Human Tumor Explants Research Articles

Abstract 910: A novel bifunctional anti-PD-1 IL-7 fusion protein to reinvigorate exhausted T cell and disarms Treg suppressive activity

Abstract Despite the clinical success of anti-PD(L)1 therapy, the majority of patient remain unresponsive or fail to develop a durable response. We explored a second generation of PD-1 antibody by fusing IL-7 cytokine to the Fc portion, called BiCKI® IL-7. The high affinity of the anti PD-1 antibody will allow the concentration/retention of the drug into the tumor microenvironment and preferentially deliver IL-7 in cis-dependent manner to PD-1+ cells. IL-7 is an optimal target for immunotherapy to preferentially stimulate effector T-cell (Teff) functions over regulatory T-cells (Treg)due to the differential expression of IL-7R and poor capacity of IL-7 to stimulate Treg proliferation. Moreover, It has been published that PD-1 blockades increase IL-7R expression and improve IL-7 signaling in exhausted T-cells rationalizing our combinatorial approach. Results Our anti PD-1/IL-7 bispecific antibody efficiently blocks the PD-1/PD-L1 and PD-L2 interactions and the PD-1-mediated inhibitory signal (pSHP1) and in parallel activates IL-7R pSTAT5 signaling into T cells. Anti PD-1/IL-7 preferentially binds in cis to T cells coexpressing PD-1 and CD127 enabling a selective activation of primed antigen- experienced T cells over PD1-negative (e.g. naïve) cells. A high affinity/avidity of the molecule was observed using biosensor when both receptor CD127 and PD-1, supporting the cis-targeting activity of the drug. Using in vitro T cell activation bioassay, we observed that IL-7 portion fused to the anti-PD-1 synergizes to enhance TCR mediated signaling (NFAT) through activation of the non-canonical pathway while IL-7 in combination with anti-PD1 (two separates product) has no additive effect. Although IL-7R expression decrease over chronic stimulation of Teff cells, we demonstrated that IL-7 efficiently activates progenitor and some fully-exhausted human T-cells (pSTAT5) and maintain their proliferation and survival capacity. This IL-7R signaling activation was associated with a significant increased IFNγ secretion using ex-vivo fresh human tumor explant culture. A significant higher IFNγ production was obtained with anti PD-1/IL-7compared to anti PD-1 treatment alone, including in non-responder patients to anti-PD1, suggesting that the anti PD-1/IL-7 bispecific can reactivate TILs that are resistant to PD-1 therapy. Knowing that Tregs have a key suppressive function, we also explored the possibility that anti-PD-1/IL-7 affect Treg functions. In a human Treg/Teff coculture assay, anti PD-1/IL-7 abrogates the Treg capacity to inhibit proliferation and IFN-g secretion of CD8+ Teff. Moreover, IL-7 and anti-PD-1/IL-7 does not stimulate Treg proliferation, in contrast to IL-2 and IL-15. Conclusion Our data validate the therapeutic potential of providing IL-7 signals to overcome PD-1 resistance. The bifunctional anti-PD1/IL-7 favors the T-cell effector over T-regulatory immune balance by stimulating Teff cells and exhausted T-cell, while disarming Tregs suppressive functions. Citation Format: Aurore Morello, Justine Durand, Virginie Thepenier, Géraldine Teppaz, Margaux Seite, Sabrina Pengam, Caroline Mary, Nicolas Poirier. A novel bifunctional anti-PD-1 IL-7 fusion protein to reinvigorate exhausted T cell and disarms Treg suppressive activity [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 910.

Silibinin down-regulates PD-L1 expression in nasopharyngeal carcinoma by interfering with tumor cell glycolytic metabolism

The upregulation of checkpoint inhibitor PD-L1 expression has recently been associated with nasopharyngeal carcinoma (NPC) resistance to therapy. The mechanism of induction of PD-L1 has also been linked to enhanced aerobic glycolysis promoted by HIF1-α dysregulation and LDH-A activity in cancer. Here, we investigated the effect of the anti-tumoral compound Silibinin on HIF-1α/LDH-A mediated cancer cell metabolism and PD-L1 expression in NPC. Our results demonstrate that exposure to Silibinin potently inhibits tumor growth and promotes a shift from aerobic glycolysis toward oxidative phosphorylation. The EBV + NPC cell line C666-1 and glycolytic human tumor explants treated with Silibinin displayed a reduction in LDH-A activity which consistently associated with a reduction in lactate levels. This effect was accompanied by an increase in intracellular citrate levels in C666-1 cells. Accordingly, expression of HIF-1α, a critical regulator of glycolysis, was down-regulated after treatment. This event associated with a down-regulation in PD-L1. Altogether, our results provide evidence that silibinin can alter PD-L1 expression by interfering with HIF-1α/LDH-A mediated cell metabolism in NPC. These results provide a new perspective for Silibinin use to overcome PD-L1 mediated NPC resistance to therapy.

