Cancer Patient-derived Xenograft Research Articles

The MURAL collection of prostate cancer patient-derived xenografts enables discovery through preclinical models of uro-oncology

Preclinical testing is a crucial step in evaluating cancer therapeutics. We aimed to establish a significant resource of patient-derived xenografts (PDXs) of prostate cancer for rapid and systematic evaluation of candidate therapies. The PDX collection comprises 59 tumors collected from 30 patients between 2012–2020, coinciding with availability of abiraterone and enzalutamide. The PDXs represent the clinico-pathological and genomic spectrum of prostate cancer, from treatment-naïve primary tumors to castration-resistant metastases. Inter- and intra-tumor heterogeneity in adenocarcinoma and neuroendocrine phenotypes is evident from bulk and single-cell RNA sequencing data. Organoids can be cultured from PDXs, providing further capabilities for preclinical studies. Using a 1 x 1 x 1 design, we rapidly identify tumors with exceptional responses to combination treatments. To govern the distribution of PDXs, we formed the Melbourne Urological Research Alliance (MURAL). This PDX collection is a substantial resource, expanding the capacity to test and prioritize effective treatments for prospective clinical trials in prostate cancer.

Constitutive BAK/MCL1 complexes predict paclitaxel and S63845 sensitivity of ovarian cancer

We previously found that preformed complexes of BAK with antiapoptotic BCL2 proteins predict BH3 mimetic sensitivities in lymphohematopoietic cells. These complexes have not previously been examined in solid tumors or in the context of conventional anticancer drugs. Here we show the relative amount of BAK found in preformed complexes with MCL1 or BCLXL varies across ovarian cancer cell lines and patient-derived xenografts (PDXs). Cells bearing BAK/MCL1 complexes were more sensitive to paclitaxel and the MCL1 antagonist S63845. Likewise, PDX models with BAK/MCL1 complexes were more likely to respond to paclitaxel. Mechanistically, BIM induced by low paclitaxel concentrations interacted preferentially with MCL1 and displaced MCL1-bound BAK. Further studies indicated that cells with preformed BAK/MCL1 complexes were sensitive to the paclitaxel/S63845 combination, while cells without BAK/MCL1 complexes were not. Our study suggested that the assessment of BAK/MCL1 complexes might be useful for predicting response to paclitaxel alone or in combination with BH3 mimetics.

Cotargeting of miR-126-3p and miR-221-3p inhibits PIK3R2 and PTEN, reducing lung cancer growth and metastasis by blocking AKT and CXCR4 signalling.

Lung cancer is the leading cause of cancer‐related death worldwide. Late diagnosis and metastatic dissemination contribute to its low survival rate. Since microRNA (miRNA) deregulation triggers lung carcinogenesis, miRNAs might represent an interesting therapeutic tool for lung cancer management. We identified seven miRNAs, including miR‐126‐3p and miR‐221‐3p, that are deregulated in tumours compared with normal tissues in a series of 38 non‐small‐cell lung cancer patients. A negative correlation between these two miRNAs was associated with poor patient survival. Concomitant miR‐126‐3p replacement and miR‐221‐3p inhibition, but not modulation of either miRNA alone, reduced lung cancer cell viability by inhibiting AKT signalling. PIK3R2 and PTEN were validated as direct targets of miR‐126‐3p and miR‐221‐3p, respectively. Simultaneous miRNA modulation reduced metastatic dissemination of lung cancer cells both in vitro and in vivo through CXCR4 inhibition. Systemic delivery of a combination of miR‐126‐3p mimic and miR‐221‐3p inhibitor encapsulated in lipid nanoparticles reduced lung cancer patient‐derived xenograft growth through blockade of the PIK3R2–AKT pathway. Our findings reveal that cotargeting miR‐126‐3p and miR‐221‐3p to hamper both tumour growth and metastasis could be a new therapeutic approach for lung cancer.

Discovery of novel KRAS‒PDEδ inhibitors with potent activity in patient-derived human pancreatic tumor xenograft models

KRAS‒PDEδ interaction is revealed as a promising target for suppressing the function of mutant KRAS. The bottleneck in clinical development of PDEδ inhibitors is the poor antitumor activity of known chemotypes. Here, we identified novel spiro-cyclic PDEδ inhibitors with potent antitumor activity both in vitro and in vivo. In particular, compound 36l (KD = 127 ± 16 nmol/L) effectively bound to PDEδ and interfered with KRAS–PDEδ interaction. It influenced the distribution of KRAS in Mia PaCa-2 cells, downregulated the phosphorylation of t-ERK and t-AKT and promoted apoptosis of the cells. The novel inhibitor 36l exhibited significant in vivo antitumor potency in pancreatic cancer patient-derived xenograft (PDX) models. It represents a promising lead compound for investigating the druggability of KRAS‒PDEδ interaction.

