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Patient-Derived Xenografts (PDXs) are tumor-in-mouse models for cancer. PDX collections, such as those supported by the NCI PDXNet program, are powerful resources for preclinical therapeutic testing. However, variations in experimental design and analysis procedures have limited interpretability. To determine the robustness of PDX studies, the PDXNet tested temozolomide drug response for three pre-validated PDX models (sensitive, resistant, and intermediate) across four blinded PDX Development and Trial Centers (PDTCs) using independently selected SOPs. Each PDTC was able to correctly identify the sensitive, resistant, and intermediate models, and statistical evaluations were concordant across all groups. We also developed and benchmarked optimized PDX informatics pipelines, and these yielded robust assessments across xenograft biological replicates. These studies show that PDX drug responses and sequence results are reproducible across diverse experimental protocols. Here we share the range of experimental procedures that maintained robustness, as well as standardized cloud-based workflows for PDX exome-seq and RNA-Seq analysis and for evaluating growth.

Cancer immunotherapy continues to make headway as a treatment for advanced stage tumors, revealing an urgent need to understand the fundamentals of anti-tumor immune responses. Noteworthy is a scarcity of data pertaining to the breadth and specificity of tumor-specific T cell responses in metastatic breast cancer. Autochthonous transgenic models of breast cancer display spontaneous metastasis in the FVB/NJ mouse strain, yet a lack of knowledge regarding tumor-bound MHC/peptide immune epitopes in this mouse model limits the characterization of tumor-specific T cell responses, and the mechanisms that regulate T cell responses in the metastatic setting. We recently generated the NetH2pan prediction tool for murine class I MHC ligands by building an FVB/NJ H-2q ligand database and combining it with public information from six other murine MHC alleles. Here, we deployed NetH2pan in combination with an advanced proteomics workflow to identify immunogenic T cell epitopes in the MMTV-PyMT transgenic model for metastatic breast cancer. Five unique MHC I/PyMT epitopes were identified. These tumor-specific epitopes were confirmed to be presented by the class I MHC of primary MMTV-PyMT tumors and their T cell immunogenicity was validated. Vaccination using a DNA construct encoding a truncated PyMT protein generated CD8 + T cell responses to these MHC class I/peptide complexes and prevented tumor development. In sum, we have established an MHC-ligand discovery pipeline in FVB/NJ mice, identified and tracked H-2Dq/PyMT neoantigen-specific T cells, and developed a vaccine that prevents tumor development in this metastatic model of breast cancer.

Great progress has been made in cancer therapeutics. However, metastasis remains the predominant cause of death from cancer. Importantly, metastasis can manifest many years after initial treatment of the primary cancer. This is because cancer cells can remain dormant before forming symptomatic metastasis. An important question is whether metastasis research should focus on the early treatment of metastases, before they are clinically evident ("overt"), or on developing treatments to stop overt metastasis (stage IV cancer). In this commentary we want to clarify why it is important that all avenues of treatment for stage IV patients are developed. Indeed, future treatments are expected to go beyond the mere shrinkage of overt metastases and will include strategies that prevent disseminated tumor cells from emerging from dormancy.

Estrogen receptor-negative (ER-negative) breast cancer is thought to be more malignant and devastating than ER-positive breast cancer. ER-negative breast cancer exhibits elevated NF-κB activity, but how this abnormally high NF-κB activity is maintained is poorly understood. The importance of linear ubiquitination, which is generated by the linear ubiquitin chain assembly complex (LUBAC), is increasingly appreciated in NF-κB signaling, which regulates cell activation and death. Here, we showed that epsin proteins, a family of ubiquitin-binding endocytic adaptors, interacted with LUBAC via its ubiquitin-interacting motif and bound LUBAC's bona fide substrate NEMO via its N-terminal homolog (ENTH) domain. Furthermore, epsins promoted NF-κB essential modulator (NEMO) linear ubiquitination and served as scaffolds for recruiting other components of the IκB kinase (IKK) complex, resulting in the heightened IKK activation and sustained NF-κB signaling essential for the development of ER-negative breast cancer. Heightened epsin levels in ER-negative human breast cancer are associated with poor relapse-free survival. We showed that transgenic and pharmacological approaches eliminating epsins potently impeded breast cancer development in both spontaneous and patient-derived xenograft breast cancer mouse models. Our findings established the pivotal role epsins played in promoting breast cancer. Thus, targeting epsins may represent a strategy to restrain NF-κB signaling and provide an important perspective into ER-negative breast cancer treatment.

Human cancer is a complex and heterogeneous collection of diseases that kills more than 18 million people every year worldwide. Despite advances in detection, diagnosis, and treatments for cancers, new strategies are needed to combat deadly cancers. Models of human cancer continue to evolve for preclinical research and have culminated in patient-derived systems that better represent the diversity and complexity of cancer. Still, no model is perfect. This Perspective attempts to address ways that we can improve the clinical translatability of models used for cancer research, from the point of view of researchers who mainly conduct cancer studies in vivo.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.

Background: Endocrine therapy resistance is common and is a leading cause of breast cancer-related death. Thus, the search for therapeutic targets based on mechanistic insights into endocrine therapy resistance continues. Kinases are important drug targets and regulators in cells, many of which are involved in tumorigenesis and the development of treatment resistance. Mass spectrometry-based kinome analysis has been impeded by the low abundance of individual kinases. Here we utilized kinase inhibitor-conjugated beads to enrich and thereby sensitively profile the kinome of estrogen receptor-positive (ER+) breast cancer patient-derived xenograft (PDX) tumors under estradiol-deprivation treatment. Experimental design and methods: We harvested tumor samples from 20 breast PDX lines with various degrees of estradiol (E2) dependence in ovariectomized SCID-beige mice with and without E2 supplementation (n=3 per PDX line per arm). The kinases in the tumor lysates were enriched using kinase inhibitor pulldown (KIP) beads and the tightly bound kinases were quantified by mass spectrometry. To identify candidates, we selected for kinases that were highly E2-regulated in the E2-dependent PDX lines but constitutively expressed in E2-independent PDX lines. Survival analysis of candidate kinases in patients with ER+ breast cancer was performed using the METABRIC dataset. Results: Each PDX line had a unique yet reproducible kinome. To seek kinases with consistent relationships with estrogen dependence, we sought kinases that were statistically differentially expressed in tumors between E2 supplied and deprived conditions. Noticeably, membrane-associated tyrosine-and threonine-specific cdc2-inhibitory kinase (PKMYT1), a WEE family kinase known to have estrogen response elements (EREs) in its regulatory region, was significantly decreased after E2 deprivation in E2-dependent PDXs (log2 fold change = -7.86, p Conclusion: Here, we analyzed the kinomes of 20 ER+ breast cancer PDX tumors with or without E2 by mass spectrometry. We discovered that PKMYT1 is a marker of hormone independent growth and poor outcome. Ongoing experiments that study the effects of PKMYT1 inhibition in both ER-dependent and independent circumstances will be presented. Citation Format: Anran Chen, Beom-Jun Kim, Doug W Chan, Purba Singh, Lacey E Dobrolecki, Jonathan T Lei, Shunqiang Li, Alana L Welm, Michael T Lewis, Matthew J Ellis. Kinome profiling of ER+ breast cancer PDXs identifies PKMYT1 as a marker of hormone independent growth and poor outcome [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD8-02.

Background: Predicting the risk of relapse in patients with non-metastatic breast cancer is important for medical decisions and patient counseling. For patients who receive neoadjuvant chemotherapy, pathologic response and especially pathologic complete response (pCR) has been associated with risk of relapse; however this association is imperfect. Our prior work in patient-derived xenograft (PDX) models indicated that tumor engraftment in mice correlated with risk of recurrence. To understand further the prognostic utility of PDX, we designed a prospective clinical trial to determine the correlation between PDX generation with residual disease following neoadjuvant chemotherapy and relapse. Preliminary data are presented. Methods: Women with newly diagnosed non-metastatic hormone receptor low-positive (HR-low, ER and/or PR ≤ 10%) or negative breast cancer planned to receive systemic chemotherapy prior to definitive surgery were eligible. Tumor tissue at diagnosis was orthotopically implanted in NOD/SCID mice. The primary objective of the study is to correlate the ability of a tumor to engraft in mice with pathologic responses and clinical outcomes. Results: Between 12/2016 and 2/2020, 58 patients enrolled (triple negative breast cancer (TNBC), n=37; HR-low or negative/Her2+, n=11; or HR-low/Her2-, n=8; mixed, n=1). PDXs were successfully established from 16 patients. Patients uniformly received intensive preoperative chemotherapy per standard of care. Among the 12 patients whose tumors engrafted in mice (PDX(+)) and underwent surgery, the pCR rate was 41.7%. In the subgroup of patients with postoperative follow up >6 months (n=9), 3 patients had achieved a pCR, 1 of whom recurred; and 6 patients had residual disease, 3 of whom recurred (overall relapse rate 44.4%). All patients who relapsed (TNBC n=3; HR-low/Her2- n=1), experienced a very early relapse ( Among the 38 patients whose tumors did not engraft (PDX(-)) and underwent surgery, the overall pCR rate was 60.6%. In the subgroup of patients with postoperative follow up >6 months (n=30), 18 patients had achieved a pCR of whom none recurred, and 12 had residual disease of whom 1 patient (with TNBC) had locoregional recurrence 35.1 months after her definitive surgery (relapse rate 3.3%). She underwent repeat surgery followed by radiation therapy and 5.5 months after her recurrence, she remains disease-free. Achievement of pCR was not statistically different between the PDX(+) and PDX(-) group. In the entire cohort, the presence of residual disease was associated with high risk of relapse (22.2%) as compared to the achievement of pCR (4.8%, p = 0.16). However, successful tumor engraftment was associated not only with a higher risk of relapse (44.4% vs 3.3%, p = 0.0068, odds ratio 20.45), but also an early and exceptionally aggressive relapse. Conclusion. Our functional studies not only identify a patient population with a high risk of relapse with greater precision than the achievement of pCR, but also identify patients whose relapse has a particularly aggressive natural history. We established models that recapitulate this aggressive disease and in-depth genomic studies are underway. These studies will lead us to better patient stratification on the basis of risk of relapse and novel therapeutic strategies focused on patients with exceptionally aggressive breast cancers that our current diagnostic methods cannot identify. Citation Format: Christos Vaklavas, Zhengtao Chu, Rachel E Factor, Alana L Welm, Bryan E Welm, Cindy B Matsen. Towards personalized medicine - patient-derived breast tumor grafts as predictors of relapse and response to therapy. Preliminary results [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-01.

