Growth Of Patient-derived Xenograft Tumors Research Articles

CombPDX: a unified statistical framework for evaluating drug synergism in patient-derived xenografts

Anticancer combination therapy has been developed to increase efficacy by enhancing synergy. Patient-derived xenografts (PDXs) have emerged as reliable preclinical models to develop effective treatments in translational cancer research. However, most PDX combination study designs focus on single dose levels, and dose–response surface models are not appropriate for testing synergism. We propose a comprehensive statistical framework to assess joint action of drug combinations from PDX tumor growth curve data. We provide various metrics and robust statistical inference procedures that locally (at a fixed time) and globally (across time) access combination effects under classical drug interaction models. Integrating genomic and pharmacological profiles in non-small-cell lung cancer (NSCLC), we have shown the utilities of combPDX in discovering effective therapeutic combinations and relevant biological mechanisms. We provide an interactive web server, combPDX (https://licaih.shinyapps.io/CombPDX/), to analyze PDX tumor growth curve data and perform power analyses.

Abstract 1798: Mechanisms of acquired resistance to palbociclib reveals pathways of response to abemaciclib

Abstract Background: CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy (ET) delay progression and improve survival in patients with hormone receptor positive (HR+), HER2 negative metastatic breast cancer (MBC). However, most patients develop resistance to CDK4/6i leading to disease progression, thus new therapies to overcome resistance are needed. CDK4/6i [palbociclib (palbo), ribociclib (ribo), and abemaciclib (abema)] have the same nominal targets but vary in their target specificity and are pharmacologically distinct. Clinical benefit was observed in patients treated with abema after progression on a prior CDK4/6i suggesting that palbo/ribo refractory tumors retain abema sensitivity. We hypothesize that mechanisms driving acquired resistance to palbo and abema are distinct, and palbo-resistant models may be responsive to abema. Models: 1) MCF-7 and T47D palbo and abema resistant cells, 2) patient derived xenografts (PDX) established from patients who progressed on ET alone (ET-resistant) or palbo + ET (palbo-resistant), 3) organoids derived from the palbo-resistant PDX, and 4) a HR+/HER2- MBC patient cohort who received abema after progression on palbo therapy. Results: RNA-sequencing and proteomic analysis revealed that palbo and abema resistant cells exhibit more than 30 differentially altered pathways. EMT, IL6/STAT3, and CSC pathways were upregulated only in palbo-resistant cells, but not in abema-resistant cells. Palbo-resistant cells also showed upregulation of G2M/Mitotic spindle and downregulation of ER pathways, whereas abema-resistant cells displayed downregulation of G2M/Mitotic spindle and upregulation of ER pathways. Mechanistic analysis revealed that palbo-resistant cells are responsive to abema with a delay in doubling time and a reduction of the proliferation marker pHH3. Further, cells accumulated in the G2M-phase with concomitant high phospho-(Y15)-CDK1 and cyclin B levels. Organoid cultures generated from palbo-resistant PDX were sensitive to abema. Likewise, abema significantly delayed the tumor growth of palbo-resistant PDX, correlating in vivo with ex vivo treatments. Moreover, we assessed response to abema after progression on palbo in the ET-resistant PDX model (CDK4/6 sensitive) demonstrating a continued survival benefit from abema treatment compared to palbo. Clinical outcome analyses of a MBC patient cohort treated with abema following prior CDK4/6i showed a median progression free survival (PFS) of 6.3 months in those treated sequentially vs a PFS of 4.0 months in non-sequential treated patients. Conclusion: Differential mechanism underlying palbo and abema acquired resistance can be exploited to overcome CDK4/6i resistance. These results provide rationale for clinical trials evaluating the benefit of abema treatment following progression on a prior CDK4/6i. Citation Format: Juliana Navarro-Yepes, Nicole M. Kettner, Tuyen Bui, Akshara S. Raghavendra, Xiayu Rao, Jing Wang, Aysegul Sahin, Senthil Damodaran, Debasish Tripathy, Kelly K. Hunt, Khandan Keyomarsi. Mechanisms of acquired resistance to palbociclib reveals pathways of response to abemaciclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1798.