MHC-restricted phosphopeptide antigens: preclinical validation and first-in-humans clinical trial in participants with high-risk melanoma

BackgroundPhosphorylated peptides presented by MHC molecules represent a new class of neoantigens expressed on cancer cells and recognized by CD8 T-cells. These peptides are promising targets for cancer immunotherapy. Previous...

Carbamate derivatives of colchicine show potent activity towards primary acute lymphoblastic leukemia and primary breast cancer cells-in vitro and ex vivo study.

Colchicine (COL) shows strong anticancer activity but due to its toxicity towards normal cells its wider application is limited. To address this issue, a library of 17 novel COL derivatives, namely N-carbamates of N-deacetyl-4-(bromo/chloro/iodo)thiocolchicine, has been tested against two types of primary cancer cells. These included acute lymphoblastic leukemia (ALL) and human breast cancer (BC) derived from two different tumor subtypes, ER+ invasive ductal carcinoma grade III (IDCG3) and metastatic carcinoma (MC). Four novel COL derivatives showed higher anti-proliferative activity than COL (IC50 = 8.6 nM) towards primary ALL cells in cell viability assays (IC50 range of 1.1-6.4 nM), and several were more potent towards primary IDCG3 (IC50 range of 0.1 to 10.3 nM) or MC (IC50 range of 2.3-9.1 nM) compared to COL (IC50 of 11.1 and 11.7 nM, respectively). In addition, several derivatives were selectively active toward primary breast cancer cells compared to normal breast epithelial cells. The most promising derivatives were subsequently tested against the NCI panel of 60 human cancer cell lines and seven derivatives were more potent than COL against leukemia, non-small-cell lung, colon, CNS and prostate cancers. Finally, COL and two of the most active derivatives were shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants. The present findings indicate that the select COL derivatives constitute promising lead compounds targeting specific types of cancer.

Combination of AMG 160, a PSMA x CD3 half-life extended bispecific T-cell engager (HLE BiTE) immune therapy, with an anti-PD-1 antibody in prostate cancer (PCa).

155 Background: AMG 160 is an HLE BiTE immune therapy that binds prostate-specific membrane antigen (PSMA) on tumor cells and CD3 on T cells, leading to T cell activation, cytokine production, and tumor cell lysis. A phase 1 study of AMG 160 in patients with metastatic castration-resistant PCa is ongoing (NCT03792841). We investigated whether the combination of AMG 160 and an anti-PD-1 antibody could increase AMG 160 cytotoxicity against PCa cells in vitro and ex vivo. Methods: C4-2B human PCa cells engineered to overexpress PD-L1 were treated with a range of AMG 160 doses (0.144–30,000 pM) +/– a fixed dose of anti-PD-1 in the presence of activated human T cells. Cell viability was assessed by Steady-Glo after 1 day. PD-1 expression on T cells was evaluated by flow cytometry. A human tumor explant model consisting of thin sections of fresh surgically resected human PCa tumors was also used to evaluate the AMG 160 + anti-PD-1 combination. Explants were incubated in media over 2 days and treated with buffer alone, AMG 160, or AMG 160 + anti-PD-1 at fixed doses. Activation of autologous T cells within the tumor slices was assayed by flow cytometry, and secretion of cytokines into culture supernatants was measured by Meso Scale Discovery (MSD). Immune-response profiles of the culture tissues were assessed by NanoString. Results: In the PCa in vitro study, AMG 160 treatment induced upregulation of PD-1 on T cells. Cotreatment with AMG 160 + anti-PD-1 increased the potency of cell killing by 2–5x and killed a greater percentage of cells than AMG 160 alone. In the tumor explant model, which preserves tumor architecture and microenvironment, AMG 160 treatment induced activation of the autologous infiltrating T cells, and this activation was increased by cotreatment with anti-PD-1. AMG 160 treatment increased cytokine production, and cotreatment of tumor explants with AMG 160 + anti-PD-1 increased cytokine production further. Conclusions: Together these data suggest that antitumor efficacy of the AMG 160 HLE BiTE immune therapy can be increased by co-treatment with anti-PD-1. These data provide a rationale for evaluating this combination in future clinical studies.