Short-form Ron (sfRon) is an understudied, alternative isoform of the full-length Ron receptor tyrosine kinase. In contrast to Ron, which has been shown to be an important player in many cancers, little is known about the role of sfRon in cancer pathogenesis. Here we report the striking discovery that sfRon expression is required for development of carcinogen-induced malignant ovarian tumors in mice. We also show that sfRon is expressed in several subtypes of human ovarian cancer including high-grade serous carcinomas, which is in contrast to no detectable expression in healthy ovaries. In addition, we report that introduction of sfRon into OVCAR3 cells resulted in epithelial-to-mesenchymal transition, activation of the PI3K and PDK1 pathway, and inhibition of the MAPK pathway. We demonstrated that sfRon confers an aggressive cancer phenotype in vitro characterized by increased proliferation and migration, and decreased adhesion of ovarian cancer cells. Moreover, the in vivo studies show that OVCAR3 tumors expressing sfRon exhibit significantly more robust growth and spreading to the abdominal cavity when compared with the parental sfRon negative OVCAR3 cells. These data suggest that sfRon plays a significant role in ovarian cancer initiation and progression, and may represent a promising therapeutic target for ovarian cancer treatment.

Purpose: Recent studies have demonstrated that short-form Ron (sfRon) kinase drives breast tumor progression and metastasis through robust activation of the PI3K pathway. We reasoned that upfront, concurrent inhibition of sfRon and PI3K might enhance the antitumor effects of Ron kinase inhibitor therapy while also preventing potential therapeutic resistance to tyrosine kinase inhibitors (TKI). Experimental Design: We used patient-derived breast tumor xenografts (PDX) as high-fidelity preclinical models to determine the efficacy of single-agent or dual Ron/PI3K inhibition. We tested the Ron kinase inhibitor ASLAN002 with and without coadministration of the PI3K inhibitor NVP-BKM120 in hormone receptor–positive [estrogen receptor (ER)+/progesterone receptor (PR)+] breast PDXs with and without PIK3CA gene mutation. Results: Breast PDX tumors harboring wild-type PIK3CA showed a robust response to ASLAN002 as a single agent. In contrast, PDX tumors harboring mutated PIK3CA demonstrated partial resistance to ASLAN002, which was overcome with addition of NVP-BKM120 to the treatment regimen. We further demonstrated that concurrent inhibition of sfRon and PI3K in breast PDX tumors with wild-type PIK3CA provided durable tumor stasis after therapy cessation, whereas discontinuation of either monotherapy facilitated tumor recurrence. Conclusions: Our work provides preclinical rationale for targeting sfRon in patients with breast cancer, with the important stipulation that tumors harboring PIK3CA mutations may be partially resistant to Ron inhibitor therapy. Our data also indicate that tumors with wild-type PIK3CA are most effectively treated with an upfront combination of Ron and PI3K inhibitors for the most durable response. Clin Cancer Res; 21(24); 5588–600. ©2015 AACR.

Although the RAF>MEK>ERK MAP kinase pathway is key to the development of many RAS-mutated cancers, efforts to target this pathway with pharmacologic inhibitors have failed to deliver clinical benefit to patients. Here we show that inhibition of RAS>RAF>MEK>ERK signaling in RAS-mutated cancer cell lines elicits autophagy, a process of cellular recycling that protects cancer cells from the potentially cytotoxic effects of RAF>MEK>ERK pathway inhibition. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic antiproliferative effects against cell lines in vitro and promotes regression of xenografted patient-derived tumors in mice. Finally, treatment of a cancer patient with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking, disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven malignancies such as melanoma, lung, and pancreatic cancer. Citation Format: Conan Kinsey, Soledad Camolotto, Amelie Boespflug, Katrin Gullien, Amanda Truong, Mona Foth, Jill Shea, Michael Seipp, Jeffrey Yap, Lance Burrell, David Lum, Jonathan Whisenant, Courtney Cavalier, Kaitren Rehbein, Stephanie Cutler, Kajsa Afotler, Alana Welm, Bryan Welm, Courtney Scaife, Eric Snyder, Martin McMahon. Targeting MEK1/2 inhibitor resistance in RAS-mutated cancers [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA25.

Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, which could impact the capacity of PDXs for faithful modeling of patient treatment response. Such results contrast with reports that have observed genomic fidelity of PDX models with respect to the originating patient tumors and from early to late passages by direct DNA measurements (DNA sequencing or SNP arrays). Here we resolve these contradicting observations by systematically evaluating CNA changes and the genes they affect during engraftment and passaging in a large, internationally collected set of PDX models, comparing both RNA and DNA-based approaches. The data collected, as part of the U.S. National Cancer Institute (NCI) PDXNet (PDX Development and Trial Centers Research Network) Consortium and EurOPDX consortium, comprises 1548 patient (PT) and PDX datasets (1451 unique samples) from 509 models derived from American, European and Asian cancer patients, spanning across 16 tumor types. By assessing copy number changes by pairwise (PT-PDX or PDX-PDX) correlation and residual analysis to control for systematic biases, our study demonstrates that prior reports of systematic copy number divergence between PTs and PDXs are incorrect, and confirms the high retention of copy number during PDX engraftment and passaging. Moreover, only a small proportion of models show large CNA discordance between the samples, suggesting that the variations observed in PDX are mainly due to rare clonal selection of individual tumors rather than murine pressures. This large scale data analysis also reveals several other findings that clarify the evolutionary process in PDXs. We do observe larger deviations between PT-PDX than in PDX-PDX comparisons, likely due to dilution of PT signal by human stromal cells. Interestingly, we found that a major contributor to the differences between PDX samples is lineage-specific drift associated with splitting of tumors into fragments during PDX propagation. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models, suggesting the lack of systematic copy number evolution driven by the PDX mouse host. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multi-region tumor samples or intra-patient samples. Thus concerns about the genetic stability of the PDX system are likely to be less important than the spatial heterogeneity of solid tumors themselves. This result is consistent with our results on lineage effects during passaging, which indicate that intratumoral spatial evolution is the major reason for genetic drift. Our in-depth tracking of CNAs throughout PDX engraftment and passaging confirms that tumors engrafted and passaged in PDX models maintain a high degree of molecular fidelity to the original patient tumors and their suitability for pre-clinical drug testing. This work also finely enumerates the copy number profiles in hundreds of publicly available models, which will enable researchers to assess the suitability of each for individualized treatment studies. Citation Format: Xing Yi Woo, Jessica Giordano, Anuj Srivastava, Zi-Ming Zhao, Michael W. Lloyd, Roebi de Bruijn, Yun-Suhk Suh, Jong-Il Kim, Han-Kwang Yang, Charles Lee, Dennis A. Dean, Brandi Davis-Dusenbery, Yvonne A. Evrard, James H. Doroshow, Alana L. Welm, Bryan Welm, Michael T. Lewis, Bingliang Fang, Jack A. Roth, Funda Meric-Bernstam, Meenhard Herlyn, Michael Davies, Li Ding, Shunqiang Li, Ramaswamy Govindan, Claudio Isella, Jeffrey A. Moscow, Livio Trusolino, Annette Byrne, Jos Jonkers, Carol J. Bult, Enzo Medico, Jeffrey H. Chuang, PDXNET consortium & EurOPDX consortium. Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1673.