Abstract 6258: Development of humanized patient-derived xenograft models for triple negative breast cancer

Abstract As a new promising in vivo platform for personalized treatment in the field of cancer research, the most improved way has been adopted by far is grafting patient-derived xenograft (PDX) to humanized mouse models which represent the unique features of interaction with human immune system and tumor microenvironment (TME). Here, we engineered triple negative breast cancer (TNBC) humanized PDX mouse (Hu-PDX) model to observe human immune system and tumor growth in TME. In addition, we analyzed gene ontology of PDX isolated from hu or non-humanized mouse model. Method: Human CD34+hematopoietic stem cells (HSCs) derived from cord blood is reconstructed into mature human leukocytes (hCD45+) in NOD.Cg-PrkdcscidIL2γgtm1 Sug (NOG) mice. TNBC PDX tumors are established it with unmatched human leukocyte antigen (HLA) genotypes, confirmed by that hCD45+ engraftment levels did not exceed 20-30% in the peripheral blood of mice. To be repopulated functional human T cells in xenograft models with severely depleted thymus, hCD34-PBMC cells are directly intravenously injected into tail vein of humanized mice followed by hCD34+cells injected without the onset of graft-versus-host disease (GVHD). The growth and gene expression of TNBC PDX tumors, as well as engrafted human immune population including T, B, natural killer (NK) cells and myeloid cells in TME, are monitored and investigated in both hu and non-hu-mice model. Results: Human immune reconstruction of cells, of the lymphoid as well as myeloid lineages, has been successfully accomplished in peripheral blood, spleen, and bone marrow over time in TME. Although the number of human mature leukocytes was gradually decreased in mice by 17 weeks, humanized mice have enabled studies of human immunity and TME in limitation period. Also, one of remaining limitations is unable to positive and negative selection of human T cells in the humanized mouse thymus and required to human major histocompatibility complex (MHC) molecules in humanized mice. To overcome this barrier, additional stimulation factor for generating functional T cells have successfully engrafted by transferring hCD34-PBMC cells. Moreover, tumor infiltrating human lymphocytes (TILs) have obviously detected in Hu-PDX model, although the differences tumor growth rate was not significantly monitored, compared with non-humanized mice. Interestingly, the analysis of gene ontology; biological process, cellular component and molecular function, ribosomal relevant proteins, not immune related genes, were highly up-regulated in TNBC PDX humanized in comparison to non-humanized mouse model. Conclusion: Humanized PDX model will contribute to studies more efficiently for the machinery underlying immunological response in breast tumor microenvironment. Furthermore, it might be an important unknown mechanism that the effect of elevated cancer-related ribosomal proteins expression within hu immune-TME. Citation Format: Yujeong Her, Hye Yeon Son, Ju Hee Kim, Woo Hang Heo, Mingji Quan, Songbin Li, Hyeong Gon Moon. Development of humanized patient-derived xenograft models for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6258.

Abstract 5333: Pancreatic cancer enhances HER2 signaling through DUSP6 to circumvent therapeutic MAPK inhibition

Abstract Background: Oncogenic KRAS is present in almost all cases of pancreatic ductal adenocarcinoma (PDAC). However, targeting KRAS or its canonical signaling cascades, especially the mitogen-activated protein kinase (MAPK) pathway, remains clinically unsuccessful. Targeting ERK kinases has recently emerged as a promising therapeutic strategy and combinatorial strategies should be developed. Aim: Identify adaptive mechanisms to ERK inhibition that can be co-targeted to achieve effective tumor inhibition in multiple patient-derived xenograft (PDX) models. Methods: Reverse-phase protein array (RPPA) was used in early-passage patient-derived cell lines (PDCLs) to identify potential resistance mechanisms. These were confirmed using RNA interference and overexpression in PDAC cell lines and PDCLs. The promising combinations were tested in 30 early-passaged PDAC PDXs. Results: RPPA showed dramatic downregulation of DUSP4 and DUSP6 phosphatases following MEK and ERK inhibition, which coincided with upregulation of phospho-HER2 and -HER3. Knockdown of DUSP6, but not DUSP4, was sufficient in phosphorylation of HER2. Conversely, overexpression of DUSP6 curbed HER2 and ERK activation. Downregulation of DUSP4 and DUSP6 induced by ulixertinib, an ERK inhibitor now in clinical development, was reversed by bortezomib, suggesting DUSP4 and DUSP6 are proteosomally degraded. Combined ulixertinib plus PI3K inhibitor copanlisib, or pan-HER inhibitor afatinib slowed but did not arrest PDX tumor growth in vivo, and addition of gemcitabine was required to achieve tumor regression or durable growth arrest. Alternatively, ulixertinib or MEK inhibitor (trametinib) in combination with trastuzumab deruxtecan (DS-8201a), an anti-HER2 conjugated topoisomerase I inhibitor, were extremely effective, leading to complete and durable tumor regression for all tested PDX models. We showed that upregulation of HER2 expression following MEK or ERK inhibitor treatment provides a conduit for enhanced internalization of DS-8201a. Conclusions: Our study provided novel mechanistic insight on how PDAC cells evade MAPK inhibition via enhancing HER2 signaling. We demonstrated that the combination of MEK or ERK inhibitor plus DS-8201a is extremely effective, leading to complete tumor regression in multiple PDAC PDX models. This combination should be advanced as a clinical trial for PDAC patients. Keys: DS-8201a, DUSP6, HER2, KRAS, ulixertinib, pancreatic ductal adenocarcinoma Citation Format: Ashenafi Shiferaw Bulle, Kuljeet Seehar, Sapana Bansod, Yali Chen, Chen Hung-Po, Paarth B. Dodhiawala, Lin Li, Vikas Somani, Jacqueline Mudd, Ryan C. Fields, Deborah Knoerzer, Andrea Wang-Gillam, Lim Kian-Huat. Pancreatic cancer enhances HER2 signaling through DUSP6 to circumvent therapeutic MAPK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5333.