Antitumor efficacy of XPO1 inhibitor Selinexor in KRAS-mutant lung adenocarcinoma patient-derived xenografts

Gain-of-function Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations occur in 25% of lung adenocarcinomas, and these tumors are challenging to treat. Some preclinical work, largely based on cell lines, suggested KRASmut lung cancers are especially dependent on the nuclear export protein exportin-1 (XPO1), while other work supports XPO1 being a broader cancer dependency. To investigate the sensitivity of KRASmut lung cancers to XPO1 inhibition in models that more closely match clinical tumors, we treated 10 independently established lung cancer patient-derived tumor xenografts (PDXs) with the clinical XPO1 inhibitor, Selinexor. Monotherapy with Selinexor reduced tumor growth in all KRASmut PDXs, which included 4 different codon mutations, and was more effective than the clinical MEK1/2 inhibitor, Trametinib. Selinexor was equally effective in KRASG12C and KRASG12D tumors, with TP53 mutations being a biomarker for a weaker drug response. By mining genome-wide dropout datasets, we identified XPO1 as a universal cancer cell dependency and confirmed this functionally in two KRASWT PDX models harboring kinase drivers. However, targeted kinase inhibitors were more effective than Selinexor in these models. Our findings support continued investigation of XPO1 inhibitors in KRASmut lung adenocarcinoma, regardless of the codon alteration.

Characterization of a RAD51C-silenced high-grade serous ovarian cancer model during development of PARP inhibitor resistance.

Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

Abstract 3020: Human leukocyte antigen (HLA) typing of a broad panel of cancer patient-derived xenograft (PDX) models for immune therapies

Abstract Background In immuno-oncology research, an appropriate microenvironment for both human tumor cells and human immune cells is an important but challenging factor for successful development of relevant preclinical models. Humanized patient-derived xenograft (PDX) models can support establishment of such an environment based on matching human leukocyte antigen (HLA) profiles of PDX and compatible human immune cell populations. In this study, we determined the individual HLA types of a broad panel of 291 established PDX models from 18 different tumor entities. Furthermore, we performed comprehensive HLA matching analyses of all PDX models and 13 peripheral blood mononuclear cell (PBMC) donors to enable personalized studies with HLA-matched PDX. Methods The HLA profile of each PDX was determined comprising HLA class I, II and non-class types in 4-digit resolution. Matching analyses were performed according to donor-recipient HLA matching criteria recommended by the Blood and Marrow Transplant Clinical Trials Network (BMT CTN). For comparative HLA profile analyses, allele frequencies of 8862 German stem cell donors provided by The Allele Frequency Net Database were downloaded. Results The individual HLA profiles of all PDX models were generated comprising HLA-A, -B and -C (class I), HLA-DQA1, -DQB1, -DRB1, -DPA1 and -DPB1 (class II) as well as HLA-H (non-class) types. Comparative analyses of HLA profiles revealed that obtained frequencies of PDX HLA alleles are comparable with frequencies of a representative population of 8862 German stem cell donors. HLA profile matching analyses performed for all PDX models and PBMC donors resulted in 15 matches regarding PDX from head and neck, lung, mammary and pancreatic carcinoma as well as from endometrial cancer, glioma, lymphoma, melanoma and sarcoma. Dependence of immune therapy efficacy on matched HLA profiles was shown exemplarily for a head and neck squamous cell cancer (HNSCC) PDX. The HNSCC PDX was humanized with PBMC from 5 donors representing different degrees of HLA matching (33 - 100 %). Treatment with the checkpoint inhibitor Nivolumab caused a more powerful tumor growth inhibition in matched HLA profiles compared to non-matched profiles. This indicated a relation between antitumoral immune therapy effect and HLA matching in preclinical models. Conclusions We generated a comprehensive HLA profile portfolio containing information of a broad panel of PDX models. Matched HLA profiles of PDX models and PBMC donors enable personalized, preclinical immune-oncology studies to support the development of novel therapeutic strategies. Citation Format: Theresia Conrad, Annika Wulf-Goldenberg, Maria Stecklum, Michael Becker, Konrad Klinghammer, Jens Hoffmann. Human leukocyte antigen (HLA) typing of a broad panel of cancer patient-derived xenograft (PDX) models for immune therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3020.