Patient-Derived Xenografts (PDXs) are preclinical models largely used to study tumor biology and drug response. Recent literature highlighted the possibility that growth of human tumors in a mouse microenvironment imposes a selection driving mouse-specific genetic evolution of PDXs, which may compromise their reliability as human cancer models. Conversely, independent studies observed a conservation of the genomic landscape during PDX engraftment and passaging. We noticed that PDX genetic evolution was particularly evident in studies based on copy number aberration (CNA) inferred from gene expression data, while it was negligible when DNA-based CNA profiles were employed. Therefore, in a joint international effort of the EurOPDX and PDXNet consortia, we assembled a dataset of 37 hepatocellular and 54 gastric carcinoma tumor or PDX samples with matched RNA-based and DNA-based CNA profiles. We found that DNA-based CNA profiles invariably yield higher concordance between patient's tumor and derived PDXs than those inferred from RNA. RNA-based profiles displayed poor concordance with matched DNA-based profiles, and much lower resolution, so that they missed many focal copy number events detected by DNA-based methods. These results revealed that CNA measurements cannot be accurately estimated by expression data and that a systematic reassessment of CNA dynamics in PDXs based on DNA data is required. To this aim, we generated CNA profiles by low-pass whole genome sequencing (WGS) of 87 colorectal and 43 breast cancer triplets, each composed of matched patient's tumor (PT) and PDX at early (PDX-early) and later (PDX-late) passage. In this way, for each tumor type, we generated three perfectly matched PT, PDX-early and PDX-late cohorts and performed CNA recurrence analysis by GISTIC in each cohort. The hypothesis was that if the mouse host induces a selective pressure capable of shaping the CNA landscape during PDX engraftment and propagation, GISTIC analysis would highlight systematic and progressive changes, from the PT to the PDX-early cohort, and then to the PDX-late cohort. Notably instead, the CNA profiles of the PT and PDX-early/late cohorts were virtually indistinguishable, with no progressive accumulation or loss of CNA during PDX passage and only minor changes not functionally related or associated to cancer-driver or actionable genes. These results were not consequence of insufficient capture of the CNA repertoire, since the GISTIC profiles recapitulated those generated by TCGA for colorectal and breast cancer. In summary, our analyses highlighted that while RNA-based CNA inferences have inadequate resolution and accuracy to study genomic evolution in PDXs, DNA-based CNA profiles confirm retention of CNAs in PTs and PDXs, excluding a systematic mouse driven selection via copy number changes. Ultimately, these results support the robustness of PDXs as preclinical models for predicting drug response. Citation Format: Jessica Giordano, Xing Yi Woo, Anuj Srivastava, Zi-Ming Zhao, Michael W. Lloyd, Roebi de Bruijn, Yun-Suhk Suh, Francesco Galimi, Andrea Bertotti, Adam Lafferty, Alice C. O'Farrell, Elodie Modave, Diether Lambrechts, Petra ter Brugge, Violeta Serra, Elisabetta Marangoni, Rania El Botty, Jong-Il Kim, Han-Kwang Yang, Charles Lee, Dennis A. Dean, Brandi Davis-Dusenbery, Yvonne A. Evrard, James H. Doroshow, Alana L. Welm, Bryan E. Welm, Michael T. Lewis, Bingliang Fang, Jack Roth, Funda Meric-Bernstam, Meenhard Herlyn, Michael Davies, Li Ding, Shunqiang Li, Ramaswamy Govindan, Jeffrey A. Moscow, Carol J. Bult, Claudio Isella, Livio Trusolino, Annette T. Byrne, Jos Jonkers, Jeffrey H. Chuang, Enzo Medico, EurOPDX consortium & PDXNET consortium. Absence of mouse-specific tumor evolution in patient-derived cancer xenografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1118.

Pancreatic ductal adenocarcinoma was responsible for ~43,000 deaths in the USA in 2017 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS. Although the clinical picture remains grim, the mechanisms by which key alterations in tumor suppressors and proto-oncogenes contribute to PDA have been dissected. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis. However, to date, pharmacologic inhibition of this pathway has provided no clinical benefit to PDA patients. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling in PDA cell lines elicits autophagy, a process of cellular recycling that protects pancreatic cancer cells from the potentially cytotoxic effects of KRAS pathway inhibition. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic antiproliferative effects against PDA cell lines in vitro, and promotes regression of xenografted patient-derived PDA tumors in mice. Finally, treatment of a KRAS-mutated PDA patient on a compassionate basis with the combination of trametinib plus hydroxychloroquine resulted in a partial but nonetheless striking disease response. These data suggest that this combination therapy may represent a new strategy to target RAS-driven cancers such as PDA. Citation Format: Conan G. Kinsey, Soledad A. Camolotto, Amelie M. Boespflug, Katrin P. Gullien, Mona Foth, Jill E. Shea, Michael T. Seipp, Jeffrey T. Yap, Lance D. Burrell, David H. Lum, Jonathan R. Whisenant, Weldon Gilcrease, Courtney C. Cavalieri, Kaitrin M. Rehbein, Stephanie L. Cutler, Kajsa E. Affolter, Alana L. Welm, Bryan E. Welm, Courtney L. Scaife, Eric L. Snyder, Martin McMahon. Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A45.

Background: Estrogen Receptor (ER) positive breast cancer is the most common subtype (70%) and associated with the most deaths from the disease. Almost all deaths from breast cancer are caused by metastasis. While metastatic ER-positive breast cancer is a leading cause of cancer-related death, metastasis studies have largely focused on ER-negative breast cancer. A major reason for such imbalance is the lack of physiologically relevant mice models for studying ER-positive breast cancer. The patient derived xenograft (PDX) technique provides us with a powerful means to study highly clinically relevant human ER-positive breast cancer in mice, but most studies using PDX models still focus on orthotopic rather than metastatic tumor growth. Here we describe studies of the metastatic process using PDX models to more comprehensively understand the value of these PDX lines for therapeutic modelling. These findings will empower us to develop therapeutics designed to control systemic disease, not just orthotopic tumor growth. Experimental design and methods: We have studied 14 ER positive PDX lines in ovariectomized SCID beige mice with and without estradiol supplementation. We have analyzed both orthotopic tumor and a variety of metastatic sites including liver, lung, brain and bone. Data is being generated using next-generation DNA and RNA sequencing, mass spectrometry-based proteomics. We use integrated bioinformatics pipelines to shed light on the metastatic process. Results: We have generated macro-metastatic maps that record sites with observable metastatic nodules of each PDX line in mice. IHC of Liver, bone, brain, lung of each mouse also generated micro-metastatic maps. The most common organ of metastasis in mice is the lung. In some PDX lines, the sites of metastasis differ between groups with and without estradiol supply. The ongoing proteogenomic profiling of orthotopic and metastatic tumors reveals potential molecular drivers of metastasis in ER+ breast cancer. Conclusion: Here, we describe an approach to the systematic investigation of the ER+ breast cancer metastasis process. This approach will allow us to identify druggable targets that may interrupt the metastatic process and/or inhibit tumor growth at metastatic sites. Citation Format: Anran Chen, Purba Singh, Beom-Jun Kim, Lacey E Dobrolecki, Alana L Welm, Michael T Lewis, Matthew J Ellis. Proteogenomic analysis of estrogen modulated breast cancer metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-20.

Metastasis is the biggest challenge in treating breast cancer, and it kills >40,000 breast cancer patients annually in the US. Aberrant expression of the RON receptor tyrosine kinase in breast tumors correlates with poor prognosis and has been shown to promote metastasis. However, the molecular mechanisms that govern how RON promotes metastasis, and how to block it, are still largely unknown. We sought to determine critical effectors of RON using a combination of mutational and pharmacologic strategies. High-throughput proteomic analysis of breast cancer cells upon activation of RON showed robust phosphorylation of ribosomal protein S6. Further analysis revealed that RON strongly signals through mTORC1/p70S6K, which is mediated predominantly by the PI3K pathway. A targeted mutation approach to modulate RON signaling validated the importance of PI3K/mTORC1 pathway for spontaneous metastasis in vivo. Finally, inhibition of mTORC1 with an FDA-approved drug, everolimus, resulted in transient shrinkage of established RON-dependent metastases, and combined blockade of mTORC1 and RON delayed progression. These studies have identified a key downstream mediator of RON-dependent metastasis in breast cancer cells and revealed that inhibition of mTORC1, or combined inhibition of mTORC1 and RON, may be effective for treatment of metastatic breast cancers with elevated expression of RON., Metastasis: Druggable target mediates metastasis-promoting signal A surface protein linked to metastasis in various cancers promotes the spread and migration of breast tumors by activating a druggable signaling pathway. Alana Welm and colleagues from the University of Utah, Salt Lake City, USA, engineered human breast cancer cells to conditionally overexpress RON, a receptor known to drive metastatic disease. They searched for proteins turned on by high RON levels, and found that one, called ribosomal protein S6 (rpS6), became highly decorated with phosphate groups, a sign of activation. Further analyses showed that an intermediary—the mechanistic target of rapamycin complex 1 (mTORC1)—relayed the activation signal to rpS6. The researchers thus inhibited mTORC1, both genetically and with an approved drug called everolimus, and observed transient shrinkage of metastatic breast tumors in mice, while combined blockade of mTORC1 and RON delayed metastatic progression.

The advent of immune checkpoint blockade as a new strategy for immunotherapy has changed the outlook for many aggressive cancers. Although complete tumor eradication is attainable in some cases, durable clinical responses are observed only in a small fraction of patients, underlining urgent need for improvement. We previously showed that RON, a receptor tyrosine kinase expressed in macrophages, suppresses antitumor immune responses, and facilitates progression and metastasis of breast cancer. Here, we investigated the molecular changes that occur downstream of RON activation in macrophages, and whether inhibition of RON can cooperate with checkpoint immunotherapy to eradicate tumors. Activation of RON by its ligand, MSP, altered the gene expression profile of macrophages drastically and upregulated surface levels of CD80 and PD-L1, ligands for T-cell checkpoint receptors CTLA-4 and PD-1. Genetic deletion or pharmacological inhibition of RON in combination with anti-CTLA-4, but not with anti-PD-1, resulted in improved clinical responses against orthotopically transplanted tumors compared to single-agent treatment groups, resulting in complete tumor eradication in 46% of the animals. Positive responses to therapy were associated with higher levels of T-cell activation markers and tumor-infiltrating lymphocytes. Importantly, co-inhibition of RON and anti-CTLA-4 was also effective in clearing metastatic breast cancer cells in lungs, resulting in clinical responses in nearly 60% of the mice. These findings suggest that RON inhibition can be a novel approach to potentiate responses to checkpoint immunotherapy in breast cancer.

