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Trop2, an oncogenic cell surface protein under investigation as a therapeutic target, is commonly overexpressed in several epithelial tumor types yet its function in tumor biology remains relatively unexplored. To investigate the role of Trop2 in epithelial carcinogenesis, we generated Trop2−/− mice, which are viable and possess a normal lifespan. Contrary to expectations, Trop2 loss fails to suppress keratinocyte transformation. Instead, ras-transformed Trop2−/− keratinocytes preferentially pass through an epithelial to mesenchymal transition (EMT) and form tumors with spindle cell histology. Furthermore, Trop2 loss renders Arf-null mice susceptible to the formation of biphasic sarcomatoid carcinomas containing both squamous and spindle cell components upon carcinogen exposure in an otherwise skin cancer–resistant strain (C57BL/6). Immortalized keratinocytes derived from Trop2−/−Arf−/− mice exhibit enhanced proliferative and migratory capacity as well as increased activation of mitogen-activated protein kinase and Src prior to transformation. The clinical relevance of these findings was supported by studying the molecular epidemiology of Trop2 in primary head and neck squamous cell carcinomas. This analysis revealed that Trop2 mRNA levels are decreased in a subset of tumors with features of EMT, and total loss of Trop2 protein expression is observed in the spindle cell component of sarcomatoid carcinomas. Therefore, while previous studies have emphasized the potential importance of Trop2 gain of function, these results uncover a role for Trop2 loss in tumorigenesis and the mesenchymal transdifferentiation observed in a subset of squamous cell carcinomas. Mol Cancer Res; 9(12); 1686–95. ©2011 AACR.

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Purpose:Pembrolizumab improved survival in patients with recurrent or metastatic head and neck squamous-cell carcinoma (HNSCC). The aims of this study were to determine if pembrolizumab would be safe, result in pathologic tumor response (pTR), and lower the relapse rate in patients with resectable human papillomavirus (HPV)–unrelated HNSCC.Patients and Methods:Neoadjuvant pembrolizumab (200 mg) was administered and followed 2 to 3 weeks later by surgical tumor ablation. Postoperative (chemo)radiation was planned. Patients with high-risk pathology (positive margins and/or extranodal extension) received adjuvant pembrolizumab. pTR was quantified as the proportion of the resection bed with tumor necrosis, keratinous debris, and giant cells/histiocytes: pTR-0 (Results:Thirty-six patients enrolled. After neoadjuvant pembrolizumab, serious (grades 3–4) adverse events and unexpected surgical delays/complications did not occur. pTR-2 occurred in eight patients (22%), and pTR-1 in eight other patients (22%). One-year relapse rate among 18 patients with high-risk pathology was 16.7% (95% confidence interval, 3.6%–41.4%). pTR ≥10% correlated with baseline tumor PD-L1, immune infiltrate, and IFNγ activity. Matched samples showed upregulation of inhibitory checkpoints in patients with pTR-0 and confirmed clonal loss in some patients.Conclusions:Among patients with locally advanced, HPV-unrelated HNSCC, pembrolizumab was safe, and any pathologic response was observed in 44% of patients with 0% pathologic complete responses. The 1-year relapse rate in patients with high-risk pathology was lower than historical.

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Supplementary Figure Legends 1-4 from Synthetic Lethality through Combined Notch–Epidermal Growth Factor Receptor Pathway Inhibition in Basal-Like Breast Cancer

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The p19ARF tumor suppressor limits ribosome biogenesis and responds to hyperproliferative signals to activate the p53 checkpoint response. Although its activation of p53 has been well characterized, the role of ARF in restraining nucleolar ribosome production is poorly understood. Here we report the use of a mass spectroscopic analysis to identify protein changes within the nucleoli of Arf-deficient mouse cells. Through this approach, we discovered that ARF limited the nucleolar localization of the RNA helicase DDX5, which promotes the synthesis and maturation of rRNA, ultimately increasing ribosome output and proliferation. ARF inhibited the interaction between DDX5 and nucleophosmin (NPM), preventing association of DDX5 with the rDNA promoter and nuclear pre-ribosomes. In addition, Arf-deficient cells transformed by oncogenic RasV12 were addicted to DDX5, because reduction of DDX5 was sufficient to impair RasV12-driven colony formation in soft agar and tumor growth in mice. Taken together, our findings indicate that DDX5 is a key p53-independent target of the ARF tumor suppressor and is a novel non-oncogene participant in ribosome biogenesis. Cancer Res; 71(21); 6708–17. ©2011 AACR.