EXOC4 Promotes Diffuse-Type Gastric Cancer Metastasis via Activating FAK Signal.

The EXOC4-FAK axis promoted DGC metastasis and could be a potential therapeutic target for patients with DGC.

Liver Endothelium Promotes HER3-Mediated Cell Survival in Colorectal Cancer with Wild-Type and Mutant KRAS.

This body of work highlighted a potential strategy of using HER3 antibodies in combination with standard chemotherapy agents for treating patients with either KRAS wild-type or KRAS mutant metastatic colorectal cancer.

Large-cohort humanized NPI mice reconstituted with CD34+ hematopoietic stem cells are feasible for evaluating preclinical cancer immunotherapy.

Cancer immunotherapy has achieved impressive therapeutic effects in many cancers, while only a small subset of patients benefit from it and some patients even have experienced severe toxicity. It is urgent to develop a feasible large-cohort humanized mouse model to evaluate the pre-clinical efficacy and safety of cancer immunotherapy. Furthermore, developing potentially effective combination therapy between cancer immunotherapy and other therapies also needs humanized mouse model to adequately mimic clinical actual setting. Herein, we established a humanized mouse model engrafted with less human CD34+ HSCs than ever before and then evaluated reconstitution efficiency and the profiles of human immune cells in this humanized mouse model. Also, this humanized mouse model was used to evaluate the preclinical efficacy and safety of cancer immunotherapy. For each batch of CD34+ HSCs humanized mouse model, a relatively-large cohort with over 25% human CD45+ cells in peripheral blood was established. This humanized mouse model could efficiently reconstitute human innate and adaptive immune cells. This humanized mouse model supported patient-derived xenograft tumor growth and tumor infiltration of PD-1+ human T cells. Furthermore, therapeutic efficacy, re-activation of tumor-infiltrated T cells, and side effects of checkpoint blockade therapy could be monitored in this humanized mouse model. Human T cells from this humanized mouse model were successfully engineered with CD19-CAR. CD19 CAR-T cells could effectively deplete B cells and suppress tumor growth of acute lymphoblastic leukemia in vivo in this humanized mouse model. This humanized mouse model also could be used to demonstrate the efficacy of bispecific antibodies, such as anti-CD19/CD3. Overall, our work provides a feasible large-cohort humanized mouse model for evaluating a variety of cancer immunotherapy approaches including checkpoint inhibitors, adoptive cell therapy, and bispecific antibody therapy, and demonstrates that human T cells from this humanized mouse model possess anti-tumor activities in vitro and in vivo.

Bevacizumab is an Efficient Therapeutic Approach with Low Side Effects in Patient-Derived Xenografts of Adenoid Cystic Carcinoma of the Lacrimal Gland.