With the advancement of personalized cancer immunotherapies, new tools are needed to identify tumor antigens and evaluate T-cell responses in model systems, specifically those that exhibit clinically relevant tumor progression. Key transgenic mouse models of breast cancer are generated and maintained on the FVB genetic background, and one such model is the mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) mouse-an immunocompetent transgenic mouse that exhibits spontaneous mammary tumor development and metastasis with high penetrance. Backcrossing the MMTV-PyMT mouse from the FVB strain onto a C57BL/6 genetic background, in order to leverage well-developed C57BL/6 immunologic tools, results in delayed tumor development and variable metastatic phenotypes.Therefore, we initiated characterization of the FVB MHC class I H-2q haplotype to establish useful immunologic tools for evaluating antigen specificity in the murine FVB strain. Our study provides the first detailed molecular and immunoproteomic characterization of the FVB H-2q MHC class I alleles, including >8,500 unique peptide ligands, a multiallele murine MHC peptide prediction tool, and in vivo validation of these data using MMTV-PyMT primary tumors. This work allows researchers to rapidly predict H-2 peptide ligands for immune testing, including, but not limited to, the MMTV-PyMT model for metastatic breast cancer. Fil: DeVette, Christa I.. Oklahoma Medical Research Foundation; Estados Unidos Fil: Andreatta, Massimo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Bardet, Wilfried. Oklahoma Medical Research Foundation; Estados Unidos Fil: Cate, Steven J.. Oklahoma Medical Research Foundation; Estados Unidos Fil: Jurtz, Vanessa I.. Technical University of Denmark; Dinamarca Fil: Jackson, Kenneth W.. Oklahoma Medical Research Foundation; Estados Unidos Fil: Welm, Alana L.. University of Utah; Estados Unidos Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca Fil: Hildebrand, William H.. Universidad Nacional de San Martín; Argentina

Triple-negative breast cancers (TNBCs) are aggressive and lack targeted therapies. Understanding how nutrients are used in TNBCs may provide new targets for therapeutic intervention. We demonstrate that the transcription factor c-Myc drives glucose metabolism in TNBC cells but does so by a previously unappreciated mechanism that involves direct repression of thioredoxin-interacting protein (TXNIP). TXNIP is a potent negative regulator of glucose uptake, aerobic glycolysis, and glycolytic gene expression; thus its repression by c-Myc provides an alternate route to c-Myc-driven glucose metabolism. c-Myc reduces TXNIP gene expression by binding to an E-box-containing region in the TXNIP promoter, possibly competing with the related transcription factor MondoA. TXNIP suppression increases glucose uptake and drives a dependence on glycolysis. Ectopic TXNIP expression decreases glucose uptake, reduces cell proliferation, and increases apoptosis. Supporting the biological significance of the reciprocal relationship between c-Myc and TXNIP, a Mychigh/TXNIPlow gene signature correlates with decreased overall survival and decreased metastasis-free survival in breast cancer. The correlation between the Mychigh/TXNIPlow gene signature and poor clinical outcome is evident only in TNBC, not in other breast cancer subclasses. Mutation of TP53, which is a defining molecular feature of TNBC, enhances the correlation between the Mychigh/TXNIPlow gene signature and death from breast cancer. Because Myc drives nutrient utilization and TXNIP restricts glucose availability, we propose that the Mychigh/TXNIPlow gene signature coordinates nutrient utilization with nutrient availability. Further, our data suggest that loss of the p53 tumor suppressor cooperates with Mychigh/TXNIPlow-driven metabolic dysregulation to drive the aggressive clinical behavior of TNBC.

Cancer cell survival is dependent on oxidative-stress defenses against reactive oxygen species (ROS) that accumulate during tumorigenesis. Here, we show a non-canonical oxidative-stress defense mechanism through TRPA1, a neuronal redox-sensing Ca2+-influx channel. In TRPA1-enriched breast and lung cancer spheroids, TRPA1 is critical for survival of inner cells that exhibit ROS accumulation. Moreover, TRPA1 promotes resistance to ROS-producing chemotherapies, and TRPA1 inhibition suppresses xenograft tumor growth and enhances chemosensitivity. TRPA1 does not affect redox status but upregulates Ca2+-dependent anti-apoptotic pathways. NRF2, an oxidant-defense transcription factor, directly controls TRPA1 expression, thus providing an orthogonal mechanism for protection against oxidative stress together with canonical ROS-neutralizing mechanisms. These findings reveal an oxidative-stress defense program involving TRPA1 that could be exploited for targeted cancer therapies.

Context College of American Pathologists (CAP) and the American Society of Clinical Oncology have emphasized the need to reduce preanalytic variables for evaluating predictive biomarker expression in breast cancer. Postoperative x-ray of excised breast tissue is commonplace, yet is a variable that has not been investigated previously. We asked whether such radiation affects expression of relevant biomarkers. Design A previous study found that human breast cancers grown in mice demonstrate the same immunohistochemical and molecular profiles as the original tumors. Thirteen patient-derived xenografts were harvested fresh and divided for specimen radiography and a matched nonirradiated control, while following CAP/ASCO guidelines for cold ischemia time and fixation. Samples were processed in a tissue microarray for immunohistochemistry. Estrogen receptor (ER), progesterone receptor (PR), p53, and Ki67 staining was evaluated using an optimized scoring algorithm performed on digitally scanned slides. Samples were also scored manually by a blinded pathologist using the H-score method, and HER2 by the CAP/ASCO 2013 protocol. Histologic scores were compared by analysis of variance. Results There was no significant difference in quantity or intensity of staining between irradiated and nonirradiated samples for estrogen receptor (P=0.28), p53 (P=0.96), and Ki67 (P=0.94). A small but statistically significant difference was observed for PR (P=0.0058). HER2 staining was similarly unchanged in the 1 tumor exhibiting 3+ staining. Conclusions Our study demonstrates that x-ray of breast carcinomas does not significantly affect the expression of predictive biomarkers, with the exception of PR for unclear reasons. It also highlights the utility of the patient-derived xenograft model for biomarker studies.

Non-classical secretory vesicles, collectively referred to as extracellular vesicles (EVs), have been implicated in different aspects of cancer cell survival and metastasis. Here, we describe how a specific class of EVs, called microvesicles (MVs), activates VEGF receptors and tumour angiogenesis through a unique 90 kDa form of VEGF (VEGF90K). We show that VEGF90K is generated by the crosslinking of VEGF165, catalysed by the enzyme tissue transglutaminase, and associates with MVs through its interaction with the chaperone Hsp90. We further demonstrate that MV-associated VEGF90K has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor activation. However, treatment with an Hsp90 inhibitor releases VEGF90K from MVs, restoring the sensitivity of VEGF90K to Bevacizumab. These findings reveal a novel mechanism by which cancer cell-derived MVs influence the tumour microenvironment and highlight the importance of recognizing their unique properties when considering drug treatment strategies., Extracellular vesicles (EVs) contain VEGF and can contribute to tumour angiogenesis, although the mechanism remains unclear. Here, the authors find that a form of VEGF (VEGF90K) resistant to Bevacizumab but sensitive to HSP90 inhibitors, associates with EVs through its interaction with Hsp90.

With an increasing body of literature covering RON receptor tyrosine kinase function in different types of human cancers, it is becoming clear that RON has prominent roles in both cancer cells and in the tumor-associated microenvironment. RON not only activates several oncogenic signaling pathways in cancer cells, leading to more aggressive behavior, but also promotes an immunosuppressive, alternatively activated phenotype in macrophages and limits the antitumor immune response. These two unique functions of this oncogene, the strong correlation between RON expression and poor outcomes in cancer, and the high tolerability of a new RON inhibitor make it an exciting therapeutic target, the blocking of which offers an advantage toward improving the survival of cancer patients. Here, we discuss recent findings on the role of RON signaling in cancer progression and its potential in cancer therapy.

Ewing sarcoma is an aggressive pediatric bone cancer. In Ewing sarcoma cells, down-regulation of focal adhesion and actin regulatory proteins disrupts cell adhesion and actin integrity. Reexpression of target proteins zyxin and α5 integrin is sufficient to restore actin cytoskeletal integrity while enhancing lung colonization in a mouse model., Ewing sarcoma is the second-most-common bone cancer in children. Driven by an oncogenic chromosomal translocation that results in the expression of an aberrant transcription factor, EWS/FLI, the disease is typically aggressive and micrometastatic upon presentation. Silencing of EWS/FLI in patient-derived tumor cells results in the altered expression of hundreds to thousands of genes and is accompanied by dramatic morphological changes in cytoarchitecture and adhesion. Genes encoding focal adhesion, extracellular matrix, and actin regulatory proteins are dominant targets of EWS/FLI-mediated transcriptional repression. Reexpression of genes encoding just two of these proteins, zyxin and α5 integrin, is sufficient to restore cell adhesion and actin cytoskeletal integrity comparable to what is observed when the EWS/FLI oncogene expression is compromised. Using an orthotopic xenograft model, we show that EWS/FLI-induced repression of α5 integrin and zyxin expression promotes tumor progression by supporting anchorage-independent cell growth. This selective advantage is paired with a tradeoff in which metastatic lung colonization is compromised.

SUMMARY Metastasis is the major cause of death in cancer patients, yet the genetic and epigenetic programs that drive metastasis are poorly understood. Here, we report an epigenetic reprogramming pathway that is required for breast cancer metastasis. Concerted differential DNA methylation is initiated by the activation of the RON receptor tyrosine kinase by its ligand, macrophage stimulating protein (MSP). Through PI3K signaling, RON/MSP promotes expression of the G:T mismatch-specific thymine glycosylase MBD4. RON/MSP and MBD4-dependent aberrant DNA methylation results in the misregulation of a specific set of genes. Knockdown of MBD4 reverses methylation at these specific loci and blocks metastasis. We also show that the MBD4 glycosylase catalytic residue is required for RON/MSP-driven metastasis. Analysis of human breast cancers revealed that this epigenetic program is significantly associated with poor clinical outcome. Furthermore, inhibition of Ron kinase activity with a pharmacological agent blocks metastasis of patient-derived breast tumor grafts in vivo.