Basal-like breast cancers (BLBC) are highly aggressive, yet selective therapies targeting the specific oncoproteins driving these tumors have not been developed. These cancers frequently express epidermal growth factor receptor (EGFR), with resistance to its inhibition being well documented, albeit poorly understood. Notch pathway activation is also common in this breast cancer subtype and can be suppressed by γ-secretase inhibitors, which effectively block receptor cleavage and activation. Herein, we show that although inhibition of either EGFR or Notch signaling alone is insufficient to suppress basal-like breast tumor cell survival and proliferation, simultaneous inhibition uncovers a synthetic lethal relationship between these two oncogenic pathways. This lethality is due in part to significant decreases in AKT activation caused by combined EGFR and Notch inhibition. Expression of the activated form of Notch1 restores AKT activity and enables cells to overcome cell death after dual-pathway blockade. Combined pathway inhibition is also dramatically more effective at suppressing tumor growth in mice than blocking EGFR or Notch signaling alone. Thus, we show that Notch pathway activation contributes to resistance to EGFR inhibition, and provide a novel treatment strategy for BLBCs. Cancer Res; 70(13); 5465–74. ©2010 AACR.

Supplementary Figure 4 from Synthetic Lethality through Combined Notch–Epidermal Growth Factor Receptor Pathway Inhibition in Basal-Like Breast Cancer

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Supplementary Figure 1 from Synthetic Lethality through Combined Notch–Epidermal Growth Factor Receptor Pathway Inhibition in Basal-Like Breast Cancer

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Background Glycoprotein-A repetitions predominant (GARP) regulates membrane-bound transforming growth factor β1 (TGFβ1), an immunosuppressive cytokine. ABBV-151 is a first-in-class monoclonal antibody (mAb) that binds to the GARP-TGFβ1 complex and blocks TGFβ1 release. Preclinical data demonstrated that targeting both GARP-TGFβ1 and programmed cell death protein 1 (PD-1) improved antitumor effects compared with anti-PD-1 alone. Combining ABBV-151 with the anti-PD-1 mAb budigalimab (ABBV-181) may enable a more effective antitumor immune response by reducing the immunosuppressive effect of TGFβ1. Trial design This is a multicenter phase 1, dose escalation and dose expansion study (NCT03821935) in patients (pts; ≥18 yr, Eastern Cooperative Oncology Group performance status 0-1) with locally advanced or metastatic solid tumors. The primary objective of dose escalation is to determine the recommended phase 2 dose (RP2D) of ABBV-151 as monotherapy or with budigalimab; dose expansion will assess the objective response rate of ABBV-151 ± budigalimab. Secondary/exploratory objectives include assessing preliminary efficacy, safety, tolerability, pharmacokinetics (PK), and evaluating potential pharmacodynamic and predictive biomarkers. Dose escalation of ABBV-151, guided by a Bayesian optimal interval design, will assess dose-limiting toxicities during the first 28-day cycle and will be utilized until the RP2D is defined. ABBV-151 + budigalimab (fixed dose) will start ≥2 dose levels below that proven safe for ABBV-151. Adverse events will be evaluated per National Cancer Institute Common Terminology Criteria v5.0. Response will be assessed using Response Evaluation Criteria In Solid Tumors (RECIST) v1.1 and iRECIST every 8 weeks. PK of ABBV-151 will be characterized. Saturation of GARP-TGFβ1 on platelets and PD-1 on CD4 T cells will be determined. Modulation of cytokines, chemokines, lymphocyte activity, and gene expression will be assessed in blood, while gene signatures and protein markers will be explored in tumor tissues. Baseline tumor characteristics will be retrospectively related to response. Enrollment initiated Mar 2019, with 37 pts enrolled as of May 2020. Citation Format: John Powderly, Toshio Shimizu, Patricia LoRusso, Albiruni Razak, Kathy Miller, Arjun Balar, Jordi Bruix, Loren Michel, Martha Blaney, Xiaowen Guan, Susan Lacy, Satwant Lally, Stacie Lambert, Rachel Leibman, Gregory Vosganian, Talia Golan, Anthony Tolcher. Phase 1 first-in-human study of ABBV-151 as monotherapy or in combination with budigalimab in patients with locally advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT207.