Abstract 3083: Role of S100A10 in the metastatic colonization of breast cancer stem cells

Abstract Breast cancer is now the most frequently diagnosed cancer and the leading global cause of cancer death in women. Despite advances in the diagnosis and treatment of human breast cancer, advanced stage, metastatic breast cancers are difficult to cure. Among the common target organs for breast cancer metastasis, post recurrence survival is worst when multiple metastasis was observed in the liver. Cancer stem cells (CSCs) are a sub-population of the cells that retain high tumorigenic capacity and, at the same time, are able to sustain the formation of tumors that recreate the cellular diversity of the parent lesions from which they have been originally isolated. We and others have shown that in epithelial malignancies such as breast and colorectal cancers, self-renewal ability of cancer cells with CSC properties is epigenetically regulated by their expression of microRNAs, such as with miR-200c, miR-142 and miR-221. Because of their highly tumorigenic properties, CSCs with are associated with metastatic progression, especially at the initial steps of metastases. We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. Comparison of gene expression profiles between the CD44+ human breast cancer cells metastasized to the liver (‘metastasized CSCs') and those in the primary site (‘primary CSCs') revealed that S100A10 was upregulated in metastasized CSCs than in primary CSCs. Knockdown of S100A10 in breast cancer cells reduced the matrix metalloproteinase activity and suppressed their invasion abilities in transwell cell invasion assays in which upper surface of transwell inserts were precoated with Matrigel. Knockdown of S100A10 suppressed, and overexpression of S100A10 enhanced the 3D organoid formation capacities of breast cancer PDX cells in vitro. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. Upregulation of S100 family protein expression is observed in many human cancers, including breast, lung, bladder and gastric cancers. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers. Citation Format: Yohei Shimono, Hisano Yanagi, Takashi Watanabe, Tatsunori Nishimura, Takanori Hayashi, Seiji Okada, Motoshi Suzuki, Kenji Kawada, Hironobu Minami, Noriko Gotoh. Role of S100A10 in the metastatic colonization of breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3083.

Abstract 1130: First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation

Abstract KAT6A is a lysine histone acetyltransferase (HAT) of the MYST family of HATs. KAT6A, and its paralog KAT6B, have been shown to acetylate histone H3K23Ac and regulate diverse biological processes, including transcription, cell-cycle progression, stem cell maintenance and development. Molecular dysregulation of KAT6A has been observed in several cancers, including amplifications in breast, lung, ovarian cancer along with oncogenic fusions in AML. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in 10-15% of the patient population, which correlates with a worse clinical outcome in the estrogen receptor+ (ER+) subtype. Here we present identification of a first-in-class potent KAT6A/KAT6B tool inhibitor CTx-648 (PF-9363), that possesses high selectivity versus other MYST family members (KAT7, KAT5, KAT8) and other KATs, demonstrating anti-tumor activity in breast cancer. Using genetic and pharmacological approaches, we have demonstrated several ER+ breast cancer cell lines including KAT6A amplified and over-expressing models, are dependent on KAT6A enzymatic function. Epigenomic profiling studies using bulk and nascent RNA-seq combined with ATAC-seq revealed CTx-648 leads to downregulation of a specific set of genes involved in ESR1 pathway, cell cycle and stem cell pathways. In vivo target validation studies showed strong anti-tumor activity of CTx-648 in several ER+ breast cancer cell line and patient-derived xenograft models, including models harboring endocrine therapy resistance ESR1 mutations, highlighting promise for this novel therapy in ER+ breast cancer population. Based on the strength of the pre-clinical data, a selective KAT6 inhibitor (PF-07248144) is now commencing a Phase 1 clinical study in Advanced or Metastatic Solid Tumors. Citation Format: Shikhar Sharma, Jay Chung, Sean Uryu, Amanda Rickard, Natalie Nady, Showkhin Khan, Zhenxiong Wang, Yong Zhang, Haikuo Zhang, Pei-Pei Kung, Eric Greenwald, Karen Maegley, Patrick Bingham, Hieu Lam, Ylva E. Bozikis, Hendrik Falk, Elizabeth Allan, Vicky M. Avery, Miriam S. Butler, Michelle A. Camerino, Catalina Carrasco-Pozo, Susan A. Charman, Melissa J. Davis, Mark A. Dawson, Dawson Sarah-Jane, Melanie de Silva, Matthew L. Dennis, Olan Dolezal, Rachel Lagiakos, Geoffrey J. Lindeman, Laura MacPherson, Stewart Nuttall, Thomas S. Peat, Bin Ren, Alexandra E. Stupple, Elliot Surgenor, Chin Wee Tan, Tim Thomas, Jane E. Visvader, Anne K. Voss, Francois Vaillant, Karen L. White, James Whittle, Yuqing Yang, Soroor Hediyeh-Zadeh, Paul A. Stupple, Ian P. Street, Brendon J. Monahan, Thomas Paul. First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1130.