Luminal breast cancers are typically estrogen receptor–positive and generally have the best prognosis. However, a subset of luminal tumors, namely luminal B cancers, frequently metastasize and recur. Unfortunately, the causal events that drive their progression are unknown, and therefore it is difficult to identify individuals who are likely to relapse and should receive escalated treatment. Here, we identify a bifunctional RasGAP tumor suppressor whose expression is lost in almost 50% of luminal B tumors. Moreover, we show that two RasGAP genes are concomitantly suppressed in the most aggressive luminal malignancies. Importantly, these genes cooperatively regulate two major oncogenic pathways, RAS and NF-κB, through distinct domains, and when inactivated drive the metastasis of luminal tumors in vivo. Finally, although the cooperative effects on RAS drive invasion, NF-κB activation triggers epithelial-to-mesenchymal transition and is required for metastasis. Collectively, these studies reveal important mechanistic insight into the pathogenesis of luminal B tumors and provide functionally relevant prognostic biomarkers that may guide treatment decisions. Significance: The lack of insight into mechanisms that underlie the aggressive behavior of luminal B breast cancers impairs treatment decisions and therapeutic advances. Here, we show that two RasGAP tumor suppressors are concomitantly suppressed in aggressive luminal B tumors and demonstrate that they drive metastasis by activating RAS and NF-κB. Cancer Discov; 7(2); 202–17. ©2016 AACR. See related commentary by Sears and Gray, p. 131. This article is highlighted in the In This Issue feature, p. 115

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational pre-clinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and “Triple-negative” (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward “credentialing” of PDX models as surrogates to represent individual patients for use in pre-clinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research.

Targeting HER2 is one of the greatest successes in oncology, and has resulted in the generation of a wide array of HER2-targeting agents. Our genomic approaches are revealing other mechanisms of HER2 activation, such as our discovery of activating HER2 mutations in different cancer types. From initial breast cancer and SUMMIT trial data, the pan-HER drug neratinib as monotherapy showed initial clinical response in ER+ breast cancer, but with frequent early relapse. This study investigates the preclinical efficacy of anti HER2 agents alone or in combination with endocrine therapy agents or in combination with CDK4/6 inhibitors using ER+/HER2 mutant cell lines and ex vivo HER2 mutant patient derived xenograft (PDX) model. ER+ breast cancer cell lines (T47D and MCF7) stably expressing HER2V777L, and ER+/HER2 mutant PDX model (HER2G778_P780 dup) were used to examine HER2 signaling. We found that MCF7/T47D cells expressing HER2V777L and HER2G778_P780 dup PDX tumors showed strongly activated autophosphorylation of HER2 and increased expression of CDK4, CDK6, phospho-Rb, and cyclin D1 as compared to MCF7/T47D cells expressing HER2WT or ER+/non-HER2mut PDX model, suggesting that HER2 mutations preferentially depend on CDK4/6 signaling for cell growth. Additionally, we showed that activating MCF7 HER2V777L cause resistance to endocrine therapy treatment (fulvestrant IC50 >5μM). Further, we show that neratinib alone is effective at higher concentrations (IC50 < 2μM) in MCF7/HER2V777L cells. Next we showed that abemaciclib alone exhibited moderate activity against MCF7 HER2V777L cells (IC50 < 0.4μM) and additional activity in combination with neratinib (IC50 < 0.06μM) was seen. Moreover, ex vivo HER2G778_P780 dup cells are relatively resistant to fulvestrant alone (IC50 < 0.2μM), neratinib alone (IC50 < 0.006μM), abemaciclib alone (IC50 < 0.04μM), and neratinib in combination with abemaciclib (IC50 < 0.005μM), suggesting that patients harboring ER+/HER2-mutant tumors may benefit from neratinib in combination with abemaciclib. Therefore, we propose that simultaneous targeting of both HER2 and the CDK4/6 axis is required for maximal inhibition of ER+ breast cancers harboring HER2 activating mutations. Citation Format: Vaishnavi Devarakonda, LaTerrica Williams, Sinem Seker, Jonathan T. Lei, Purba Singh, Airi Han, Meenakshi Anurag, Kimberly R. Holloway, Alana L. Welm, Matthew J. Ellis, Shyam M. Kavuri. Evaluating preclinical efficacy of anti-HER2 drug combinations using ER+/HER2 mutant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 850.

Pancreatic ductal adenocarcinoma was responsible for ~43,000 deaths in the USA in 2017 and is the epitome of a recalcitrant cancer driven by an, as yet, pharmacologically intractable oncoprotein, KRAS. Although the clinical picture remains grim, the mechanisms by which key alterations in tumor suppressors and proto-oncogenes contribute to PDA have been dissected. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis. However, to date, pharmacological inhibition of this pathway has provided no clinical benefit to PDA patients. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling in PDA cell lines elicits autophagy, a process of cellular recycling that protects pancreatic cancer cells from the potentially cytotoxic effects of KRAS pathway inhibition. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro, and promotes regression of xenografted patient-derived PDA tumors in mice. Finally, treatment of a PDA patient with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers such as PDA. Citation Format: Conan G. Kinsey, Soledad A. Camolotto, Amelie M. Boespflug, Katrin P. Guillen, Mona Foth, Amanda Truong, Sophia S. Schuman, Jill E. Shea, Michael T. Seipp, Jeffrey T. Yap, Lance D. Burrell, David H. Lum, Jonathan R. Whisenant, G. Weldon Gilcrease, Courtney C. Cavalieri, Kaitrin M. Rehbein, Stephanie L. Cutler, Kajsa E. Affolter, Alana L. Welm, Bryan E. Welm, Courtney L. Scaife, Eric L. Snyder, Martin McMahon. Protective autophagy elicited by RAF®MEK®ERK inhibition suggests a treatment strategy for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2183.

In the version of this article initially published, the label over the bottom schematic in Fig. 1a was “pH > 5.0”; it should have been “pH < 5.0”. Further, the original article misspelt the surname of Katrin P. Guillen as “Gullien”. These errors have been corrected in the print, PDF and HTML versions of the article.

Invasive lobular carcinoma (ILC) represents ∼10% of newly diagnosed breast tumors, or ∼30,000 cases annually in the US. However, ILC-specific signaling and endocrine responsiveness are not well characterized. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients with similar biomarkers, and that ILC patients may not benefit from adjuvant tamoxifen. We hypothesize that estrogen receptor-alpha (ER) regulated gene expression is unique in ILC cells and drives endocrine resistance. The ER-positive ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of cell growth and ER-regulated gene expression in response to estradiol (E2). To examine the ER-regulated transcriptome, we performed gene expression microarray analyses and ER ChIP-Seq following E2 treatment. In parallel, E2 response was assessed in vivo in the primary ILC xenograft HCI-013. Response to endocrine therapies, tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI), were also examined in ILC cell lines. We observed that E2 induced growth and ER target gene expression in MDA MB 134VI and SUM44PE. We compared our ILC microarray data to published data from ER-positive IDC cell lines (MCF-7, T47D, BT474), and identified 254 genes that were E2-regulated in all 5 cell lines (e.g. GREB1, MYC). 915 genes were E2-regulated only in both ILC cell lines. Consistent with this, roughly half of ER binding sites identified in MDA MB 134VI ChIP-Seq were unique versus published MCF-7 data. We chose a subset of ILC-specific and common E2-regulated genes (n = 107) to assess in vivo using Nanostring gene expression analyses of HCI-013. E2-regulation was observed for 32/107 target genes (30%), suggesting that these genes may be E2-regulated in vivo in ILC patient tumors. Consistent with clinical data, both ILC cell lines presented de novo tamoxifen resistance. SUM44PE were growth-inhibited by ICI, but unaffected by Tam, 4OHT, or Bx. Similarly, ICI blocked E2-induced growth in MDA MB 134VI, but Tam, 4OHT, and Bx acted as partial agonists, inducing ∼25% growth. Partial agonism was not limited to tamoxifen, as other SERMs (e.g. raloxifene) also induced growth. We then measured ER-regulated gene expression in MDA MB 134VI following tamoxifen treatment. Tam, 4OHT, and Bx acted as agonists for 38/107 genes, whereas ICI acted as an antagonist. All 38 genes were E2-repressed targets (e.g. CCNG2), suggesting that ER-mediated gene repression may be critical to tamoxifen-resistance in ILC. Finally, we observed that FGFR1, frequently amplified in ILC, may be critical for ILC cell survival in the presence of tamoxifen. These data support the hypothesis that unique ER-mediated gene expression in ILC cells drives endocrine resistance. The de novo tamoxifen resistance observed in ILC cells may correlate with the worse outcomes in ILC patients recently reported. We hypothesize that genes regulated by tamoxifen as an agonist may play a role in tamoxifen-induced growth or serve as biomarkers of resistance. Targeting growth factor signaling using FGFR1 inhibitors may block survival pathways required by ILC cells and reverse tamoxifen resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-03.