Basal-like breast cancers (BLBC) are highly aggressive, yet selective therapies targeting the specific oncoproteins driving these tumors have not been developed. These cancers frequently express epidermal growth factor receptor (EGFR), with resistance to its inhibition being well documented, albeit poorly understood. Notch pathway activation is also common in this breast cancer subtype and can be suppressed by γ-secretase inhibitors, which effectively block receptor cleavage and activation. Herein, we show that although inhibition of either EGFR or Notch signaling alone is insufficient to suppress basal-like breast tumor cell survival and proliferation, simultaneous inhibition uncovers a synthetic lethal relationship between these two oncogenic pathways. This lethality is due in part to significant decreases in AKT activation caused by combined EGFR and Notch inhibition. Expression of the activated form of Notch1 restores AKT activity and enables cells to overcome cell death after dual-pathway blockade. Combined pathway inhibition is also dramatically more effective at suppressing tumor growth in mice than blocking EGFR or Notch signaling alone. Thus, we show that Notch pathway activation contributes to resistance to EGFR inhibition, and provide a novel treatment strategy for BLBCs. Cancer Res; 70(13); 5465–74. ©2010 AACR.

Significant clinical and pre-clinical research has shown that PAMPs- Pathogen Associated Molecular Patterns- can trigger an integrated anti-cancer immune response involving both innate and adaptive immunity. Imprime PGG is a soluble yeast β-1,3/1,6 glucan currently in multiple phase 2 clinical studies in combination with the immune checkpoint inhibitor (CPI), pembrolizumab. Preclinical mechanistic research has shown that Imprime PGG can re-polarize immunosuppressive myeloid cells in the tumor microenvironment and activate antigen presenting cells to prime antigen-specific CD8 T cells thereby boosting effector T cell function and expansion. Based on this ability to activate dendritic cells and induce type I interferon, we sought to explore the use of Imprime as an immune activating/antigen-directing scaffold onto which we could attach tumor antigen to drive an antigen-specific T cell based immune response. Imprime PGG has been safely administered intravenously to more than 400 human subjects and thus represents a safe starting point for the development of a cancer vaccine. Imprime’s 1,3/1,6 β-glucan polymeric structure allows for the straightforward attachment of peptides and proteins via three different conjugation routes. To explore this function we covalently linked the chicken ovalbumin (OVA) protein to Imprime to generate a β-glucan-protein conjugate (Imprime-OVA). Using T cell receptor transgenic OT-I CD8 and OT-II CD4 T cells to track responses to OVA, we treated mice with Imprime-OVA intravenously and examined the expansion and functional quality of the T cell response 7 days later at the peak of expansion. Following Imprime-OVA treatment, both OVA-specific CD8 and CD4 T cells underwent vigorous expansion. OT-I CD8 T cells upregulated the transcription factor Tbet, which is central to developing effector functions, and were highly multifunctional in their ability to produce IFN-γ, TNF-α and IL-2. OT-I T cells responded similarly to vaccination with the minimal H-2Kb/OVA257-264 peptide covalently attached to Imprime. By comparison, vaccination with unconjugated OVA (protein or peptide) and Imprime was much less effective in driving T cell expansion and differentiation. The CD8 T cell response required Batf3-dependent cross-presenting DCs whereas the CD4 T cell response did not, and both CD4 and CD8 responses required the C-type lectin receptor Dectin-1. These data show that an Imprime PGG-protein conjugate can effectively elicit the expansion and functional activation of cytotoxic T cells and may have utility as a potential cancer vaccine platform. These data provide evidence that Imprime PGG not only serves as a combination therapy for CPIs, anti-angiogenics, and tumor targeting antibodies but may also be a robust platform for multiple tumor vaccine strategies. Citation Format: Kyle S. Michel, Ross B. Fulton, Steven M. Leonardo, Keith B. Gorden, Jeremy R. Graff, Michael E. Danielson. A novel tumor vaccine platform: direct conjugation of antigens to the β glucan PAMP Imprime PGG enhances antigen presentation and T cell priming [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 1703. doi:10.1158/1538-7445.AM2017-1703