Abstract 4316: Targeting LIFR overcomes HDAC inhibitor resistance in ovarian cancer

Abstract Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States and a critical need exists for the development of novel therapies for the treatment of OCa. Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Recent clinical studies showed that cancer cells elucidate feedback activation of LIFR that limits response to histone deacetylase (HDAC) inhibitors. Recently, we developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIF-LIFR interactions. Here, we examined whether LIFR inhibitor EC359 abrogate the side effects of histone deacetylase inhibitor SAHA (Vorinostat) for the treatment of OCa. Methods: The effect of EC359 and SAHA as a combination therapy on OCa cell viability was examined by MTT assays. The efficacy of combination therapy on cell survival and apoptosis was determined using clonogenic assays and caspase3/7 assays respectively. The efficacy of combination therapy on OCa stem cells was determined using extreme limiting dilution assays. Mechanistic studies were performed using western blotting, qRT-PCR and Mass Spectrometry analyses. The effect of combination therapy on STAT3 signaling was examined using reporter gene assays. The in vivo efficacy of combination therapy on tumors was examined using ex vivopatient derived explants and mouse xenograft models. Results: EC359 significantly enhanced the efficacy of SAHA in reducing cell viability, colony formation ability, and apoptosis compared to monotherapy of SAHA in multiple established and primary OCa cells. Further, EC359 enhanced SAHA ability to reduce self-renewal of OCa stem cells. As expected in STAT3 reporter assays, SAHA treatment activated STAT3 reporter and EC359 addition abrogated SAHA mediated STAT3 activation. Mechanistic studies using multiple OCa models and western blot analysis confirmed activation of LIFR signaling pathway upon SAHA treatment and its blockage by EC359 treatment. Treatment of human primary OCa tumor explants with EC359 enhanced ability of SAHA to decrease the proliferation (Ki-67 positivity) compared to monotherapy treated tumors. DIA based Mass Spectrometry analyses identified unique pathways modulated by combination therapy. Treatment of OCa xenografts with EC359 enhanced the ability of SAHA to reduce in vivotumor growth compared to monotherapy treated tumors. Conclusions: Our results suggest that EC359 has therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDAC inhibitors in treating OCa. Citation Format: Mengxing Li, Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Bindu Santhamma, Hui Yan, Zhenming Xu, Edward Kost, Rajeshwar Rao Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Targeting LIFR overcomes HDAC inhibitor resistance in ovarian 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 4316.

Chinese‐German Cooperation Group Tumor Immunology: Another inspiring Meeting in Deidesheim

Chinese‐German Cooperation Group Tumor Immunology: Another inspiring Meeting in Deidesheim

Carbamate Derivatives of Colchicine Show Potent Activity toward Primary Acute Lymphoblastic Leukemia and Primary Breast Cancer Cells: In Vitro and Ex Vivo Study

Colchicine (COL) is the major alkaloid isolated from the plant Colchicum autumnale. It shows strong therapeutic effects towards various types of cancer. Its biological activity is related to its ability to bind to tubulin causing microtubule depolymerization, mitotic arrest, and cell death. The colchicine binding site is an attractive target for potential tubulin polymerization destabilizers. Indeed, a large number of molecules interacting with the colchicine binding site have been designed and synthesized with significant structural diversity. The identification of new therapeutics based on COL structure is warranted due to the toxicity of COL towards normal cells. In order to increase potency and reduce toxicity against normal cells, while retaining the activities in the nanomolar range, a library of novel COL analogs, namely N‐carbamates of N‐deacetyl‐4‐(bromo/chloro/iodo)thiocolchicine, has been synthesized (Fig. 1).COL and several of the derivatives arrested MCF‐7 cells in mitosis, and caused microtubule depolymerization. Compounds were then tested against two types of primary cancer cells; adult acute lymphoblastic leukemia (ALL) cells, and human breast cancer (BC) cells derived from newly excised human tumor tissue. These represent more clinically relevant drug screening models compared to established cell lines. Four novel colchicine derivatives showed higher activity towards primary ALL cells (IC50 = 1.1 ± 0.5 to 6.4 ± 1.4 nM) and nine were more potent towards primary BC cells (IC50 = 2.3 ± 0.0 to 10.3 ± 4.6 nM) compared to COL (IC50 = 8.6 ± 0.2 nM and 11.7 ± 3.1 nM, respectively) in cell viability assays. COL and two of the most active derivatives were also shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants derived from residual invasive ductal carcinoma and invasive mucinous carcinoma. The present findings indicate that the COL derivatives described here constitute promising lead compounds for targeting acute lymphoblastic leukemia and different subtypes of breast cancer.Support or Funding InformationThe present study was funded by a grant (to TCC) from the Arkansas Breast Cancer Research Program. Financial support by grant (to AH. UM, GK) of the Polish National Science Centre (NCN) – No. 2016/21/B/ST5/00111 is gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract B050: Identification of PD-1T TILs and CXCL13 as determinants for response to anti-PD-1 treatment using human tumor explants

