergistically inhibited cell viability, suppressed PI3K signaling, induced G2/M arrest and apoptosis in PI3K-activated cell lines. In the three PDX models, the combination demonstrated superior anti-tumor activity compared to the single agents. Pharmacodynamic studies confirmed the inhibition of both PLK1 and PI3K activity and pronounced apoptosis in the combination-treated tumors. Our findings support that targeting PLK1 and PI3Kα with onvansertib and alpelisib, respectively, may be a promising strategy for patients with PIK3CA-mutant HR+ breast cancer failing ET + CDK4/6i therapies and warrant clinical evaluation.

Investigation of the α9-nicotinic receptor single nucleotide polymorphisms induced oncogenic properties and molecular mechanisms in breast cancer.

α9-nAChR, a subtype of nicotinic acetylcholine receptor, is significantly overexpressed in female breast cancer tumor tissues compared to normal tissues. Previous studies have proposed that specific single nucleotide polymorphisms (SNPs) in the CHRNA9 (α9-nAChR) gene are associated with an increased risk of breast cancer in interaction with smoking. The study conducted a breast cancer risk assessment of the α9-nAChR SNP rs10009228 (NM_017581.4:c.1325A > G) in the Taiwanese female population, including 308 breast cancer patients and 198 healthy controls revealed that individuals with the heterozygous A/G or A/A wild genotype have an increased susceptibility to developing breast cancer in the presence of smoking compared to carriers of the G/G variant genotype. Our investigation confirmed the presence of this missense variation, resulting in an alteration of the amino acid sequence from asparagine (N442) to serine (S442) to facilitate phosphorylation within the α9-nAchR protein. Additionally, overexpression of N442 (A/A) in breast cancer cells significantly enhanced cell survival, migration, and cancer stemness compared to S442 (G/G). Four-line triple-negative breast cancer patient-derived xenograft (TNBC-PDX) models with distinct α9-nAChR rs10009228 SNP genotypes (A/A, A/G, G/G) further demonstrated that chronic nicotine exposure accelerated tumor growth through sustained activation of the α9-nAChR downstream oncogenic AKT/ERK/STAT3 pathway, particularly in individuals with the A/G or A/A genotype. Collectively, our study established the links between genetic variations in α9-nAChR and smoking exposure in promoting breast tumor development. This emphasizes the need to consider gene-environment interactions carefully while developing effective breast cancer prevention and treatment strategies.

Mapping the transcriptional evolution of human metastatic breast cancer.

Many aspects of breast cancer metastasis remain poorly understood, despite its clinical importance. In this issue of the JCI, Winkler et al. have applied an elegant patient-derived xenograft (PDX) model to map the transcriptomes of single cells in matched primary tumors and lung metastases across 13 breast cancer PDX models. They identified distinct transcriptional changes associated with metastatic evolution in lowly and highly metastatic primary tumors. Furthermore, by classifying the "epithelial-mesenchymal plasticity" (EMP) state of single cells, they revealed that considerable EMP heterogeneity exists among primary and metastatic human breast cancer cells. However, the EMP profile of a tumor does not change substantially upon metastasis. These findings give an unprecedentedly detailed view into the transcriptional heterogeneity and evolution of metastatic human breast cancer.

Unsupervised Segmentation of 3D Microvascular Photoacoustic Images Using Deep Generative Learning.

Mesoscopic photoacoustic imaging (PAI) enables label-free visualization of vascular networks in tissues with high contrast and resolution. Segmenting these networks from 3D PAI data and interpreting their physiological and pathological significance is crucial yet challenging due to the time-consuming and error-prone nature of current methods. Deep learning offers a potential solution; however, supervised analysis frameworks typically require human-annotated ground-truth labels. To address this, an unsupervised image-to-image translation deep learning model is introduced, the Vessel Segmentation Generative Adversarial Network (VAN-GAN). VAN-GAN integrates synthetic blood vessel networks that closely resemble real-life anatomy into its training process and learns to replicate the underlying physics of the PAI system in order to learn how to segment vasculature from 3D photoacoustic images. Applied to a diverse range of in silico, in vitro, and in vivo data, including patient-derived breast cancer xenograft models and 3D clinical angiograms, VAN-GAN demonstrates its capability to facilitate accurate and unbiased segmentation of 3D vascular networks. By leveraging synthetic data, VAN-GAN reduces the reliance on manual labeling, thus lowering the barrier to entry for high-quality blood vessel segmentation (F1 score: VAN-GAN vs. U-Net = 0.84 vs. 0.87) and enhancing preclinical and clinical research into vascular structure andfunction.