PurposeAdenoid cystic carcinoma (ACC) of the lacrimal gland (LGACC) is an aggressive malignant lacrimal gland tumor with a generally poor prognosis. Survival rates for LGACC are 56% at 5 years and 49% at 10 years. Recent studies have indicated that anti-vascular endothelial growth factor (VEGF) therapy can inhibit angiogenesis in ACC cells. This study was designed to explore the efficacy of the antiangiogenic drug bevacizumab in a LGACC patient-derived xenograft (PDX) animal model.MethodsThe histological structure of PDX was determined by hematoxylin-eosin staining to confirm successful xenografting. Immunohistochemistry (IHC) was used to detect the expression of neovascularization-related genes in LGACC patients and in the PDX model, including VEGF, VEGFR1, and FGFR. In order to compare the efficacy of antiangiogenic drug and traditional chemotherapy drug, PDX models were treated with bevacizumab and cisplatin respectively, and body weight was evaluated. Subsequently, the neovascularization-related proteins VEGF, VEGFR2, and CD34, tumor suppressor P53 and proliferation-related protein Ki67 were analyzed by IHC. Quantitative real-time PCR was employed to examine the mRNA expression of apoptosis-related genes BAD and Caspase 9, and of HIF1α.ResultsVEGF, VEGFR1, and FGFR were highly expressed in patients with LGACC and PDX models. Both bevacizumab and cisplatin treatment inhibited PDX tumor growth. The body weight of PDX models treated with cisplatin significantly decreased from day 15, while those treated with bevacizumab did not markedly change. Bevacizumab reduced the expression of VEGF, CD34, and Ki67 in PDX tumors; whereas, bevacizumab upregulated P53 and downregulated HIF1α levels.ConclusionThe present study indicates that antiangiogenic drugs may be a promising treatment strategy for LGACC.

LCDR regulates the integrity of lysosomal membrane by hnRNP K–stabilized LAPTM5 transcript and promotes cell survival

Lysosome plays important roles in cellular homeostasis, and its dysregulation contributes to tumor growth and survival. However, the understanding of regulation and the underlying mechanism of lysosome in cancer survival is incomplete. Here, we reveal a role for a histone acetylation-regulated long noncoding RNA termed lysosome cell death regulator (LCDR) in lung cancer cell survival, in which its knockdown promotes apoptosis. Mechanistically, LCDR binds to heterogenous nuclear ribonucleoprotein K (hnRNP K) to regulate the stability of the lysosomal-associated protein transmembrane 5 (LAPTM5) transcript that maintains the integrity of the lysosomal membrane. Knockdown of LCDR, hnRNP K, or LAPTM5 promotes lysosomal membrane permeabilization and lysosomal cell death, thus consequently resulting in apoptosis. LAPTM5 overexpression or cathepsin B inhibitor partially restores the effects of this axis on lysosomal cell death invitro and invivo. Similarly, targeting LCDR significantly decreased tumor growth of patient-derived xenografts of lung adenocarcinoma (LUAD) and had significant cell death using nanoparticles (NPs)-mediated systematic short interfering RNA delivery. Moreover, LCDR/hnRNP K/LAPTM5 are up-regulated in LUAD tissues, and coexpression of this axis shows the increased diagnostic value for LUAD. Collectively, we identified a long noncoding RNA that regulates lysosome function at the posttranscriptional level. These findings shed light on LCDR/hnRNP K/LAPTM5 as potential therapeutic targets, and targeting lysosome is a promising strategy in cancer treatment.

Ethyl Ferulate Suppresses Esophageal Squamous Cell Carcinoma Tumor Growth Through Inhibiting the mTOR Signaling Pathway.

Ethyl ferulate is a phenylpropanoid compound isolated from the medicinal herb Ferula. Although ethyl ferulate has anti-inflammatory, antioxidant, and neuroprotective activities with potential use in the nutraceutical and pharmaceutical industry, its anticancer effects and underlying molecular mechanisms against esophageal squamous cell carcinoma (ESCC) have not been investigated. This study investigates the anticancer activity and molecular mechanism of ethyl ferulate in ESCC. MTT, focus formation, soft agar, and cell cycle analysis were used to determine the effect of ethyl ferulate on cell proliferation and cell cycle. Potential candidate proteins were screened and verified via Western blotting, in vitro kinase assay, and in vitro pull-down assay. Mammalian target of rapamycin (mTOR) knockdown cell lines were established by lentiviral infection with shmTOR. The effect of ethyl ferulate on tumor growth was assessed using ESCC patient-derived xenograft models. Ethyl ferulate significantly inhibited cell growth and induced G1 phase cell cycle arrest in ESCC cells. Ethyl ferulate reduced the activity of mTOR in vitro. The inhibition of ESCC cell growth by ethyl ferulate is dependent on mTOR expression. In addition, ethyl ferulate strongly reduced ESCC patient-derived xenograft tumor growth in an in vivo mouse model. Ethyl ferulate is an mTOR inhibitor that can suppress ESCC progression and may be a novel candidate compound for esophageal cancer chemoprevention.