Abstract 2877: LUZP1 is an understudied transcriptional regulator in triple negative breast cancer (TNBC)

Abstract Introduction: TNBC is associated with poorer prognosis compared to other types of breast cancer due to poorly understood aggressive metastatic behavior. Proteomic profiling prioritized Leucine-Zipper Protein 1 (LUZP1) as an understudied TNBC-specific protein from an analysis of breast cancer patient-derived xenografts (PDXs) [PMC5379071]. Approach: RNA-seq data and isobaric tags for relative and absolute quantitation (iTRAQ) data from 16 PDXs were used to identify differentially expressed genes in basal-like and claudin-low breast cancers, compared to luminal and HER2-enriched subtypes. Multiple datasets (DepMap, METABRIC, and TCGA) were used to validate the enrichment of LUZP1 in TNBC. T-test was applied to identify differential genes/proteins. Pearson correlation analysis was used to identify RNAs that are correlated with LUZP1 protein level in CTPAC proteogenomic clinical sample data [PMC5102256 and Cell in press]. Gene Set Enrichment Analysis (GSEA) was used to identify enriched pathways. Cell fractionation was used to determine the subcellular expression of LUZP1. qPCR was used to validate differential expression observed in RNA-seq. Results: Leucine-Zipper Protein 1 (LUZP1) was differentially elevated in basal-like and claudin-low PDX models versus luminal and HER2-enriched at the mRNA and protein levels across 16 PDXs. Furthermore, LUZP1 mRNA was significantly enriched in TNBCs, relative to luminal and HER2-enriched subtypes, in breast cancer cell lines (data from DepMap), and human breast tumors (data from METABRIC and TCGA). LUZP1 has three putative nuclear localization signals and three leucine-zipper motifs, thought to mediate its protein dimerization and DNA-binding properties. Fractionation of TNBC cells showed that LUZP1 was predominately present in the nucleus. RNA-sequencing of control and MDA-MB-231 LUZP1 knockdown cells revealed that several extracellular matrix (ECM)-related pathways and epithelial-to-mesenchymal transition (EMT) were regulated by LUZP1. In particular, expression of migration, invasion, and metastasis genes such as matrix metalloproteinases (MMPs), Bone Morphogenetic Protein 2 (BMP2), and Transforming Growth Factor Beta 2 (TGFB2) were found to be LUZP1 regulated. Additionally, in CPTAC breast cancer proteogenomic data, LUZP1-correlated proteins and transcripts were enriched in ECM-related pathways and EMT, confirming the pathophysiological relevance of the cell-line knock-down data. Conclusion: LUZP1 is a novel TNBC-enriched protein in breast cancer PDXs, cell lines, and human tumors data. Gene silencing experiments demonstrated that LUZP1 transcriptionally regulates metastasis-related genes, and thus may drive a transcriptional program responsible for invasion, metastasis and EMT in TNBC. Functional data will be presented. Citation Format: Junkai Wang, Doug Chan, Beom-Jun Kim, Gloria V. Echeverria, Eric Jaehnig, Bing Zhang, Matthew J. Ellis. LUZP1 is an understudied transcriptional regulator in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2877.

Abstract 2038: A non-canonical, cell-autonomous STING function protects breast cancer cells from intrinsic and genotoxic-induced DNA instability