Abstract Reinvigoration of tumor-specific T-cells by cancer immunotherapies, in particular PD-1/PD-L1 blocking agents, has greatly improved clinical outcome in multiple cancer types. Nevertheless, durable clinical benefit is currently limited to a small number of patients. To achieve a better understanding of the immunologic determinants of response to anti-PD-1 therapy, we assessed transcriptional and functional profiles of tumor-infiltrating lymphocyte (TIL) subsets from non-small cell lung cancer specimens. Thereby, we identified a transcriptionally distinct CD8 TIL pool with enriched capacity for tumor recognition. This TIL pool, termed PD-1T TILs, is characterized by bright PD-1 expression and constitutive CXCL13 secretion, which can mediate immune cell recruitment to tertiary lymphoid structures. Notably, the presence of PD-1T TILs correlates with response and survival in a small cohort of lung cancer patients treated with PD-1 blockade. To assess the role of PD-1T TILs and CXCL13 for response to PD-1 blockade in other cancer types, we developed a platform using human tumor explants to visualize immunologic responses to anti-PD-1 on a patient-specific level. Analysis of the cellular and soluble tumor microenvironment composition as well as of treatment-induced changes in tumor-infiltrating immune cells revealed immunologic responses to anti-PD-1 in 5 different cancer types. Of note, responding tumors in different tumor types were characterized by a clear enrichment in both PD-1T TILs and CXCL13 production. Collectively, our data reveal a distinct state of PD-1 bright lymphocytes that are enriched for tumor-reactivity in human cancer, making them an attractive proxy for the antitumor potential of the intratumoral T-cell pool. Furthermore, we established technology using human tumor explants to measure the immunologic response to T-cell checkpoint inhibition on a personalized basis. Finally, with this approach we identified PD-1T TILs and CXCL13 as determinants for response to anti-PD-1 in multiple cancer types, opening potential new avenues for therapeutic intervention and improved patient selection. Citation Format: Daniela S. Thommen, Viktor H. Koelzer, Petra Herzig, Marjolein de Bruijn, Paula Voabil, Marlous van den Braber, Karlijn Hummelink, Kim Monkhorst, Kirsten D. Mertz, Alfred Zippelius, John B.A.G. Haanen, Ton N.M. Schumacher. Identification of PD-1T TILs and CXCL13 as determinants for response to anti-PD-1 treatment using human tumor explants [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B050.