Evaluation of Copanlisib in Combination with Eribulin in Triple-negative Breast Cancer Patient-derived Xenograft Models.

In this research, we demonstrated that the pan-PI3K inhibitor copanlisib enhanced the cytotoxicity of eribulin in a panel of TNBC PDX models. The improved tumor growth inhibition was irrespective of PI3K pathway alteration and was corroborated by the enhanced mitotic arrest and apoptotic induction observed in PDX tumors after combination therapy compared with each drug alone. These data provide the preclinical rationale for the clinical testing in TNBC.

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 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 742: PYX-201, a stroma-targeting ADC composed of an anti-EDB+FN antibody conjugated to Auristatin0101, demonstrates strong anti-tumor efficacy across multiple human cancer indications in pre-clinical PDX tumor models

Abstract Stroma is crucial to support solid tumor growth, metastasis and resistance to treatment. While most antibody-drug conjugates (ADCs) directly bind to cancer cells, PYX-201, an investigational drug, is designed to target tumor stroma by binding to the extra-domain B splice variant of fibronectin (EDB+FN), a matrix protein abundantly expressed in the tumor microenvironment of many solid tumors with absent or low expression in normal adult tissues. In vitro studies demonstrated PYX-201 was strongly cytotoxic to EDB+FN positive Caki2 cells, but not to EDB+FN negative HT29 cells. In addition, PYX-201 was 30-times more potent than a non-targeted ADC control, demonstrating PYX-201 cytotoxic effects were EDB+FN dependent. Furthermore, when Caki2 and HT29-luciferase cancer cells were co-cultured, the luciferase activity was used to quantify the amount of EDB+FN negative HT29 cells only. Interestingly, PYX-201 was able to kill EDB+FN negative cancer cells in a co-culture assay as shown by a decrease in luciferase activity, demonstrating PYX-201 bystander activity. Pre-clinical mouse model studies were conducted to evaluate PYX-201 anti-tumor efficacy and pharmacokinetic (PK) properties. First, a comprehensive patient-derived xenograft (PDX) mouse model trial was designed to evaluate the anti-tumor efficacy of PYX-201. To fully represent the diversity and characteristics of human tumors, PDX models were pre-selected from ten solid tumor indications based on different levels of EDB+FN protein expression and stromal density. PYX-201 (Q4D × 4 at 3mg/kg) was well tolerated, and strong anti-tumor responses (% Tumor Growth Inhibition [TGI] >90%) were observed in PDX models across various cancer indications. Potential correlations between EDB+FN protein expression with anti-tumor activity will be evaluated. Remarkably, long-term anti-tumor efficacy of PYX-201 was observed as tumors did not relapse even after >100 days in a breast cancer PDX model. Additionally, plasma samples were analyzed from a cell line derived xenograft mouse model after a single dose of PYX-201 to determine its PK profile. At 3 mg/kg, the maximum concentration (Cmax) was 28.1 μg/mL, area under the curve (AUC) was 1,732.7 h*μg/mL, with a low clearance of 1.5 mL/h/kg, and the half-life of the mAb from PYX-201 was calculated at 81.2 h. These PK parameters were associated with strong PYX-201 anti-tumor efficacy (% TGI >90%) in the H1975 xenograft model. Altogether, PYX-201 was well tolerated and demonstrated strong anti-tumor efficacy in a variety of pre-clinical human PDX models. PYX-201 is a promising and innovative ADC which is currently under investigation in a Phase I clinical trial (NCT05720117). Citation Format: Nicolas Severe, Amanda Facklam, Liz McMichael, Biplab Das, Sara Lewandowski, Justin Trickett, Liyang Diao, Chengfeng Merriman, Chuan Shen, Jianwen Feng, Shawn Harriman, Marsha Crochiere, Philipp Steiner, Jan Pinkas. PYX-201, a stroma-targeting ADC composed of an anti-EDB+FN antibody conjugated to Auristatin0101, demonstrates strong anti-tumor efficacy across multiple human cancer indications in pre-clinical PDX tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 742.