Abstract Several studies have recently indicated the activation of the immune system against tumor cells as well as the targeting of cancer cell DNA damage repair mechanisms as effective strategies to target tumor growth. STING is a well-known DNA sensor of innate immunity mostly characterized as a transmembrane protein of various cytoplasmic organelles that senses cytosolic DNA as a danger signal and triggers inflammatory responses. A current cancer immunotherapy strategy relies on the use of STING agonists to boost the patient's immune system through a cytokine-mediated recruitment of immune cells that infiltrate and kill tumor cells. However, the role of the STING pathway in cancer is far to be fully understood as there is otherwise accumulating evidence that activation of the cGAS-STING pathway can have a deleterious outcome. We recently showed that genotoxic treatment of breast cancer PDXs and cell lines triggered the STING pathway. Genetic inhibition of this pathway in MCF7 cells increased genotoxic treatment efficacy by promoting cell death and delaying cell colony regrowth, indicating that STING pathway intrinsically promotes cell resistance to treatment. In this study, we show that STING silencing decreased cell viability in a panel of classical or PDX-derived breast cancer cell lines irrespective of their ER status and of the genotoxic treatment received. Cell fractionation indicates that part of the STING pool intrinsically resides in the nucleus of various malignant and non-malignant cells. Fluorescence and electron microscopy show that STING partly resides at the inner membrane of the nucleus, and mass-spectrometry analysis revealed that STING interacts with core proteins of the non-homologous end joining (NHEJ) DNA damage repair (DDR) complex. STING promotes NEHJ-related protein assembly with chromatin, and its silencing decreases DDR and cell viability, while STING overexpression protects cancer cells from genotoxic treatment. STING involvement in DDR is independent of the classical STING-TBK1-IFN inflammatory response, thus identifying a new functional pathway for STING. STING nuclear localization was confirmed in a panel of breast cancer patient-derived xenografts and in surgical samples from breast cancer patients that received neoadjuvant chemotherapy. Evaluation of the impact of STING expression on patient outcome via the Kaplan Meier plotter show that overall STING expression level is positively correlated with favorable outcome in breast cancer patients, however high STING expression in breast and ovarian cancer patients treated with adjuvant chemotherapy is associated with poor prognosis. These findings place STING at the crossroad of DDR and immune surveillance, two major pathways for tumorigenesis and tumor survival. Citation Format: Laura Cheradame, Ida Chiara Guerrera, Julie Gaston, Alain Schmitt, Vincent Jung, Marion Pouillard, Nina Radosevic-Robin, Mauro Modesti, Jean-Gabriel Judde, Vincent Goffin, Stefano Cairo. A non-canonical, cell-autonomous STING function protects breast cancer cells from intrinsic and genotoxic-induced DNA instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2038.

Abstract 1399: Tumor-specific molecular changes in luminal B breast cancer patient-derived xenografts (PDXs) with acquired resistance to endocrine treatment

Abstract Worldwide, numerous preclinical agents that enter oncology clinical trials fail to demonstrate sufficient efficacy in patients to gain regulatory approval. This failure rate reflects both a poor understanding of the complexity of human cancers as well as the weak predictive value of existing preclinical models. PDXs are now widely embraced as better preserving characteristics of the original tumor and thus providing better tools for translational research. However, tumor collection for PDX generation usually coincides with initial surgery or biopsy, meaning that PDXs typically reflect the state of patient tumors at the times of collection. Unfortunately, this may not reflect changes that occur after treatment, particularly those associated with acquisition of resistance. Although analysis of changes associated with acquisition of resistance would be of great value for translational purposes, opportunities to obtain paired initial primary and later resistant samples are limited in current practice. In this study we describe establishment of a luminal breast cancer PDX model with acquired resistance to hormonotherapy. Starting from a luminal A breast cancer PDX that was both estrogen-dependent and fulvestrant-sensitive, we derived delayed outgrowth tumors under in vivo selection conditions of estrogen deprivation and/or fulvestrant treatment, thus corresponding to acquisition of resistance following hormonotherapy in the clinical setting. To understand phenotypic changes associated with resistance acquisition, resistant PDXs were analyzed for estrogen and progesterone receptor expression and subjected to whole exome sequencing, RNA sequencing, and DNA methylation analysis comparatively with the parental PDX. Intriguingly, all derived resistant PDXs showed complete loss of PR expression by immunohistochemistry regardless of selection method, whereas ER expression was only decreased in resistant PDXs that had been selected by fulvestrant treatment. Genetic analyses showed that resistant PDX tumors fell into two different transcriptomic signatures, depending on whether resistance was driven by estrogen deprivation or fulvestrant administration. Compared to the naïve parental tumor, the cluster corresponding to fulvestrant-selected PDX showed up-regulated and down-regulated pathways associated with genomic alterations related to endocrine therapy and KRAS pathways. Interestingly, the other cluster in which PDX were selected without fulvestrant showed only partial genomic alterations. Furthermore, whole exome sequencing revealed that similar driver gene mutations existed in both derived resistant and sensitive parental PDX. Finally, we confirmed pharmacological resistance to fulvestrant in the derived resistant PDX. New experiments investigating drug combinations in the context of endocrine resistance are ongoing. Citation Format: Marc Hillairet De Boisferon, Ismahène Benzaid, Elodie Marie Dit Chatel, Nicolas Hoffmann, Bruce A. Littlefield. Tumor-specific molecular changes in luminal B breast cancer patient-derived xenografts (PDXs) with acquired resistance to endocrine treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1399.