RA10.02: A NOVEL ROLE FOR THE COMPLEMENT CASCADE IN CHEMORADIATION THERAPY RESISTANT OESOPHAGEAL ADENOCARCINOMA

Abstract Background Resistance to chemoradiation therapy (CRT) in oesophageal adenocarcinoma (OAC) is an important clinical challenge. Elucidating underlying mechanisms of treatment resistance is crucial to improving survival rates. Methods OAC tumour biopsies were collected prior to neoadjuvant CRT. Tumour conditioned media was generated by culturing human explants for 24 h. Response to treatment was determined pathologically using the Mandard Grading System. C3, CFB and MCP-1 mRNA expression was assessed by qPCR. Secreted C3, C3a and MCP-1 were measured by ELISA. An isogenic model of radioresistant OAC was established by chronically irradiating OE33 cells with clinically-relevant doses of 2 Gy X-ray radiation. C5aR and C3aR expression was determined by flow cytometry. Results We demonstrate, for the first time, that the central factor of the complement cascade, complement component 3 (C3), is expressed in OAC tumours and is increased in pre-treatment OAC biopsies from patients (n = 13) who have a subsequent poor response to neoadjuvant CRT (P < 0.05). In addition, C3 is secreted from ex vivo human OAC tumour explants (n = 13) and correlates with levels of the monocyte chemoattractant MCP-1 (P < 0.008) and complement factor B (CFB) a component of the alternative pathway of complement activation (P < 0.0001). In vitro, radioresistant OE33 R cells had increased mRNA levels of C3 (P < 0.01), CFB (P < 0.05) and MCP-1 (P < 0.05) compared to radiosensitive OE33 P cells. OEE3 R cells expressed increased levels of secreted C3 and the anaphylatoxin C3a, compared to radiosensitive OE33P cells (both P < 0.001). Furthermore, we demonstrate for the first time that the anaphylatoxin receptor C5aR is expressed by OAC cells, suggesting that OAC cells can respond to complement anaphylatoxins. Conclusion This study highlights, for the first time, a novel role for the complement cascade in the resistance of OAC to CRT and highlights C3 as a novel predictive marker of response to CRT in OAC. Disclosure All authors have declared no conflicts of interest.

Helicase-Driven Activation of NFκB-COX2 Pathway Mediates the Immunosuppressive Component of dsRNA-Driven Inflammation in the Human Tumor Microenvironment.

Presence of cytotoxic CD8+ T cells (CTL) in tumor microenvironments (TME) is critical for the effectiveness of immune therapies and patients' outcome, whereas regulatory T(reg) cells promote cancer progression. Immune adjuvants, including double-stranded (ds)RNAs, which signal via Toll-like receptor-3 (TLR3) and helicase (RIG-I/MDA5) pathways, all induce intratumoral production of CTL-attractants, but also Treg attractants and suppressive factors, raising the question of whether induction of these opposing groups of immune mediators can be separated. Here, we use human tumor explant cultures and cell culture models to show that the (ds) RNA Sendai Virus (SeV), poly-I:C, and rintatolimod (poly-I:C12U) all activate the TLR3 pathway involving TRAF3 and IRF3, and induce IFNα, ISG-60, and CXCL10 to promote CTL chemotaxis to ex vivo-treated tumors. However, in contrast with SeV and poly I:C, rintatolimod did not activate the MAVS/helicase pathway, thus avoiding NFκB- and TNFα-dependent induction of COX2, COX2/PGE2-dependent induction of IDO, IL10, CCL22, and CXCL12, and eliminating Treg attraction. Induction of CTL-attractants by either poly I:C or rintatolimod was further enhanced by exogenous IFNα (enhancer of TLR3 expression), whereas COX2 inhibition enhanced the response to poly-I:C only. Our data identify the helicase/NFκB/TNFα/COX2 axis as the key suppressive pathway of dsRNA signaling in human TME and suggest that selective targeting of TLR3 or elimination of NFκB/TNFα/COX2-driven suppression may allow for selective enhancement of type-1 immunity.Significance: This study characterizes two different poly-I:C-induced signaling pathways in their induction of immunostimulatory and suppressive factors and suggests improved ways to reprogram the TME to enhance the antitumor efficacy of immunotherapies. Cancer Res; 78(15); 4292-302. ©2018 AACR.

CAMP regulated EPAC1 supports microvascular density, angiogenic and metastatic properties in a model of triple negative breast cancer

Breast cancer is a leading cause of cancer-related mortality in women. Triple-negative breast cancer (TNBC; HER2-, ER-/PR-) is an aggressive subtype prone to drug resistance and metastasis, which is characterized by high intratumor microvascular density (iMVD) resulting from angiogenesis. However, the mechanisms contributing to the aggressive phenotypes of TNBC remain elusive. We recently reported that down-regulation of exchange factor directly activated by cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in metastatic properties including proliferation and cell migration in TNBC cell lines. Here, we report that EPAC1 supports TNBC-induced angiogenesis, tumor cell migration and invasiveness as well as pro-metastatic phenotypes in endothelial cells induced through the tumor secretome. Using an approach that integrates proteomics with bioinformatics and gene ontologies, we elucidate that EPAC1 supports a tumor-secreted network of angiogenic, cell adhesion and cell migratory pathways. Using confocal microscopy, we show that signaling molecules involved in focal adhesion, including Paxillin and MENA, are down-regulated in the absence of EPAC1, and electric cell substrate impedance sensing technique confirmed a role for EPAC1 on TNBC-induced endothelial cell permeability. Finally, to provide a translational bridge, we studied iMVD and therapy response using a primary human tumor explant assay, CANscriptTM, which suggests a link between therapy-modulated neovascularization and drug sensitivity. These data provide mechanistic insight into the role of EPAC1 in regulating the tumor microenvironment, iMVD and cancer cell-induced angiogenesis, a dynamic mechanism under drug pressure that may associate to treatment failure.