Abstract 5911: In vivo efficacy of a novel peptide-conjugated drug in patient-derived xenograft models of breast cancer

Abstract Introduction: Antibody and peptide-conjugated drugs are rapidly entering the clinic for the treatment of patients with early and advanced breast cancer. These drugs are designed to achieve more targeted delivery of cytotoxic agents to tumor cells with robust clinical activity. ARB-1-6, a sortilin (SORT1) targeted peptide conjugated to the cytotoxic agent MMAE, has demonstrated pre-clinical activity and tolerability in cell line xenografts of breast cancer. To further characterize its spectrum of antitumor activity and biomarker correlates we evaluated ARB-1-6 in a collection of clinically and genomically annotated breast cancer patient-derived xenografts (PDX). Methods: Breast cancer PDX models were generated and propagated under IRB approved protocols [14-8358]. ARB-1-6 was dosed in tumor-bearing SCID mice by tail vein injection at 3 mg/kg weekly x 4 doses. Tumor volume was measured twice weekly until day 35 or until models reached humane endpoints, after which tumors were collected. Animals were weighed regularly and assessed for treatment-related toxicity. Results: 7 models (5 TN and 2 HR) have completed efficacy assessment to date. 3/7 models were derived from treatment naïve patients while 4/7 were derived from pre-treated patients (Table 1). Complete response (mRECIST) was seen in 5/7 (71.4%) models with stable disease occurring in 2/7 (28.6%). ARB-1-6 was well tolerated with no weight loss observed. Complete tumor regression was seen in a PDX derived from a patient with clinical resistance to a TROP2 antibody drug conjugate (ADC). Antitumor activity was observed in models with low SORT1 RNA expression; SORT1 IHC and other correlatives studies are underway. Conclusion: ARB-1-6 demonstrates substantial preclinical activity in PDX models at doses that are well tolerated in mice. Treatment of additional models is ongoing. Full results and correlative analyses including relationships between antitumor activity and target protein expression will be presented. PDX Model Characteristics and Patient Treatment PDX Subtype Pt. Treatment 1 TN AC-T 2 TN FEC-D, Gem+Carbo 3 TN AC-T 4 TN No 5 TN No 6 HR No 7 HR TC, ET, Cape, Gem+Cis, TROP2-ADC Citation Format: Mitchell J. Elliott, Meghan Mcguire, Jennifer Silvester, Chantal Tobin, Samah El Ghamrasni, Francine Lui, Andrew Zhai, Ozge Yoluk, Aron Broom, Tracy Stone, Glenn Butterfoss, Serban Popa, Tianyu Lu, Chris Ing, David White, David W. Cescon. In vivo efficacy of a novel peptide-conjugated drug in patient-derived xenograft models of 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 5911.

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 907: ScreenDL: A transfer learning framework integrating tumor omics and functional drug screening for personalized clinical drug response prediction