Abstract P233: TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations

Abstract Fusions involving RET receptor tyrosine kinase are a common driver of tumors across different tissue types, such as lung, thyroid, colorectal, soft tissue and others. TAS0953/HM06 (hereby referred to as HM06) is a novel 2ndgeneration RET-specific inhibitor that is effective against RET solvent front (G810) and gatekeeper (V804) mutations. Here, we evaluated the efficacy of HM06 in lung and thyroid carcinomas, and soft-tissue sarcoma cell lines and PDXs derived from RET inhibitor-naive tumor samples or from tumors with acquired resistance to selpercatinib. HM06 was more effective than the RET multi-kinase inhibitors cabozantinib and vandetanib, and as effective as selpercatinib and pralsetinib in inhibiting growth of patient-derived and isogenic lung, thyroid and sarcoma cell lines (IC50=0.02-0.1 µM) harboring different RET fusions (KIF5B-RET, CCDC6-RET, TRIM33-RET, SPECCL1-RET) or activating mutations (RET C634W). Growth of non-tumor cells was up to 80-fold less sensitive to HM06 (IC50= 1.6 µM). Treatment of RET fusion-positive lung cancer cells with HM06 resulted in a dose-dependent inhibition of RET phosphorylation (Y905 and Y1062) and the downstream effectors AKT, ERK1/2, p70S6K and S6. Caspase 3/7 activity and markers of apoptosis (BIM, cleaved PARP) were induced by HM06 to a similar extent as pralsetinib and selpercatinib (dose range: 0.05-1 µM). HM06 induced changes in the core mediators of cell cycle regulation (upregulation of p27, downregulation of CCND1) and suppressed expression of MYC and ETV5. In vivo, HM06 blocked tumor growth and/or induced regression of up to 65% in seven patient-derived xenograft (PDX) models with RET fusions (five NSCLC PDXs, one sarcoma PDX and one NSCLC cell-line xenograft) to a similar extent as pralsetinib and selpercatinib. However, 6 weeks after cessation of treatment of the SPECCL1-RET-driven sarcoma PDX model, growth of tumors treated with HM06 was suppressed completely, whereas 3/5 pralsetinib-treated tumors and 1/5 selpercatinib-treated tumor regrew. Combination of HM06 and the MET inhibitor capmatinib effectively blocked growth of PDX tumors in a model that was derived from a patient sample that expressed RET fusion and METamplification, and was resistant to selpercatinib. These results suggest that HM06 may be an effective therapy for RET-driven tumors in a tissue-type agnostic manner and can effectively address common on-target and off-target resistance mechanisms such as RET G810X and V804X mutations. HM06 is currently in a phase 1 and 2 clinical trial for patients with advanced solid tumors with RET gene abnormalities (margaRET, NCT 04683250). Citation Format: Igor Odintsov, Renate I. Kurth, Kota Ishizawa, Lukas Delasos, Allan J.W. Lui, Inna Khodos, Connor J. Hagen, Qing Chang, Marissa S. Mattar, Morana Vojnic, Siddharth Kunte, Annalisa Bonifacio, Claudio Giuliano, Elisa De Stanchina, Emily Cheng, Emanuela Lovati, Marc Ladanyi, Romel Somwar. TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P233.