Abstract 2992: Proteogenomic characterization of triple-negative breast cancer patient-derived xenografts reveals molecular correlates of differential chemotherapy response and potential therapeutic targets to overcome resistance

Abstract Background: Chemotherapy is essential for the management of patients with triple-negative breast cancer (TNBC). Identification of biomarkers that may indicate treatment efficacy will be critical to improve patient stratification prior to treatment. To elucidate molecular determinants underlying chemotherapy response, we conducted a proteogenomic study using TNBC patient-derived xenografts (PDXs) treated with chemotherapy. Approach: 50 TNBC PDXs were treated with either docetaxel or carboplatin. Changes in tumor volume after 4 weeks from baseline were evaluated. Genomic, transcriptomic, and mass-spectrometry-based proteomic profiling were performed on baseline tumors prior to treatment to identify associations with chemotherapy response. Fisher's exact tests were used to test for significant enrichment of mutation and copy number events (p<0.05). Gene Set Enrichment Analysis was performed for pathway analyses. Results: At the DNA level, genomic aberrations in BRCA2 and BCL2 were enriched in carboplatin-responsive PDXs, while ARID1B aberrations were enriched in docetaxel-responsive PDXs. Gene-drug response correlations supported by both mRNA and protein-based measurements, but not mRNA or protein alone, for both carboplatin and docetaxel treatment in PDXs were associated with prognosis from basal and claudin-low human breast tumors in receipt of any chemotherapy from the METABRIC dataset. These data suggest that the combination of mRNA and protein data increased power to identify genes related to clinical outcome in TNBC. Some of the top genes overexpressed at both mRNA and protein levels in chemoresistant PDXs are targets of approved drugs, many of which have not been evaluated for their ability to augment response to taxane- or platinum-based chemotherapies. These genes are being investigated as therapeutic targets as well as markers of chemotherapy response. At the pathway level, both RNA and protein data associated models resistant to both agents with enhanced oxidative phosphorylation and translation regulation. Protein data further associated resistant models with elevated cytoplasmic ribosomal proteins. In contrast, both RNA and protein data associated tumors sensitive to both agents with genes involved in the E2F-Rb axis and cell cycle progression. Moreover, DNA mismatch repair and mRNA processing pathways were uniquely associated with carboplatin and docetaxel sensitivity, respectively, while amino acid metabolism and MAPK signaling pathways were uniquely associated with carboplatin and docetaxel resistance, respectively. Conclusion: Taken together, proteogenomic analysis of PDX tumors identifies diverse genes and pathways associated with chemotherapy response and further suggests potential therapeutic opportunities in TNBC. Citation Format: Jonathan T. Lei, Chen Huang, Ramakrishnan R. Srinivasan, Suhas Vasaikar, Lacey E. Dobrolecki, Alaina N. Lewis, Christina Sallas, Susan G. Hilsenbeck, C Kent Osborne, Mothaffar F. Rimawi, Matthew J. Ellis, Varduhi Petrosyan, Alexander B. Saltzman, Anna Malovannaya, Gerburg Wulf, Daniel C. Kraushaar, Tao Wang, Gloria V. Echeverria, Bing Zhang, Michael T. Lewis. Proteogenomic characterization of triple-negative breast cancer patient-derived xenografts reveals molecular correlates of differential chemotherapy response and potential therapeutic targets to overcome resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2992.

Anticancer effect of nucleoline-aptamer-conjugated gemcitabine loaded atellocollagen (IO401) in pancreatic cancer patient-derived orthotropic xenograft model

We investigated the anticancer effect and systemic effect of the atelocollagen (AC) patch coated nucleoline-aptamer-conjugated Gemcitabine (IO401 patch) by directly implanting to the tumor cell in pancreatic cancer patient-derived xenograft (PDX) model to purpose a future potential adjuvant surgical strategy during curative pancreatic resection for pancreatic cancer. Pancreatic cancer PDX model was established. Animals were grouped randomly (7 mice per group) into three types of patch transplantation groups: G1 = Null AC patch, G2 = Gemcitabine AC patch, G3 = IO401 patch. Tumor volume (length × width2, mm3), Tumor weight (mg), and Tumor inhibition rate [1-(Ti-To)/(average tumor volume of group) × 100, Ti = endpoint tumor volume, To = start tumor volume] were calculated. Anticancer therapy-related toxicity was evaluated by hematologic and histological findings. G3 (IO401 patch) showed the most significant reduction of tumor growth and tumor weight comparing with G1 (Null AC patch) and G2 (Gemcitabine AC patch) (p = 0.014, p = 0.018). G3 also showed the most significant tumor inhibition rate comparing with G1 and G2 (p = 0.011). G2 and G3 has the low necrosis proportion in histological finding comparing with G1 (p = 0.005, p < 0.05). Moreover, no leukopenia, no anemia, and no neutropenia were observed in G3. We demonstrated the anticancer effect of the IO401 patch by directly implanting to tumor cell in pancreatic cancer PDX model. This directaly implantable aptaber-drug conjugate system on tumor cell is expected to be a new surgical strategy to further increase the oncological importance of margin negative resection in pancreatic cancer surgery. Further research will be needed.