Abstract Precision oncology hinges on accurate prediction of patient-specific treatment response from tumor molecular inputs. While deep learning (DL) models achieve state-of-the-art response prediction in cell lines, existing methods do not readily translate to the clinic where training data is limited. Here, we have developed and validated ScreenDL, a novel DL framework designed explicitly for use in clinical precision oncology applications. The underlying architecture of ScreenDL consists of two fully connected branches dedicated to learning rich embeddings of a drug’s chemical structure and a tumor’s transcriptomic profile respectively. These tumor omic and drug molecular embeddings are fed into a shared response prediction subnetwork which outputs predicted ln(IC50) values. The ScreenDL training schema incorporates three phases, each designed to improve clinical response prediction: (1) an initial pretraining phase leverages large-scale cell line pharmacogenomic datasets to extract patterns/relationships linking tumor omics and drug response; (2) subsequent transfer learning integrates pharmacogenomic data from patient-derived models of cancer, adapting the pretrained model to a more patient-relevant context; (3) patient-level omics and drug screening data (e.g., from matched patient-derived organoid (PDO) models) is integrated through patient-specific fine-tuning, generating a patient-specific response prediction model. Importantly, biomaterial from surgical tumor resection is often available for organoid establishment and patient-specific drug screening in practical clinical scenarios. To assess the utility of our ScreenDL framework for clinical response prediction, we applied ScreenDL to predict treatment response in 50 advanced/metastatic triple negative breast cancer (TNBC) patient-derived xenograft models (PDX). We leveraged tumor omics profiles for each PDX and drug screening data from matched PDX-derived organoids (PDxOs), mirroring the combination of patient-level tumor omic characterization and functional drug screening in matched PDOs, a protocol currently being piloted in Functional Precision Oncology trials at the University of Utah Huntsman Cancer Institute. After cell line pretraining, ScreenDL achieves a modest median Pearson correlation per drug of 0.15 relative to 0.03 for the leading compared model. However, transfer learning and subsequent PDX-specific fine-tuning significantly improve performance, producing median Pearson correlations per drug of 0.39 and 0.51, respectively. These results demonstrate the ability of our ScreenDL framework to dramatically improve response prediction in clinically relevant domains, bringing DL-based precision treatment selection closer to clinical application. Citation Format: Casey Sederman, Tonya Di Sera, Yi Qiao, Xiaomeng Huang, Bryan E. Welm, Alana L. Welm, Gabor Marth. ScreenDL: A transfer learning framework integrating tumor omics and functional drug screening for personalized clinical drug response prediction [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 907.

Preclinical Efficacy of Cabazitaxel Loaded Poly(2-alkyl cyanoacrylate) Nanoparticle Variants.

Biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs) are receiving increasing attention in anti-cancer nanomedicine development not only for targeted cancer chemotherapy, but also for modulation of the tumor microenvironment. We previously reported promising results with cabazitaxel (CBZ) loaded poly(2-ethylbutyl cyanoacrylate) NPs (PEBCA-CBZ NPs) in a patient derived xenograft (PDX) model of triple-negative breast cancer, and this was associated with a decrease in M2 macrophages. The present study aims at comparing two endotoxin-free PACA NP variants (PEBCA and poly(2-ethylhexyl cyanoacrylate); PEHCA), loaded with CBZ and test whether conjugation with folate would improve their effect. Cytotoxicity assays and cellular uptake of NPs by flow cytometry were performed in different breast cancer cells. Biodistribution and efficacy studies were performed in PDX models of breast cancer. Tumor associated immune cells were analyzed by multiparametric flow cytometry. In vitro studies showed similar NP-induced cytotoxicity patterns despite difference in early NP internalization. On intravenous injection, the liver cleared the majority of NPs. Efficacy studies in the HBCx39 PDX model demonstrated an enhanced effect of drug-loaded PEBCA variants compared with free drug and PEHCA NPs. Furthermore, the folate conjugated PEBCA variant did not show any enhanced effects compared with the unconjugated counterpart which might be due to unfavorable orientation of folate on the NPs. Finally, analyses of the immune cell populations in tumors revealed that treatment with drug loaded PEBCA variants affected the myeloid cells, especially macrophages, contributing to an inflammatory, immune activated tumor microenvironment. We report for the first time, comparative efficacy of PEBCA and PEHCA NP variants in triple negative breast cancer models and show that CBZ-loaded PEBCA NPs exhibit a combined effect on tumor cells and on the tumor associated myeloid compartment, which may boost the anti-tumor response.

PROM2 overexpression induces metastatic potential through epithelial-to-mesenchymal transition and ferroptosis resistance in human cancers.

Despite considerable therapeutic advances in the last 20 years, metastatic cancers remain a major cause of death. We previously identified prominin-2 (PROM2) as a biomarker predictive of distant metastases and decreased survival, thus providing a promising bio-target. In this translational study, we set out to decipher the biological roles of PROM2 during the metastatic process and resistance to cell death, in particular for metastatic melanoma. Methods and results: We demonstrated that PROM2 overexpression was closely linked to an increased metastatic potential through the increase of epithelial-to-mesenchymal transition (EMT) marker expression and ferroptosis resistance. This was also found in renal cell carcinoma and triple negative breast cancer patient-derived xenograft models. Using an oligonucleotide anti-sense anti-PROM2, we efficaciously decreased PROM2 expression and prevented metastases in melanoma xenografts. We also demonstrated that PROM2 was implicated in an aggravation loop, contributing to increase the metastatic burden both in murine metastatic models and in patients with metastatic melanoma. The metastatic burden is closely linked to PROM2 expression through the expression of EMT markers and ferroptosis cell death resistance in a deterioration loop. Our results open the way for further studies using PROM2 as a bio-target in resort situations in human metastatic melanoma and also in other cancer types.