Integrating of lipophilic platinum(IV) prodrug into liposomes for cancer therapy on patient-derived xenograft model

Platinum-based anticancer agents such as cisplatin and its analogues are widely used for treating multiple cancers. However, due to the inferior water-solubility, chemoresistance and consequent adverse side effects, their clinical applications are limited. Herein, cholesPt(IV), a lipophilic platinum(IV) prodrug was synthesized for manufacture of CholesPt(IV)-Liposomes aiming to resolve the predefined obstacles encountered by platinum drugs. Following systematic screening, CholesPt(IV)-Liposomes showed a small particle size (105.6 nm), the rapid release of platinum (Pt) ions, and notable apoptosis of cancer cells. In addition, according to the fluidity and safety results of animal experiments in mice, CholesPt(IV)-Liposomes also showed better therapeutic effect, which significantly inhibited the growth of patient-derived xenograft tumors of hepatocellular carcinoma with an inhibition ratio of 80.7%, and effectively alleviated the drug toxicity brought by traditional platinum drugs. Overall, this study provides a promising route to enhance the therapeutic efficiency of platinum drugs in cancer treatment.

Dendrimeric nanosystem consistently circumvents heterogeneous drug response and resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with no efficacious treatment. The application of nanomedicine is expected to bring new hope to PDAC treatment. In this study, we report a novel supramolecular dendrimeric nanosystem carrying the anticancer drug doxorubicin, which demonstrated potent anticancer activity, markedly overcoming the heterogeneity of drug response and resistance of primary cultured tumor cells derived from PDAC patients. This dendrimer nanodrug was constructed with a fluorinated amphiphilic dendrimer, which self-assembled into micelles nanostructure and encapsulated doxorubicin with high loading. Because of the fine nanosize, stable formulation and acid-promoted drug release, this dendrimeric nanosystem effectively accumulated in tumor, with deep penetration in tumor tissue and rapid drug uptake/release profile in cells, ultimately resulting in potent anticancer activity and complete suppression of tumor growth in patient-derived xenografts. Most importantly, this dendrimer nanodrug generated uniform and effective response when treating 35 primary pancreatic cancer cell lines issued from patient samples as a robust platform for preclinical drug efficacy testing. In addition, this dendrimer nanodrug formulation was devoid of adverse effects and showed excellent tolerability. Given all these uniquely advantageous features, this simple and convenient dendrimer nanodrug holds great promise as a potential candidate to treat the deadly PDAC.

Pharmaceutical Interference of the EWS-FLI1-driven Transcriptome By Cotargeting H3K27ac and RNA Polymerase Activity in Ewing Sarcoma.

The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the hallmark genomic alteration of Ewing sarcoma, a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five-year survival rate. EWS-FLI1 is currently not clinically druggable, driving the need for more effective targeted therapies. Treatment with the H3K27 demethylase inhibitor, GSK-J4, leads to an increase in H3K27me and a decrease in H3K27ac, a significant event in Ewing sarcoma because H3K27ac associates strongly with EWS-FLI1 binding at enhancers and promoters and subsequent activity of EWS-FLI1 target genes. We were able to identify targets of EWS-FLI1 tumorigenesis directly inhibited by GSK-J4. GSK-J4 disruption of EWS-FLI1-driven transcription was toxic to Ewing sarcoma cells and slowed tumor growth in patient-derived xenografts (PDX) of Ewing sarcoma. Responses were markedly exacerbated by cotreatment with a disruptor of RNA polymerase II activity, the CDK7 inhibitor THZ1. This combination together suppressed EWS-FLI1 target genes and viability of ex vivo PDX Ewing sarcoma cells in a synergistic manner. In PDX models of Ewing Sarcoma, the combination shrank tumors. We present a new therapeutic strategy to treat Ewing sarcoma by decreasing H3K27ac at EWS-FLI1-driven transcripts, exacerbated by blocking phosphorylation of the C-terminal domain of RNA polymerase II to further hinder the EWS-FLI1-driven transcriptome.

Abstract 1388: ATM inhibitor AZD1390 sensitizes breast cancer brain metastasis to radiation therapy