Purpose: Ras/MEK/ERK pathway activation is common in oral cavity squamous cell carcinoma (OCSCC). To determine biomarker and clinical tumor response of MEK inhibition in patients with OCSCC, we performed a neoadjuvant window of opportunity trial in which the MEK inhibitor trametinib was administered before surgery (NCT01553851). Patients and Methods: Patients with untreated Stage II-IV OCSCC were scheduled to receive trametinib 2 mg/day orally for 7-14 days prior to surgery (last dose 24 hours before surgery). Tumor specimens from the primary site obtained before and after trametinib underwent immunohistochemistry staining for p-ERK1/2 (a marker of Ras/MEK/ERK activation) and CD44 (a protein upregulated by ERK activation), which represented the primary endpoint. Secondary endpoints included comparison of changes in pre- and post-trametinib tumor measurement by clinical examination and in metabolic activity (SUVmax) by FDG-PET/CT (partial response: >25% reduction). Adverse events (AE) and surgical/wound complications were evaluated. Results: Of the 20 enrolled patients, 17 (85%) completed the study as planned. Three patients withdrew from the study due to AE, two (nausea; duodenal perforation) related to trametinib and one (constipation) related to narcotics. The most common drug-related AE was mild rash (9/20 patients, 45%). Nineteen patients (95%) underwent surgery and neck dissection with no unexpected surgical/wound complications. Fifteen patients (75%) were evaluable for the primary biomarker endpoint. 5 (25%) patients either had insufficient pre- or post-treatment biopsies or did not complete the trial. Reduction in p-ERK1/2 expression occurred in 7/15 evaluable patients (47%), whereas a reduction in CD44 occurred in 3/15 (20%). Reduction in tumor size (median 40%, range -74 to +17%) assessed by clinical examination was observed in 12/17 (71%) evaluable patients, and partial metabolic tumor response assessed by FDG-PET/CT was observed in 5/13 (38%) patients. Conclusions: Trametinib was safe to administer as a neoadjuvant treatment in patients with OCSCC and several patients displayed significant reduction in Ras/MEK/ERK pathway activation, and in clinical and metabolic tumor responses. Further exploration of trametinib response in OCSCC patients is warranted. Citation Format: Ravindra Uppaluri, Ashley Winkler, Tianxiang Lin, Jonathan Law, Bruce Haughey, Brian Nussenbaum, Randal Paniello, Jason Rich, Jason Diaz, Loren Michel, Tanya Wildes, Gavin Dunn, Dorina Kallogjeri, Paul Zolkind, Farrokh Dehdashti, Barry Siegel, Rebecca Chernock, James S. Lewis, Jay Piccirillo, Douglas Adkins. Biomarker and clinical response of oral cavity squamous cell carcinoma to the MEK 1/2 inhibitor trametinib: A phase II neoadjuvant window of opportunity clinical trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT070.

Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in both objective tumor response and survival. Mechanistic work has revealed that Imprime acts to stimulate a coordinated, anti-cancer immune response in conjunction with antibody therapy. Imprime represents a pathogen associated molecular pattern (PAMP) and, as such, can be efficiently and effectively recognized by cells of the innate immune system, triggering macrophage repolarization, neutrophil activation, monocyte-derived dendritic cell maturation, NK cell activation and, via cross-talk with the adaptive immune system, expansion of CD4 and CD8 T cells. As PAMPs have been shown to stimulate dendritic cells (DCs), which are critical for generating robust and durable anti-tumor responses, we sought to better understand the effects of Imprime on DCs. Here we demonstrate that Imprime binds to various DC subsets, stimulating the critical antigen presenting functional activity of these DC subsets. Following i.v. administration in mice, Imprime bound both classical splenic DCs, including CD8α+ cross-presenting DCs, and migratory DC subsets within peripheral lymph nodes. In vitro treatment of human whole blood showed binding in the inflammatory DC subset (Lin-HLA-DR+CD11c+CD16+) as well as the classical (Lin-HLA-DR+CD11c+CD1c+) DC subset. Binding of Imprime to both mouse and human DCs resulted in the upregulation of MHC class II and the co-stimulatory molecules CD80/86 that are critical for antigen presentation and T cell activation. Furthermore, in vivo treatment of mice with Imprime in combination with H-2Kb-restricted OVA257-264 peptide resulted in enhanced expansion of adoptively transferred OVA-specific OT-I CD8 T cells. In contrast to mice immunized with OVA peptide alone, which did not generate a functional CD8 T cell response, Imprime co-administration resulted in functional OT-I capable of ex vivo degranulation and production of the cytokines IFN-γ and IL-2. These data suggest that, in addition to previous studies showing Imprime primes monocytes, macrophages, and neutrophils, Imprime also enhances T cell activation and expansion by directly stimulating dendritic cell maturation and efficient antigen presentation. These data demonstrate that Imprime PGG treatment may enhance the adaptive immune response necessary for durable tumor control and, together with previously published data, indicate that Imprime PGG treatment triggers an orchestrated anti-cancer immune response involving both the innate and adaptive immune systems. Citation Format: Ross B. Fulton, Steven M. Leonardo, Kyle S. Michel, Michael E. Danielson, Keith B. Gorden, Jeremy R. Graff. Imprime PGG, a soluble β-glucan, binds to and activates dendritic cells resulting in enhanced T cell priming, expansion, and cytokine production. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B019.

Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in combination with monoclonal antibody therapy. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in both objective tumor response and survival. Mechanistic work has revealed that Imprime acts as a pathogen-associated molecular pattern (PAMP) to broadly activate the innate immune system, resulting in priming of monocytes and neutrophils, inhibition of immunosuppressive M2 macrophage polarization, and enhanced NK cell activity. Here, we demonstrate that Imprime also binds to various dendritic cell (DC) subsets and promotes DC maturation as well as subsequent T cell activation and acquisition of effector functions. In humans, in vitro treatment of whole blood with Imprime shows binding to CD16+ inflammatory DCs as well as CD1c+ classical DCs. Following i.v. administration in mice, Imprime binds resident and migratory DC subsets within secondary lymphoid organs. In both human and mouse, Imprime increases DC expression of the co-stimulatory molecules CD80/86 as well as MHC class II. Importantly, Imprime-mediated maturation of DCs enhances T cell responses: in mice, immunization with antigen in the presence of Imprime increases the magnitude of the antigen-specific CD8 T cell response as well as improves their ability to degranulate and produce IFN-γ and IL-2. Additionally, we show that Imprime imparts long-lasting effects on human monocytes that sensitize them to subsequent danger signals. Monocytes that are exposed to Imprime in human whole blood prior to their isolation and week-long differentiation into monocyte-derived DCs with GM-CSF and IL-4 show increased upregulation of CD83, CD86, and MHC class II after being matured with LPS and TNF-α. This increased maturation state translates into improved priming and expansion of allogeneic CD4 and CD8 T cells in a mixed-lymphocyte reaction. Altogether, we demonstrate that Imprime behaves as a PAMP to bridge the innate and adaptive immune systems. In a cancer setting, these data suggest that Imprime treatment in conjugation with agents that relieve immunosuppression (e.g. checkpoint inhibitors) could result in superior anti-tumor immunity. Citation Format: Ross B. Fulton, Anissa SH Chan, Steven M. Leonardo, Adria B. Jonas, Xiaohong Qiu, Nadine C. Ottoson, Takashi O. Kangas, Kyle S. Michel, Michael E. Danielson, Jeremy R. Graff, Nandita Bose, Keith Gorden. Imprime PGG, a soluble yeast β-glucan, induces dendritic cell maturation, upregulating co-stimulatory and activation markers to enhance antigen presentation and T cell priming. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A99.