DSBSO-Based XL-MS Analysis of Breast Cancer PDX Tissues to Delineate Protein Interaction Network in Clinical Samples.

Protein-protein interactions (PPIs) are fundamental to understanding biological systems as protein complexes are the active molecular modules critical for carrying out cellular functions. Dysfunctional PPIs have been associated with various diseases including cancer. Systems-wide PPI analysis not only sheds light on pathological mechanisms, but also represents a paradigm in identifying potential therapeutic targets. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for defining endogenous PPIs of cellular networks. While proteome-wide studies have been performed in cell lysates, intact cells and tissues, applications of XL-MS in clinical samples have not been reported. In this study, we adopted a DSBSO-based in vivo XL-MS platform to map interaction landscapes from two breast cancer patient-derived xenograft (PDX) models. As a result, we have generated a PDX interaction network comprising 2,557 human proteins and identified interactions unique to breast cancer subtypes. Interestingly, most of the observed differences in PPIs correlated well with protein abundance changes determined by TMT-based proteome quantitation. Collectively, this work has demonstrated the feasibility of XL-MS analysis in clinical samples, and established an analytical workflow for tissue cross-linking that can be generalized for mapping PPIs from patient samples in the future to dissect disease-relevant cellular networks.

Doublet and Triplet Combinations of Eribulin, Fulvestrant, and Palbociclib in Preclinical Breast Cancer Models.

This study investigated in vivo synergism between eribulin and palbociclib in a breast cancer patient-derived xenograft (PDX) model, with expanded scope to include fulvestrant as a third drug. Eribulin plus palbociclib combinations were tested in vitro in six cell lines each of estrogen receptor positive and triple-negative breast cancer, and in vivo in the OD-BRE-0192 PDX model using weekly eribulin plus 5×/week or 7×/week palbociclib (holiday or no-holiday schedules, respectively). When included as a third drug, fulvestrant was dosed weekly. In vitro, combining palbociclib with eribulin led to increased eribulin IC50s in 11 of 12 cell lines, suggesting that the drugs antagonized each other due to mutual exclusion of the mitotic and G1/S cell cycle block points for eribulin and palbociclib. An in vivo study in the OD-BRE-0192 PDX model compared weekly eribulin plus either palbociclib holiday or no-holiday schedules to gauge the importance of post-palbociclib cell cycle synchronization. Results showed no advantage of holiday over no-holiday schedules, arguing that differing pharmacokinetics of the drugs were sufficient to overcome cell cycle-based mechanistic antagonism. In vivo comparisons of doublet and triplet combinations of eribulin, palbociclib, and fulvestrant showed that all three doublets were superior to individual monotherapies, and that the triplet combination was markedly superior to all three doublets, being the only group to show tumor regression in 100% of the mice. Results show complex synergistic interactions between eribulin, fulvestrant, and palbociclib, and point to a particularly robust synergy when combining all three drugs.

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.

ABCB1 Regulates Immune Genes in Breast Cancer.