Many “nonmetastatic” cancers have spawned undetectable metastases before diagnosis. Eventual outgrowth of these microscopic lesions causes metastatic relapse and death, yet the events that dictate when and how micrometastases convert to overt metastases are largely unknown. We report that macrophage-stimulating protein and its receptor, Ron, are key mediators in conversion of micrometastases to bona fide metastatic lesions through immune suppression. Genetic deletion of Ron tyrosine kinase activity specifically in the host profoundly blocked metastasis. Our data show that loss of Ron function promotes an effective antitumor CD8+ T-cell response, which specifically inhibits outgrowth of seeded metastatic colonies. Treatment of mice with a Ron-selective kinase inhibitor prevented outgrowth of lung metastasis, even when administered after micrometastatic colonies had already been established. Our findings indicate that Ron inhibitors may hold potential to specifically prevent outgrowth of micrometastases in patients with cancer in the adjuvant setting. Significance: Our data shed new light on an understudied, yet critically important aspect of metastasis: the conversion of clinically undetectable micrometastatic tumor cells to overt metastases that eventually cause death of the patient. Our work shows that Ron inhibition can significantly reduce metastatic outgrowth, even when administered after metastatic colonies are established. Cancer Discov; 3(7); 751–60. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 705

Triple-negative breast cancer (TNBC) is associated with poor survival. Chemotherapy is the only standard treatment for TNBC. The prevalence of BRCA1 inactivation in TNBC has rationalized clinical trials of poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors. Similarly, the overexpression of epidermal growth factor receptor (EGFR) rationalized anti-EGFR therapies in this disease. However, clinical trials using these 2 strategies have not reached their promise. In this study, we used EGFR as a target for radioimmunotherapy and hypothesized that EGFR-directed radioimmunotherapy can deliver a continuous lethal radiation dose to residual tumors that are radiosensitized by PARP inhibitors and chemotherapy. Methods:We analyzed EGFR messenger RNA in published gene expression array studies and investigated EGFR protein expression by immunohistochemistry in a cohort of breast cancer patients to confirm EGFR as a target in TNBC. Preclinically, using orthotopic and metastatic xenograft models of EGFR-positive TNBC,we investigated the effect of the novel combination of 177 Lu-labeled anti-EGFR monoclonal antibody, chemotherapy, and PARP inhibitors on cell death and the survival of breast cancer stem cells. Results: In this first preclinical study of anti-EGFR radioimmunotherapy in breast cancer, we found that anti-EGFR radioimmunotherapy is safe and that TNBC orthotopic tumors and established metastases were eradicated in mice treated with anti-EGFR radioimmunotherapy combined with chemotherapy and PARP inhibitors. We showed that the superior response to this triple-agent combination therapy was associated with apoptosis and eradication of putative breast cancer stem cells. Conclusion: Our data support further preclinical investigations toward the development of combination therapies using systemic anti-EGFR radioimmunotherapy for the treatment of recurrent and metastatic TNBC. Refereed/Peer-reviewed

Background microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. Methods Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. Results Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. Conclusions Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0753-x) contains supplementary material, which is available to authorized users.

Purpose: The current study evaluated associative effects of breast cancer cells with the tumor microenvironment and its influence on tumor behavior. Experimental Design: Formalin-fixed, paraffin-embedded tissue and matched protein lysates were evaluated from two independent breast cancer patient datasets (TCGA and MD Anderson). Reverse-phase protein arrays (RPPA) were utilized to create a proteomics signature to define breast tumor subtypes. Expression patterns of cell lines and normal breast tissues were utilized to determine markers that were differentially expressed in stroma and cancer cells. Protein localization and stromal contents were evaluated for matched cases by imaging. Results: A subtype of breast cancers designated “Reactive,” previously identified by RPPA that was not predicted by mRNA profiling, was extensively characterized. These tumors were primarily estrogen receptor (ER)-positive/human EGF receptor (HER)2-negative, low-risk cancers as determined by enrichment of low-grade nuclei, lobular or tubular histopathology, and the luminal A subtype by PAM50. Reactive breast cancers contained high numbers of stromal cells and the highest extracellular matrix content typically without infiltration of immune cells. For ER-positive/HER2-negative cancers, the Reactive classification predicted favorable clinical outcomes in the TCGA cohort (HR, 0.36; P < 0.05). Conclusions: A protein stromal signature in breast cancers is associated with a highly differentiated phenotype. The stromal compartment content and proteins are an extended phenotype not predicted by mRNA expression that could be utilized to subclassify ER-positive/HER2-negative breast cancers. Clin Cancer Res; 22(20); 5068–78. ©2016 AACR.

SummaryReversal of promoter DNA hypermethylation and associated gene silencing is an attractive cancer therapy approach. The DNA methylation inhibitors decitabine and azacitidine are efficacious for hematological neoplasms at lower, less toxic, doses. Experimentally, high doses induce rapid DNA damage and cytotoxicity, which do not explain the prolonged time to response observed in patients. We show that transient exposure of cultured and primary leukemic and epithelial tumor cells to clinically relevant nanomolar doses, without causing immediate cytotoxicity, produce an antitumor “memory” response, including inhibition of subpopulations of cancer stem-like cells. These effects are accompanied by sustained decreases in genomewide promoter DNA methylation, gene reexpression, and antitumor changes in key cellular regulatory pathways. Low-dose decitabine and azacitidine may have broad applicability for cancer management.

Ewing sarcoma is a tumor of the bone and soft tissue caused by the expression of a translocation-derived oncogenic transcription factor, EWS/FLI. Overt metastases are associated with a poor prognosis in Ewing sarcoma, but patients without overt metastases frequently harbor micrometastatic disease at presentation. This suggests that the metastatic potential of Ewing sarcoma exists at an early stage during tumor development. We have therefore explored whether the inciting oncogenic event in Ewing sarcoma, EWS/FLI, directly modulates tumor cell features that support metastasis, such as cell adhesion, cell migration, and cytoarchitecture. We used an RNAi-based approach in patient-derived Ewing sarcoma cell lines. Although we hypothesized that EWS/FLI might induce classic metastatic features, such as increased cell adhesion, migration, and invasion (similar to the phenotypes observed when epithelial malignancies undergo an epithelial-to-mesenchymal transition during the process of metastasis), surprisingly, we found the opposite. Thus, EWS/FLI expression inhibited the adhesion of isolated cells in culture and prevented adhesion in an in vivo mouse lung assay. Cell migration was similarly inhibited by EWS/FLI expression. Furthermore, EWS/FLI expression caused a striking loss of organized actin stress fibers and focal adhesions and a concomitant loss of cell spreading, suggesting that EWS/FLI disrupts the mesenchymal phenotype of a putative tumor cell-of-origin. These data suggest a new paradigm for the dissemination and metastasis of mesenchymally derived tumors: these tumors may disseminate via a "passive/stochastic" model rather than via an "active" epithelial-to-mesenchymal type transition. In the case of Ewing sarcoma, it appears that the loss of cell adhesion needed to promote tumor cell dissemination might be induced by the EWS/FLI oncogene itself rather than via an accumulation of stepwise mutations.

Rationale: Netrin-4 regulates vascular development. Identity of netrin-4 endothelial receptor and its subsequent cell functions is controversial. We previously demonstrated that the inhibition of netrin-1 canonical receptors, Unc5B and neogenin, expressed by lymphatic endothelial cells, do not suppress netrin-4-induced cell signaling and functions. Netrin family members were shown to signal through a range of receptors, including integrins (such as α3β1, α6β1, and α6β4) in nonendothelial cells. Objective: We tested whether integrins are netrin-4 receptors in the endothelium. Methods and Results: The α6β1 integrin is expressed by endothelial cells, and binds netrin-4 in a dose-dependent manner. Inhibition of α6 or β1 integrin subunits suppresses netrin-4-induced endothelial cell migration, adhesion, and focal adhesion contact. Netrin-4-stimulated phosphorylation of Src kinase family, effectors of endothelial cell migration, is also abolished by α6 or β1 inhibition. Finally, netrin-4 and α6β1 integrin expression colocalize in mouse embryonic, intestine, and tumor vasculature. Conclusions: The α6β1 integrin is a netrin-4 receptor in lymphatic endothelium and consequently represents a potential target to inhibit netrin-4-induced metastatic dissemination.

Metastasis is the cause of death for nearly all types of cancer, including breast cancer. An exciting new area of research in metastatic breast cancer centers on immune therapy. Although new immune checkpoint blockade therapies have provided benefit for a fraction of patients tested so far, the majority of patients still do not respond to these drugs. A better understanding of how the immune system can be harnessed against metastatic breast cancer is required in order to improve patient outcomes in this area. We previously discovered that macrophage Ron receptor tyrosine kinase promotes breast cancer metastasis by inhibiting CD8+ cytotoxic T lymphocyte activity. We hypothesized that dual blockade of Ron activity and existing immune checkpoint molecules would unleash a more effective CD8+ T cell response to control or eliminate metastatic breast cancer. Our strategy would simultaneously disable tumor-mediated immune evasion mechanisms on both the innate and adaptive immune systems. To test the potential of combination therapy, the Ron inhibitor BMS-777607 and/or the immune checkpoint blocking agents anti-PD1 or anti-CTLA4, were administered in our mouse mammary tumor model to examine the effects on breast tumor progression. To examine Ron-specific effects of BMS-777607, we also examined the effect of genetic deletion of host Ron signaling activity in combination with immunotherapy. The anti-tumor immune response was comprehensively examined by multi-color flow cytometry and immunohistochemical staining for tumor-infiltrating lymphocytes (TILs). Our data suggest that the combination of Ron inhibition with immune checkpoint blockade may be an effective therapy in breast cancer. Citation Format: Shu Chin Alicia Lai, Atakan Ekiz, Alana Welm. Ron kinase inhibition to improve immunotherapy for breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A30.