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.

Establishment and characterization of immortalized human breast cancer cell lines from breast cancer patient-derived xenografts (PDX)

The application of patient-derived xenografts (PDX) in drug screening and testing is a costly and time-consuming endeavor. While cell lines permit extensive mechanistic studies, many human breast cancer cell lines lack patient characteristics and clinical treatment information. Establishing cell lines that retain patient’s genetic and drug response information would enable greater drug screening and mechanistic studies. Therefore, we utilized breast cancer PDX from the Mayo Breast Cancer Genome Guided Therapy Study (BEAUTY) to establish two immortalized, genomically unique breast cancer cell lines. Through extensive genetic and therapeutic testing, the cell lines were found to retain the same clinical subtype, major somatic alterations, and drug response phenotypes as their corresponding PDX and patient tumor. Our findings demonstrate PDX can be utilized to develop immortalized breast cancer cell lines and provide a valuable tool for understanding the molecular mechanism of drug resistance and exploring novel treatment strategies.

Reovirus mutant jin-3 exhibits lytic and immune-stimulatory effects in preclinical human prostate cancer models

Treatment of castration-resistant prostate cancer remains a challenging clinical problem. Despite the promising effects of immunotherapy in other solid cancers, prostate cancer has remained largely unresponsive. Oncolytic viruses represent a promising therapeutic avenue, as oncolytic virus treatment combines tumour cell lysis with activation of the immune system and mounting of effective anti-tumour responses. Mammalian Orthoreoviruses are non-pathogenic human viruses with a preference of lytic replication in human tumour cells. In this study, we evaluated the oncolytic efficacy of the bioselected oncolytic reovirus mutant jin-3 in multiple human prostate cancer models. The jin-3 reovirus displayed efficient infection, replication, and anti-cancer responses in 2D and 3D prostate cancer models, as well as in ex vivo cultured human tumour slices. In addition, the jin-3 reovirus markedly reduced the viability and growth of human cancer cell lines and patient-derived xenografts. The infection induced the expression of mediators of immunogenic cell death, interferon-stimulated genes, and inflammatory cytokines. Taken together, our data demonstrate that the reovirus mutant jin-3 displays tumour tropism, and induces potent oncolytic and immunomodulatory responses in human prostate cancer models. Therefore, jin-3 reovirus represents an attractive candidate for further development as oncolytic agent for treatment of patients with aggressive localised or advanced prostate cancer.

Diversity of Epithelial-Mesenchymal Phenotypes in Circulating Tumour Cells from Prostate Cancer Patient-Derived Xenograft Models.