Abstract Metastasis to the central nervous system (CNS) has remained a major source of morbidity and mortality for patients with breast cancer. This is mainly driven by the lack of therapeutic options, as traditional chemotherapy and targeted therapies have been shown to have limited efficacy in the brain. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR.AZD1390 is a potent orally bioavailable ATM inhibitor, specifically optimized for blood brain barrier penetration and has shown to be an effective radiosensitizer in gliomas. We have shown that there are frequent alterations in DNA damage response (DDR) pathways in CNS metastasis (CM), which include frequent mutations in TP53. Incorrect repair of DNA lesions often leads to genomic instability. ATM, a core component of the DNA repair system, is activated to enhance the homologous recombination (HR) repair pathway upon DNA double-strand breaks. Here we show that AZD1390, when used in combination with radiation therapy, can inhibit the growth of breast cancer CM in patient derived xenograft (PDX) tumors. Three PDX models (2 Her2+ and one triple negative), all of which harbor TP53 mutations and other DDR alterations, were implanted into the flank or brain of immunocompromised mice. Pre-treatment with AZD1390 followed by a fractionated regime of radiation therapy (2Gy/day for 5 days) significantly inhibited growth of flank PDX tumors by an average of 81% (85% and 80% in the HER2+ and 77% in the triple negative PDX, p-value < 0.05). This is compared to an average of 7% (8%, 13% and 0% respectively) for tumors treated with AZD1390 alone or 40% (39%, 21% and 58% respectively) for radiation therapy alone in all PDX models. Growth inhibition was sustained until the experiment endpoint at 90 days in 75% of the tumors, and 25% of the tumors regressed below their size at the beginning of treatment. About 31% of tumors across three models started to grow back and gene expression analysis comparing these tumors with complete responders demonstrated changes in oxidative phosphorylation, sirtuin signaling, and ERK/MAPK signaling. Adjuvant therapy of AZD1390 did not further decrease tumor growth. Our results demonstrate that AZD1390 can sensitize breast cancer CM with DDR pathway alterations to radiation therapy. This points to the potential of AZD1390 as a potential novel therapeutic agent for patients with breast cancer CM. Citation Format: Ben Yi Tew, Stephen Durant, Bodour Salhia. ATM inhibitor AZD1390 sensitizes breast cancer brain metastasis to radiation therapy [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 1388.

Combination of Epithelial Growth Factor Receptor Blockers and CDK4/6 Inhibitor for Nasopharyngeal Carcinoma Treatment.

Simple SummaryOur findings indicated that the EGF-EGFR pathway was highly activated in very young patients with recurrent or metastatic NPC. High EGFR expression in patients with metastatic NPC resulted in poor clinical outcomes. To examine whether the EGFR pathway serves as a therapeutic target for NPC, NPC patient-derived xenograft (PDX) and NPC cell lines were treated with EGFR inhibitors (EGFRi) and a cell cycle blocker. Either EGFRi or cell cycle blocker treatment alone could reduce NPC cell growth and PDX tumor growth. Furthermore, combination treatment exerted an additive suppression effect on PDX tumor growth. This study provides promising evidence that EGFRi used in combination with a cell cycle blocker may be used to treat patients with NPC.Background: Nasopharyngeal carcinoma (NPC) involves host genetics, environmental and viral factors. In clinical observations, patients of young and old ages were found to have higher recurrence and metastatic rates. Methods: Cytokine array was employed to screen druggable target(s). The candidate target(s) were confirmed through patient-derived xenografts (PDXs) and a new EBV-positive cell line, NPC-B13. Results: Overexpression of epithelial growth factor (EGF) and EGF receptor (EGFR) was detected in young patients than in older patients. The growth of NPC PDX tumors and cell lines was inhibited by EGFR inhibitors (EGFRi) cetuximab and afatinib when used separately or in combination with the cell cycle blocker palbociclib. Western blot analysis of these drug-treated PDXs demonstrated that the blockade of the EGF signaling pathway was associated with a decrease in the p-EGFR level and reduction in PDX tumor size. RNA sequencing results of PDX tumors elucidated that cell cycle-related pathways were suppressed in response to drug treatments. High EGFR expression (IHC score ≥ grade 3) was correlated with poor survival in metastatic patients (p = 0.008). Conclusions: Our results provide encouraging preliminary data related to the combination treatment of EGFRi and palbociclib in patients with NPC.

Ipriflavone Suppresses Growth of Esophageal Squamous Cell Carcinoma Through Inhibiting mTOR In Vitro and In Vivo.