Imprime PGG is a soluble yeast-derived β-1,3/1,6 glucan innate immune cell modulator that is in phase 3 and multiple phase 2 clinical trials in combination with complement activating monoclonal antibodies (e.g. bevacizumab, cetuximab). We have previously shown that Imprime binds to complement receptors and modulates the function of a variety of immune cells, including monocytes, neutrophils, and B cells. We have also demonstrated that Imprime PGG binds to various subsets of dendritic cells (DCs) and can cause intermediate upregulation of MHC class II and the co-stimulatory molecules CD80/86 critical for antigen presentation and T cell activation. Based on these findings, we hypothesized that Imprime PGG conjugated to a protein would efficiently deliver antigen to DCs and prime a cytotoxic CD8+ T cell response. To test this hypothesis, we employed the model antigen chicken ovalbumin (OVA) in a C57BL/6 mouse model to examine the generation of OVA-specific CD8+ T cell responses. We covalently linked OVA to Imprime PGG to generate a β-glucan/protein conjugate (Imprime-OVA). Using T cell receptor transgenic OT-I CD8+ T cells to track responses to OVA, we treated mice with Imprime-OVA intravenously and examined the expansion and functional quality of the T cell response 7 days later at the peak of expansion. Following Imprime-OVA treatment, OVA-specific CD8 T cells underwent vigorous expansion, upregulated the transcription factor Tbet, which is central to developing effector functions, and gained the ability to produce the cytokines IFN-γ, TNF-α and IL-2. By comparison, OVA alone did not generate a functional CD8+ T cell response and instead induced anergy. To determine if cross-presenting DCs are required for CD8+ T cell activation, we used mice deficient in the transcription factor Batf3, which selectively eliminates CD8α+ cross-presenting DCs. Following treatment with the Imprime-OVA conjugate in these Batf3-/- mice, the expansion of OVA-specific CD8+ T cells was more than 10-fold reduced compared to that in wild-type mice. Further, these cells failed to develop effector functions, indicating that CD8α+ cross-presenting DCs are crucial for this response. Together, these data show that an Imprime PGG-protein conjugate can effectively elicit the expansion and functional activation of cytotoxic T cells and may have utility as a potential cancer vaccine platform. Citation Format: Ross B. Fulton, Steven Leonardo, Kyle Michel, Lindsay Wurst, Trinda Phelon, Mike Danielson, Keith Gorden. Imprime PGG conjugated directly to protein enables cross-presentation of antigen that generates multifunctional cytotoxic T cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-236. doi:10.1158/1538-7445.AM2015-LB-236

Biothera is developing Imprime PGG® (Imprime PGG) for cancer that is synergistic with multiple anti-tumor monoclonal antibodies, demonstrating the potential to improve patient outcomes in a wide range of cancer indications. Although, the antitumor potential of Imprime PGG has now been well documented in several preclinical models, its immunomodulatory potential has not been fully elucidated. Here, we studied the in vivo effects of Imprime PGG, a pharmaceutical grade soluble yeast-derived β-1,3/1,6 glucan, on immune cell binding and function, using both tumor bearing and non-tumor bearing mice. Immunodeficient nude mice, with or without tumor (i.e. MiaPaCa-2 human pancreatic tumor), were administered a single dose of Imprime PGG (i.v.) and 3 days later, immune cells in peripheral blood, spleen, and/or tumor were isolated and evaluated for Imprime PGG binding and function. Using flow cytometric methods, Imprime PGG was found to bind B cells, macrophages, and neutrophils in whole blood and in addition, bound to NK cells in spleen. Notably, in mice bearing tumor, Imprime PGG administration lead to a relatively high percentage of Imprime PGG-bound macrophages and neutrophils in the tumor microenvironment. Furthermore, for the first time, we were able to demonstrate that Imprime PGG administered in vivo had the ability to “prime” immune cells and enhance their antitumor function. Administration of Imprime PGG increased the cytotoxic ability of immune cells, such as macrophages and NK cells, and lead to higher antibody mediated cytotoxicity and increased sensitivity to non-specific stimuli. Likewise, immune cells primed in vivo with Imprime PGG demonstrated increased cytokine responses (such as KC; a mouse isoform of human IL-8, IL-6, G-CSF, and MIP-1α and preliminary cytokine data on tumor infiltrating effector cells support an antitumor profile. Collectively, these results indicate that Imprime PGG when administered in vivo “primes” immune effector cells for enhanced antitumor activity. These observations further validate the potential benefit of combining this unique immunomodulatory drug with antitumor monoclonal antibodies as a positive treatment strategy in cancer. Citation Format: Yumi Yokoyama, Mariana I. Nelson, Nadine C. Ottoson, Mark N. Kirstein, Richard Walsh, Kyle S. Michel, Michael E. Danielson, Mary A. Antonysamy. Immune cell priming and potentiation of anti-tumor effects by Imprime PGG®. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B65.