Simple SummarySpread of prostate cancer to other parts of the body is responsible for the majority of deaths. Tumour cell epithelial mesenchymal plasticity (EMP) increases their metastatic potential and facilitates their survival in the blood as circulating tumour cells (CTCs). The aim of this study was to molecularly characterise CTCs in a panel of prostate cancer patient-derived xenografts using genes associated with epithelial and mesenchymal phenotypes, and to compare the EMP status of CTCs with their matched primary tumours. The study highlights high heterogeneity in CTC enumeration and EMP gene expression between tumour-bearing mice and within individual blood samples, and therefore caution should be taken when interpreting pooled CTC analyses. Critically, tumour cells were present in the epithelial-mesenchymal hybrid state in the circulation. The study also demonstrates that there is high variation in CTC size, which would introduce sample bias to size-based CTC isolation techniques.Metastasis is the leading cause of cancer-related deaths worldwide. The epithelial-mesenchymal plasticity (EMP) status of primary tumours has relevance to metastatic potential and therapy resistance. Circulating tumour cells (CTCs) provide a window into the metastatic process, and molecular characterisation of CTCs in comparison to their primary tumours could lead to a better understanding of the mechanisms involved in the metastatic cascade. In this study, paired blood and tumour samples were collected from four prostate cancer patient-derived xenograft (PDX) models (BM18, LuCaP70, LuCaP96, LuCaP105) and assessed using an EMP-focused, 42 gene human-specific, nested quantitative RT-PCR assay. CTC burden varied amongst the various xenograft models with LuCaP96 having the highest number of CTCs per mouse (mean: 704; median: 31) followed by BM18 (mean: 101; median: 21), LuCaP70 (mean: 73; median: 16) and LuCaP105 (mean: 57; median: 6). A significant relationship was observed between tumour size and CTC number (p = 0.0058). Decreased levels of kallikrein-related peptidase 3 (KLK3) mRNA (which encodes prostate-specific antigen; PSA) were observed in CTC samples from all four models compared to their primary tumours. Both epithelial- and mesenchymal-associated genes were commonly expressed at higher levels in CTCs compared to the bulk primary tumour, although some common EMT-associated genes (CDH1, VIM, EGFR, EPCAM) remained unchanged. Immunofluorescence co-staining for pan-cytokeratin (KRT) and vimentin (VIM) indicated variable proportions of CTCs across the full EMP axis, even in the same model. EMP hybrids predominated in the BM18 and LuCaP96 models, but were not detected in the LuCaP105 model, and variable numbers of KRT+ and human VIM+ cells were observed in each model. SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), was enriched at the RNA level in CTCs compared to primary tumours and was the most commonly expressed mesenchymal gene in the CTCs. Co-staining for SERPINE1 and KRT revealed SERPINE1+ cells in 7/11 samples, six of which had SERPINE+KRT+ CTCs. Cell size variation was observed in CTCs. The majority of samples (8/11) contained larger CTCs ranging from 15.3 to 37.8 µm, whilst smaller cells (10.7 ± 4.1 µm, similar in size to peripheral blood mononuclear cells (PBMCs)) were identified in 6 of 11 samples. CTC clusters were also identified in 9/11 samples, containing 2–100 CTCs per cluster. Where CTC heterogeneity was observed in the clusters, epithelial-like cells (KRT+VIM−) were located on the periphery of the cluster, forming a layer around hybrid (KRT+VIM+) or mesenchymal-like (KRT−VIM+) cells. The CTC heterogeneity observed in these models emphasises the complexity in CTC isolation and classification and supports the increasingly recognised importance of the epithelial-mesenchymal hybrid state in cancer progression and metastasis.

RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence

SummaryRecent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer.

Generation of Non-Small Cell Lung Cancer Patient-Derived Xenografts to Study Intratumor Heterogeneity.

Simple SummaryIt is widely thought that tumors are composed of different subpopulations of cancer cells carrying genetic alterations with some of them being common among all cells while others are unique for each subpopulation. This variable genetic profile of tumor cells is a component of what is collectively described as intratumor heterogeneity (ITH). Surviving the immune system and therapies, and establishing metastases are forces of natural selection that act upon ITH and drive tumor evolution and, eventually, the clinical presentation of patients. The aim of this prospective study was to investigate ITH in early-stage operable non-small cell lung cancer. We directly grafted human tumors in immunosuppressed mice and compared the genetic profile of the tumors grown in mice with that of the original human tumors. We identified clinical factors that affected the ability of human tumors to grow as mouse xenografts.Recent advances in sequencing technologies have allowed the in-depth molecular study of tumors, even at the single cell level. Sequencing efforts have uncovered a previously unappreciated heterogeneity among tumor cells, which has been postulated to be the driving force of tumor evolution and to facilitate recurrence, metastasis, and drug resistance. In the current study, focused on early-stage operable non-small cell lung cancer, we used tumor growth in patient-derived xenograft (PDX) models in mice as a fast-forward tumor evolution process to investigate the molecular characteristics of tumor cells that grow in mice, as well as the parameters that affect the grafting efficiency. We found that squamous cell carcinomas grafted significantly more efficiently compared with adenocarcinomas. Advanced stage, patient age and primary tumor size were positively correlated with grafting. Additionally, we isolated and characterized circulating tumor cells (CTC) from patients’ peripheral blood and found that the presence of CTCs expressing epithelial-to-mesenchymal (EMT) markers correlated with the grafting potential. Interestingly, exome sequencing of the PDX tumor identified genetic alterations in DNA repair and genome integrity genes that were under-represented in the human primary counterpart. In conclusion, through the generation of a PDX biobank of NSCLC, we identified the clinical and molecular properties of tumors that affected growth in mice.