Ipriflavone, a synthetic isoflavone that inhibits osteoclastic bone resorption, has been used clinically for the treatment of osteoporosis. However, the anticancer activity of Ipriflavone and its molecular mechanisms in the context of esophageal squamous cell carcinoma (ESCC) have not been investigated. In this study, we report that Ipriflavone is a novel mammalian target of rapamycin (mTOR) inhibitor that suppresses cell proliferation and induces cell apoptosis in ESCC cells. Ipriflavone inhibited anchorage-dependent and -independent growth of ESCC cells. Ipriflavone induced G1 phase cell cycle arrest and intrinsic cell apoptosis by activating caspase 3 and increasing the expression of cytochrome c. Based on the results of in vitro screening and cell-based assays, Ipriflavone inhibited mTOR signaling pathway through directly targeting mTOR. Knockdown of mTOR strongly inhibited the growth of ESCC cells, and the cell growth inhibitory effect exerted by Ipriflavone was found to be dependent upon mTOR signaling pathway. Remarkably, Ipriflavone strongly inhibited ESCC patient-derived xenograft tumor growth in an in vivo mouse model. Our findings suggest that Ipriflavone is an mTOR inhibitor that could be potentially useful for treating ESCC.

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.

VERU-111: An Oral Tubulin Inhibitor That Suppresses Taxane-Sensitive and Taxane-Resistant Breast Cancer

Abstract Triple negative breast cancer (TNBC) patients have poorer overall prognosis relative to patients diagnosed with other molecular subtypes due to rapid onset of drug resistance to conventional chemotherapies and increased risk of visceral metastases. The microtubule inhibitor paclitaxel (Taxol, a taxane) is a frontline therapy for advanced breast cancer. We evaluated in TNBC models the preclinical safety and efficacy of a novel, potent, and orally bioavailable tubulin inhibitor, VERU-111, a tubulin inhibitor targeting the colchicine binding site. VERU-111 showed potent anti-proliferative and anti-migratory activity against several taxane-sensitive and taxane-resistant TNBC breast cancer cell lines. Based on these observations, taxane-resistant HER2+ cell lines were generated, and were also found to be responsive to VERU-111 treatment. In vivo, orally administered VERU-111 inhibited MDA-MB-231 tumor growth in a dose-dependent manner with antitumor potency similar to paclitaxel, and repressed metastases originating from the mammary fat pad or following tail vein injection. In contrast, in a MDA-MB-231 paclitaxel-resistant (TxR) subline, tumor growth was refractory to paclitaxel whereas VERU-111 significantly inhibited primary tumor growth and reduced lung and liver metastases. VERU-111 was then tested in a luciferase-labeled, multidrug resistant patient-derived xenograft (PDX) TNBC model. VERU-111 significantly inhibited HCI-10 PDX tumor growth and suppressed the expansion of axillary lymph node metastases present prior to initiation of therapy while suppressing lung, liver, bone and kidney metastases at study endpoint. Moreover, in contrast to paclitaxel, VERU-111 therapy did not cause a significant decrease in mouse body weight during treatment. Evaluation of efficacy of VERU-111 in taxane-sensitive and -resistant HER2+ xenograft models is in progress. Overall, we conclude that VERU-111 is a new generation orally bioavailable tubulin inhibitor that potently inhibits the growth of taxane-sensitive and taxane-resistant breast cancers with reduced adverse side effects relative to paclitaxel. Importantly, VERU-111 is well-tolerated in patients as evaluated in phase I/II clinical trials for advanced prostate cancer patients (NCT03752099). We propose that VERU-111 will be an effective second line therapy for patients with advanced breast cancer who progress on taxane-based therapeutic regimens.

MiR-146 connects stem cell identity with metabolism and pharmacological resistance in breast cancer.

Although ectopic overexpression of miRNAs can influence mammary normal and cancer stem cells (SCs/CSCs), their physiological relevance remains uncertain. Here, we show that miR-146 is relevant for SC/CSC activity. MiR-146a/b expression is high in SCs/CSCs from human/mouse primary mammary tissues, correlates with the basal-like breast cancer subtype, which typically has a high CSC content, and specifically distinguishes cells with SC/CSC identity. Loss of miR-146 reduces SC/CSC self-renewal in vitro and compromises patient-derived xenograft tumor growth in vivo, decreasing the number of tumor-initiating cells, thus supporting its pro-oncogenic function. Transcriptional analysis in mammary SC-like cells revealed that miR-146 has pleiotropic effects, reducing adaptive response mechanisms and activating the exit from quiescent state, through a complex network of finely regulated miRNA targets related to quiescence, transcription, and one-carbon pool metabolism. Consistent with these findings, SCs/CSCs display innate resistance to anti-folate chemotherapies either in vitro or in vivo that can be reversed by miR-146 depletion, unmasking a "hidden vulnerability" exploitable for the development of anti-CSC therapies.