Deeper understanding the mechanisms by which luminal-type breast cancer develops resistance to endocrine therapy and development of novel strategies to treat these patients requires model systems recapitulate human breast cancer as accurately as possible. An increasing body of work suggests patient derived xenografts (PDX) may represent an informative model for development of novel therapeutics. We therefore established seven xenograft tumor lines from late-stage breast cancer patients with estrogen positive (ER+) disease. To date five ER+ PDX lines have been tested for responses to estradiol treatment in overiectomized NOD/SCID mice. Three showed estradiol independent-growth, one estrogen-stimulated growth and in one estradiol-induced a regression. These patterns mimicked the clinical phenotypes of each patient, tracking survival and responses to serial endocrine treatments. To define new mechanisms for resistance, whole genome DNA sequencing, RNA sequencing and Reverse Phase Protein Assay analysis was conducted. These studies identified an ESR1/YAP1 balanced translocation in a PDX model and tumor of origin showing low levels of ER, paradoxical high level expression form luminal genes and extreme ET resistance. The ESR1 YAP1 fusion maintained the N terminal DNA binding motif of ESR1, but the hormone binding and AF2 motifs were replaced with the C terminal transactivation domain of YAP1. Expression ESR1 YAP1 in ER+ breast cancer models down-regulated ER and induced estrogen independent growth. PDX endocrine phenotypes parallel tumor of origin responses to endocrine therapy and revel novel mechanism for endocrine therapy resistance. Citation Format: Matthew J. Ellis, Shunqiang Li, Dong Shen, Li Ding, Robert Crowder, Jeiya Shao, Rodrigo Goncalves, Yu Tao, Jingqin Luo, Aleix Prat, Wenbin Liu, Ana Maria Gonzalez-Angulo, Shuying Liu, Joshua F. McMichael, Chris Miller, Dave Larson, Robert S. Fulton, Tom Mooney, Jeremy Hoog, Li Lin, Therese Giuntoli, Caroline Bumb, Crystal Cooper, Rebecca Aft, Robert T. Kitchens, Stephen N. Johnson, Chanpheng Phommaly, Megha Shiyam Kavuri, Katherine DeSchryver, Austin Lin, YiYu Dong, Cynthia X. Ma, Timothy Pluard, Michael Naughton, Ron Bose, Rama Suresh, Reida G. McDowell, Loren Michel, Richard Wilson, Shaomeng Wang, Christopher Maher, Gordon B. Mills, Charles Perou, Elaine R. Mardis. Patient-derived xenografts from advanced luminal-type breast cancer: insights into endocrine therapy resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-265. doi:10.1158/1538-7445.AM2013-LB-265

Trop2 is expressed on many epithelial tumors and stem cells of the prostate and liver. Its association with aggressive cancer has led to the speculation that it might be a therapeutic target. We have previously shown a role for Trop2 in colon tumorigenesis and also found that antibodies against Trop2 can block tumor cell invasion. Consistent with this data, a monoclonal antibody against Trop2 is in pre-clinical development against several tumor types. However, the expression pattern and role of Trop2 in breast cancer has not been examined. Here, we show heterogeneous expression of Trop2 in normal mammary gland, primary breast tumors and cancer cells lines. After purification of Trop2 expressing tumor cell populations by fluorescence activated cell sorting, we find that Trop2hi cells are preferentially invasive and tumorigenic compared to the Trop2lo. RNAi mediated suppression of Trop2 in breast cancer cells arrests their proliferation. Even more importantly, a monoclonal antibody against Trop2 shows anti-tumor activity in vivo. Therefore, Trop2 represents a novel and unexplored therapeutic target in this common malignancy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C167.