Patient-derived xenografts (PDX) are valuable, clinically-relevant models of cancer. Their close genomic, phenotypic, and temporal association with patient tumors makes them well-suited for pre-clinical and co-clinical studies that assess the potential of new therapeutics. However, PDX models are not amenable to large-scale drug sensitivity studies due to their high cost and low throughput capacity. To address this, we established and characterized long-term PDX organoid (PDxO) cultures from breast cancer (BC) PDXs and evaluated their utility in therapeutic studies. To establish PDxO culture conditions, we extensively tested medium supplements, including growth factors, kinase inhibitors, conditioned medium, and antioxidants, along with extracellular matrix composition in 3D gels. Using optimized culture conditions for each BC subtype, we established PDxOs from 16 PDX lines in the HCI series, with a PDX to PDxO success rate of 85%. Around 20% of PDxOs contained aggressive mouse stroma, which had to be eliminated by FACS for long-term culture success. PDxOs were maintained for over 1 year, during which we tracked viability, doubling time, organoid size, genomics, epithelial character, and tumorigenicity. Doubling times stabilized after 60 days of initial culture, with a mean of 6.4±1.7 days for triple negative (TN) lines and 7.2±1 days for ER+ lines. Mean organoid size remained stable, with ER+ PDxOs significantly smaller than TN PDxOs, at 91 and 262 cells/organoid respectively (p=0.0012). PDxOs histologically resembled their PDX counterparts when stained for H&E, Vimentin, and CAM 5.2. RNA-Seq and copy-number variation analyses showed that PDxOs clustered with their PDXs and patient tumors. To assess if culturing affected tumorigenicity, we re-implanted PDxOs into mice following early and late passaging. 5/5 early passage and 5/6 late passage PDxOs engrafted and formed tumors. Resulting tumors showed similar gene expression profiles as their original PDXs by RNA-Seq. These data suggest that PDxOs generally recapitulate the molecular and genomic features of their originating PDXs and patient tumors. Having established PDxO cultures, we evaluated their utility as a therapeutic screening platform. We developed a screen to differentiate compounds with cytotoxic and cytostatic effects. NCI CTEP and clinically-approved BC therapies (n=40) were screened in quadruplicate 8-point dose response curves on all 16 PDxOs across three biological replicates. Drug response profiles were stable across biological replicates, spanning up to 1 year in culture. PDxOs exhibited diverse responses to therapies targeting cIAP1, PI3K, and CHK1/2. Initial work to evaluate concordance between PDxO and in vivo PDX responses returned trending linear correlations between PDxO dose response data and change in PDX growth rate following treatment (R2 = 0.52-0.77; p = 0.045-0.12, across 3 PDXs and 9 compounds). Ongoing work aims to confirm concordance between PDX and PDxO models. Our work demonstrates that PDxO models are cost-efficient, easy to maintain, and grow indefinitely - making them renewable and accessible cancer models. PDxOs are a powerful parallel resource to PDX models, especially useful for efficient determination of PDX drug response. We are currently expanding our PDxO bank to include 100 models which will be deposited with the NCI as part of the PDXNet effort. Citation Format: Katrin P. Guillen, Sandra D. Scherer, Yi Qiao, Satya S. Pathi, James M. Graham, Maihi Fujita, Yoko S. DeRose, Jason Gertz, Gabor T. Marth, Katherine E. Varley, Alana L. Welm, Bryan E. Welm. Predicting breast cancer therapy response using a patient-derived xenograft organoid screening platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-038.

Netrin-4, a laminin-related secreted protein is an axon guidance cue recently shown essential outside of the nervous system, regulating mammary and lung morphogenesis as well as blood vascular development. Here, we show that Netrin-4, at physiologic doses, induces proliferation, migration, adhesion, tube formation and survival of human lymphatic endothelial cells in vitro comparable to well-characterized lymphangiogenic factors fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF), vascular endothelial growth factor-A (VEGF-A), and vascular endothelial growth factor-C (VEGF-C). Netrin-4 stimulates phosphorylation of intracellular signaling components Akt, Erk and S6, and their specific inhibition antagonizes Netrin-4–induced proliferation. Although Netrin receptors Unc5B and neogenin, are expressed by human lymphatic endothelial cells, suppression of either or both does not suppress Netrin-4–promoted in vitro effects. In vivo, Netrin-4 induces growth of lymphatic and blood vessels in the skin of transgenic mice and in breast tumors. Its overexpression in human and mouse mammary carcinoma cancer cells leads to enhanced metastasis. Finally, Netrin-4 stimulates in vitro and in vivo lymphatic permeability by activating small GTPases and Src family kinases/FAK, and down-regulating tight junction proteins. Together, these data provide evidence that Netrin-4 is a lymphangiogenic factor contributing to tumor dissemination and represents a potential target to inhibit metastasis formation.

Inappropriate activation of developmental pathways is a well-recognized tumor-promoting mechanism. Here we show that overexpression of the homeoprotein Six1, normally a developmentally restricted transcriptional regulator, increases TGF-β signaling in human breast cancer cells and induces an epithelial-mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-β signaling. TGF-β signaling and EMT have been implicated in metastatic dissemination of carcinoma. Accordingly, we used spontaneous and experimental metastasis mouse models to demonstrate that Six1 overexpression promotes breast cancer metastasis. In addition, we show that, like its induction of EMT, Six1-induced experimental metastasis is dependent on its ability to activate TGF-β signaling. Importantly, in human breast cancers Six1 correlated with nuclear Smad3 and thus increased TGF-β signaling. Further, breast cancer patients whose tumors overexpressed Six1 had a shortened time to relapse and metastasis and an overall decrease in survival. Finally, we show that the effects of Six1 on tumor progression likely extend beyond breast cancer, since its overexpression correlated with adverse outcomes in numerous other cancers including brain, cervical, prostate, colon, kidney, and liver. Our findings indicate that Six1, acting through TGF-β signaling and EMT, is a powerful and global promoter of cancer metastasis.

A multidisciplinary method combining transcriptional data, specificity profiling, and biological characterization of an enzyme may be used to predict novel substrates. By integrating protease substrate profiling with microarray gene coexpression data from nearly 2,000 human normal and cancerous tissue samples, three fundamental components of a protease-activated signaling pathway were identified. We find that MT-SP1 mediates extracellular signaling by regulating the local activation of the prometastatic growth factor MSP-1. We demonstrate MT-SP1 expression in peritoneal macrophages, and biochemical methods confirm the ability of MT-SP1 to cleave and activate pro-MSP-1 in vitro and in a cellular context. MT-SP1 induced the ability of MSP-1 to inhibit nitric oxide production in bone marrow macrophages. Addition of HAI-1 or an MT-SP1-specific antibody inhibitor blocked the proteolytic activation of MSP-1 at the cell surface of peritoneal macrophages. Taken together, our work indicates that MT-SP1 is sufficient for MSP-1 activation and that MT-SP1, MSP-1, and the previously shown MSP-1 tyrosine kinase receptor RON are required for peritoneal macrophage activation. This work shows that this triad of growth factor, growth factor activator protease, and growth factor receptor is a protease-activated signaling pathway. Individually, MT-SP1 and RON overexpression have been implicated in cancer progression and metastasis. Transcriptional coexpression of these genes suggests that this signaling pathway may be involved in several human cancers.

Bone destruction occurs in aging and numerous diseases, including osteoporosis and cancer. Many cancer patients have bone osteolysis that is refractory to state-of-the-art treatments, which block osteoclast activity with bisphosphonates or by inhibiting the receptor activator of nuclear factor κB ligand (RANKL) pathway. We previously showed that macrophage-stimulating protein (MSP) signaling, which is elevated in about 40% of breast cancers, promotes osteolytic bone metastasis by activation of the MSP signaling pathway in tumor cells or in the bone microenvironment. We show that MSP signals through its receptor, RON tyrosine kinase, expressed on host cells, to activate osteoclasts directly by a previously undescribed pathway that is complementary to RANKL signaling and converges on proto-oncogene, non-receptor tyrosine kinase SRC (SRC). Genetic or pharmacologic inhibition of RON kinase blocked cancer-mediated bone destruction and osteoporosis in several mouse models. Furthermore, the RON kinase inhibitor BMS-777607/ASLAN002 altered markers of bone turnover in a first-in-human clinical cancer study, indicating the inhibitor's potential for normalizing bone loss in patients. These findings uncover a new therapeutic target for pathogenic bone loss and provide a rationale for treatment of bone destruction in various diseases with RON inhibitors.

In human breast cancer, overexpression of the protooncogene MET is strongly associated with poor prognosis and high risk of metastasis. It stands out as a reliable prognostic indicator of survival and defines a set of tumors exclusive of those that express HER2 or hormone receptors. Studies have shown that overexpression of mutant forms of MET cause cancer in mice. However, MET mutations have not been found in human breast cancer, and the consequences of overexpression of normal MET are unknown. To investigate the role of MET and other putative oncogenes in breast cancer, we developed an experimental system that involves retroviral delivery of genes into primary mammary epithelial cells, followed by transplantation of the transduced cells into mammary fat pads. Using this approach, we found that overexpression of wild-type MET leads to the development of nonprogressive neoplasms. The lesions progressed to mammary adenocarcinoma when a second protooncogene, MYC , was overexpressed, indicating that MET and MYC cooperate in mammary tumorigenesis. Both the nonprogressive neoplasms and adenocarcinomas display characteristics consistent with transformation and expansion of mammary progenitor cells. The approach described here should provide a useful model with which to efficiently test effects of various genes on tumor development in the breast.

Aim Class I MHC mediated immune responses to tumors are complex and poorly understood, and enhanced tools for studying tumor immunity are needed. One such tool is the MMTV-PyMT mouse – a breast cancer model in an immune-competent FVB strain. PyMT is a viral oncogene expressed as a transgene under a mammary gland-selective promoter. MMTV-PyMT mice exhibit clinically relevant mammary adenocarcinoma development and spontaneous metastasis. Here, we develop an MHC class I presented tumor-antigen vaccine in these mice that enables us to track T cell responses during primary tumor growth and utilize this knowledge to guide T cell immune-therapy development. Methods The MMTV-PyMT mouse vaccine model is composed of the following: (1) PyMT DNA vaccination and anti-tumor efficacy, (2) ex vivo PyMT epitope mapping and H2-q characterization, and (3) in vivo validation of H-2q/PyMT peptide epitopes in vaccination and checkpoint blockade settings. The FVB mice have MHCI molecules of the uncharacterized H-2 “q” haplotype, thus we use soluble MHCI to identify H-2D q -presented PyMT peptides. PyMT and soluble H2-Dq were co-transfected into an immortalized cell line. Secreted MHCI were purified and PyMT-specific peptides directly eluted from the Class I. Following mass spectrometry identification, PyMT peptides were validated in vaccinated FVB mice via IFN- γ ELISpot. Candidate peptides will be tested in MMTV-PyMT mice treated with the checkpoint inhibitors α -CTLA4/PD1. Results DNA vaccination with PyMT and an adjuvant significantly inhibited tumor growth. PyMT MHCI epitope discovery using soluble H2-D q identified >6000 peptides, including 2 PyMT-specific peptides. Peptides are currently being tested on vaccinated or checkpoint blockade treated animals. Together, these data have generated the first H-2D q binding motif and refined current ligand prediction tools for the “q” haplotype. Conclusions Identification of immunodominant MHCI PyMT peptides in vaccinated and checkpoint-inhibitor treated, tumor-bearing mice uniquely allows tracking of antigen-specific responses in FVB mice. Completion of this project generates the first model antigen vaccine in a highly utilized, spontaneously metastatic model of breast cancer, empowering cancer biologists to study antigen-specific T cell responses in breast cancer.