Resistance to standard chemotherapy is a critical problem for breast cancer patients. The ATP-binding cassette (ABC) superfamily transporters actively pump out drugs and play an important role in chemoresistance. ABCB1 (ABC subfamily B, member 1, also named as multidrug resistance protein 1, MDR1) and suppressive myeloid-derived suppressor cells (MDSCs) potentially involve in chemoresistance of breast cancer. The relationship between ABCB1 and immune genes in breast cancer has not been widely studied. Microarray and RNA sequencing data were obtained from The Cancer Genome Atlas Breast Invasive Carcinoma in Genomic Data Commons Data Portal and Gene Expression Omnibus database. A patient-derived xenograft (PDX) model of HER2+ breast cancer was established to investigate the association between ABCB1 and immune genes in breast cancer. Expression of ABCB1 increased in doxorubicin-selected MCF-7/ADR cells. High expression of ABCB1 mRNA is correlated with lymph-node metastasis and worse overall survival in patients with breast cancer. ABCB1 is positively correlated with IL6, CSF1, CSF3, and PTGS2. In the HER2+ stage IIA breast cancer PDX model, both doxorubicin and paclitaxel suppressed growth of P2 tumors. IL6, CSF1, CSF3, and PTGS2 expression were suppressed by paclitaxel but not doxorubicin. Intrasplenic MDSCs, including CD11b+Ly6G+ and CD11b+Ly6C+ cells, were more abundant than intratumor MDSCs in PDX-carrying nude mice. Clinically, the patient developed cancer recurrence after adjuvant chemotherapy with doxorubicin-based regimen and was well controlled after paclitaxel-trastuzumab combined therapy. ABCB1 was a poor predictor of HER2+ LN- breast cancer. Regulation of immune genes by ABCB1 contributed to cancer recurrence and treatment effect. The PDX model was suitable for investigation the expression of target genes and expansion of immune cells.

Engineering liquid metal-based nanozyme for enhancing microwave dynamic therapy in breast cancer PDX model

BackgroundsThe novel concept of microwave dynamic therapy (MDT) solves the problem of incomplete tumor eradication caused by non-selective heating and uneven temperature distribution of microwave thermal therapy (MWTT) in clinic, but the poor delivery of microwave sensitizer and the obstacle of tumor hypoxic microenvironment limit the effectiveness of MDT.ResultsHerein, we engineer a liquid metal-based nanozyme LM@ZIF@HA (LZH) with eutectic Gallium Indium (EGaIn) as the core, which is coated with CoNi-bimetallic zeolite imidazole framework (ZIF) and hyaluronic acid (HA). The flexibility of the liquid metal and the targeting of HA enable the nanozyme to be effectively endocytosed by tumor cells, solving the problem of poor delivery of microwave sensitizers. Due to the catalase-like activity, the nanozyme catalyze excess H2O2 in the tumor microenvironment to generate O2, alleviating the restriction of the tumor hypoxic microenvironment and promoting the production of ROS under microwave irradiation. In vitro cell experiments, the nanozyme has remarkable targeting effect, oxygen production capacity, and microwave dynamic effect, which effectively solves the defects of MDT. In the constructed patient-derived xenograft (PDX) model, the nanozyme achieves excellent MDT effect, despite the heterogeneity and complexity of the tumor model that is similar to the histological and pathological features of the patient. The tumor volume in the LZH + MW group is only about 1/20 of that in the control group, and the tumor inhibition rate is as high as 95%.ConclusionThe synthesized nanozyme effectively solves the defects of MDT, improves the targeted delivery of microwave sensitizers while regulating the hypoxic microenvironment of tumors, and achieves excellent MDT effect in the constructed PDX model, providing a new strategy for clinical cancer treatment.

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.

Riluzole Suppresses Growth and Enhances Response to Endocrine Therapy in ER+ Breast Cancer.

Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a significant clinical problem. Riluzole is FDA-approved for the treatment of amyotrophic lateral sclerosis. A benzothiazole-based glutamate release inhibitor with several context-dependent mechanism(s) of action, riluzole has shown antitumor activity in multiple malignancies, including melanoma, glioblastoma, and breast cancer. We previously reported that the acquisition of tamoxifen resistance in a cellular model of invasive lobular breast cancer is accompanied by the upregulation of GRM mRNA expression and growth inhibition by riluzole. We tested the ability of riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated invasive lobular breast cancer patient-derived xenograft model HCI-013EI. Single-agent riluzole suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing a histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). Riluzole induced apoptosis and ferroptosis and reduced phosphorylation of multiple prosurvival signaling molecules, including Akt/mTOR, CREB, and Fak/Src family kinases. Riluzole, in combination with either fulvestrant or 4-hydroxytamoxifen, additively suppressed ER+ breast cancer cell growth in vitro. Single-agent riluzole significantly inhibited HCI-013EI patient-derived xenograft growth in vivo, and the combination of riluzole plus fulvestrant significantly reduced proliferation in ex vivo primary breast tumor explant cultures. Riluzole may offer therapeutic benefits in diverse ER+ breast cancers, including lobular breast cancer.