Bone destruction occurs in aging and numerous diseases, including osteoporosis and cancer. Many cancer patients have bone osteolysis that is refractory to state-of-the-art treatments, which block osteoclast activity with bisphosphonates or by inhibiting the RANKL pathway. We previously showed that macrophage-stimulating protein (MSP) signaling, which is elevated in approximately 40% of breast cancers, promotes osteolytic bone metastasis, but it was unknown whether this was due to activation of the MSP signaling pathway in the tumor cells or in the bone microenvironment. Here we show that MSP signals through host expression of its receptor, RON tyrosine kinase, to directly activate osteoclasts by a previously undescribed pathway that is complementary to RANKL signaling and converges on SRC. Genetic or pharmacologic inhibition of RON kinase blocked cancer-mediated bone destruction and osteoporosis in several mouse models. Furthermore, the RON kinase inhibitor BMS-777607/ASLAN002 altered markers of bone turnover in a first-in-human clinical cancer study, indicating potential for normalizing bone loss in patients. These findings uncover a new therapeutic target for pathogenic bone loss and provide a rationale for treatment of bone destruction in various diseases with RON inhibitors. Citation Format: Kelsi Andrade, Jaime Fornetti, Ling Zhao, Scott C. Miller, Lor R. Randall, Neysi Anderson, Susan E. Waltz, Mark McHale, Alana L. Welm. RON kinase: a target for treatment of cancer-induced bone destruction and osteoporosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-209. doi:10.1158/1538-7445.AM2017-LB-209

Cell lines grown in 2-dimensional (2D) plastic surfaces have historically been the main models to identify cancer cell vulnerabilities. In contrast, malignant tumors in vivo grow as 3-dimensional (3D) cellular masses and manifest remarkably different drug-response patterns compared to cell lines. We have shown that breast cancer (BrCa) cells in 3D extracellular matrices acquire distinct phenotypic properties, compared to the 2D counterparts. Notably, serum-free culture medium supplemented with defined cocktails of developmental morphogens was sufficient to support the growth of BrCa cell lines in 3D, but not in 2D conditions; indicating a key role of 3D architecture in in vivo growth regulation. CRISPR/Cas9-based gene editing screens in BrCa cells indicated that distinct gene subset govern the growth in 3D vs. 2D conditions. Using similar culture conditions, we expanded several molecularly annotated BrCa patient-derived 3D Organoids (3D PDOs), adapted them into a high-throughput miniaturized drug screening platform, and interrogated them against a panel of ~500 target-annotated kinase inhibitors and FDA-approved anti-cancer agents. This pharmacological screen revealed high sensitivity to distinct classes of agents (eg. PI3K inhibitors), but also variable responses to others (eg. EGFR inhibitors); and provided insight into potentially driver vs. passenger mutations in the respective patients. Co-culture of PDOs with non-malignant stromal cells from bone metastatic niche reduced sensitivity to some classes of drugs (eg. PLK inhibitors). Importantly, long-term (up to 3-4 weeks) treatment of 3D organoids did not eradicate the cells in culture (in contrast to conventional 2D assays), but generated a cell subpopulation which remained viable for the duration of the experiment, reminiscent of the residual disease after initial clinical partial responses to a drug. This residual disease phenotype was also confirmed in vivo, when the respective PD-Xenografts were treated with the same agents. The majority of transcripts with differential expression in residual PDX tumor cells were also concordantly up- or down-regulated in the respective 3D PDO model. These transcripts included canonical regulators of cell cycle, chromatin signaling and epigenetic mechanisms, cytoskeletal function and metabolic pathways; as well as other transcripts not previously linked to drug resistance in conventional 2D models. To our knowledge this is the first description of an in vitro personalized cancer model that simulates both molecularly and phenotypically the post-treatment residual disease in vivo. Our high-throughput functional platform can potentially complement genomic approaches in personalized medicine and help discover novel drugs that overcome tumor drug resistance. Citation Format: Eugen Dhimolea, Ricardo De Matos Simoes, Yoko DeRose, Pallavi Awate, Xiang Weng, Huihui Tang, Aedin Culhane, Alana Welm, Constantine Mitsiades. High-throughput patient-derived 3-dimensional organoid cultures as personalized models to assess drug response and post-treatment residual disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5016. doi:10.1158/1538-7445.AM2017-5016

Generation of reactive oxygen species (ROS), a natural byproduct of oxygen metabolism, occurs in all aerobic organisms at a controlled rate. Cancer cells are subjected to numerous cellular insults, including dysregulated oncogenes and dissociation from their natural extracellular matrix (ECM) niches, leading to the generation of high levels of ROS. Although cancer cells possess enhanced canonical antioxidant programs that neutralize ROS, they nevertheless exhibit prominent ROS levels in response to these insults, suggesting the existence of additional programs that allow cancer cells to tolerate elevated ROS. In this study, we provide evidence for an unconventional mechanism for redox adaptation involving the ROS-activated, Ca2+-permeable TRPA1 channel, which normally functions as an irritant receptor in sensory neurons but is highly upregulated in breast, lung, malignant neural sheath, and other tumors. We found that TRPA1 overexpression enhances survival and proliferation of MCF-10A cells under conditions of matrix-detachment and this effect was dependent on TRPA1 activation by ROS generated as loss of matrix anchorage. In MCF-10A acini, TRPA1 expression induced Ca2+ entry in ECM-deprived cells in the luminal space and promoted their survival and proliferation. Conversely, TRPA1 knockdown inhibited Ca2+ responses to ROS generated through ECM detachment and induced clearance of cells from the luminal space in breast cancer spheroids. TRPA1 was also activated by ROS-inducing chemotherapies and drove chemoresistance in breast, lung and malignant peripheral nerve sheath tumor cells, and its downregulation suppressed breast xenograft tumor growth and enhanced chemosensitivity. TRPA1 mediated these effects independent of antioxidant responses, but through activation of cellular survival and anti-apoptotic programs involving the RAS-ERK/AKT/mTOR pathways. Together, our findings describe an unexpected mechanism whereby cancer cells co-opt the neuronal TRPA1 channel in order to adapt to oxidative environments. This TRPA1-induced response is distinct from canonical redox adaptation mechanisms that rely on antioxidant program. As TRPA1 inhibitors are currently in clinical trials for pain and respiratory therapies, these studies raise the possibility of using these inhibitors as therapeutic chemosensitizers in TRPA1-enriched tumors. Citation Format: Nobuaki Takahashi, Hsing-Yu Chen, Isaac Harris, Daniel Stover, Roderick Bronson, Thomas Deraedt, Karen Cichowski, Alana Welm, Gordon Mills, Joan Brugge. The TRPA1 Ca2+-permeable channel mediates a non-canonical redox adaptation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5478. doi:10.1158/1538-7445.AM2017-5478

BACKGROUND: Although 70–80% of women respond to standard platinum-based chemotherapy, a majority of patients will develop recurrent platinum-resistant disease. Novel treatment approaches directed against key oncogenic drivers and/or the use of novel drug combinations to prevent/overcome platinum resistance are promising opportunities to combat this disease. sfRon is an understudied, alternative isoform of the full-length Ron receptor tyrosine kinase, the latter of which has been investigated in cancer for several years. Unlike full-length Ron, the sfRon protein lacks the extracellular ligand-binding domain, but organizes into a constitutively active transmembrane protein. Our in vitro and in vivo studies indicate that sfRon expression is associated with the acquisition of aggressive and invasive ovarian tumor phenotype. Such data carries additional importance because sfRon does not require ligand binding for activity, it may not be targetable using standard approaches such as antibody inhibitors. Instead, our data suggest that inhibition of Ron kinase activity, which would block both Ron and sfRon, might have great therapeutic benefit in ovarian tumors that express sfRon. RESULTS: Our lab has recently made the striking and unexpected discovery that sfRon expression is necessary for development of carcinogen-driven ovarian tumors in mice. Our study involved treating wild type mice vs. mice with a specific genetic deletion of sfRon (ΔsfRon) with DMBA, a known carcinogen. The study revealed that lack of sfRon completely protected the mice from ovarian cancer. The frequency of ovarian tumors was 25% in wild-type mice vs. 0% in ΔsfRon mice (p CONCLUSION: Our data implicate sfRon in ovarian cancer pathogenesis, and indicate that Ron kinase inhibitors targeting both Ron and sfRon may not only suppress ovarian tumor progression, but also have potential to overcome resistance to chemotherapy. These data could have significant impact on future clinical trials for both Ron kinase inhibitors and platinum compounds, which has high potential to impact patients in the short-term. Citation Format: Magdalena Bieniasz, Katherine Moxley, Luyao Wang, Alana L. Welm. SHORT-FORM RON KINASE AS A NOVEL THERAPEUTIC TARGET IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-048.