Abstract 2045: Mechanistic insights into the role of cAMP-regulated EPAC1 in tumor-induced angiogenesis and metastatic potential in triple-negative breast cancer

Abstract Background: Breast cancer is the leading cause of cancer-related mortality in women. Triple negative breast cancer (TNBC) is one of most aggressive subtypes, which is often accompanied by metastasis, a feature that requires development of new blood vessels (neovasculature) to disseminate tumor cells throughout the body. We recently reported that down-regulation of exchange factor directly activated by Cyclic AMP (cAMP), also known as EPAC1, leads to a reduction in proliferation and inhibition of cell migration. Methods: Here, using gene knockdown by siRNA and a 3-dimensional in-vitro TNBC co-culture model with Human Vascular Endothelial Cells (HUVECs), we studied microvascular density and the role of EPAC1. Angiogenesis proteome profiling was employed to examine protein networks that were regulated by EPAC1 in the TNBC cell line, MDA-MB-231. Immunofluorescence along with Electric cell-substrate impedance sensing (ECIS) was used to study vascular permeability. To confirm these findings in a translational context, we employed a human tumor explant assay that predicts clinical response to therapy (CANscriptTM) and studied CD34+ nodes to determine how neovasculature associates to drug response. Results: We determined that downregulation of EPAC1 in the TNBC cell line, MDA-MB-231, leads to reduction of proteins involved in cell migration, metastasis, angiogenesis and adhesion. Interestingly, we determined that loss of EPAC1 leads to increased expression of negative regulators in these same pathways. Immunofluorescence imaging showed that EPAC1 downregulation in MDA-MB-231 cells leads to diminished expression of proteins such as Paxillin, MENA, MMP-9 and tubulin. indicated that EPAC1 role in vascular permeability. Finally, CANscriptTM demonstrated a link between response to therapy and microvascular density, which was reduced under drug pressure in patients that were predicted to respond to treatment. Conclusion: Our results suggest that EPAC1 is a driver of microvascular density in the tumor microenvironment, a feature that may play a key role in distant metastasis and therapy failure. Ex-vivo modeling of neovasculature may be a novel strategy to predict clinical response and distant metastasis. Citation Format: Muthu Dhandapani, Aaron Goldman, Naveen Kumar, Peeyush Prasad, Eshna Jash, Itender Singh, Kodaganur S. Gopinath, Krishnamurthy S, Prabhusankar P, Seema Sehrawat. Mechanistic insights into the role of cAMP-regulated EPAC1 in tumor-induced angiogenesis and metastatic potential in triple-negative breast cancer [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 2045.

Unleashing the therapeutic potential of oncolytic viruses.

Oncolytic viruses (OVs) are a versatile new class of therapeutic agents based on native or genetically modified viruses that selectively replicate in tumor cells and can express therapeutic transgenes designed to target cells within the tumor microenvironment and/or host immunity. To date, however, confirmation of the underlying mechanism of action and an understanding of innate and acquired drug resistance for most OVs have been limited. In this issue of the JCI, Zamarin et al. report a comprehensive analysis of an oncolytic Newcastle disease virus (NDV) using both murine melanoma tumor models and human tumor explants to explore how the virus promotes tumor eradication and details of the mechanisms involved. These findings have implications for the optimization of oncolytic immunotherapy, at least that based on NDV, and further confirm that specific combinatorial approaches are promising for clinical development.

Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells.

Malignant mesothelioma (MM) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal. Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) that demonstrate heightened sensitivity to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). This association is observed across human early passage MM cultures, mouse xenografts and human tumour explants. We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex (PR-DUB) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism. Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker. We identify BAP1 loss-of-function mutations, which are frequent in MM, as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications.

Perfused Three-dimensional Organotypic Culture of Human Cancer Cells for Therapeutic Evaluation

Pharmaceutical research requires pre-clinical testing of new therapeutics using both in-vitro and in-vivo models. However, the species specificity of non-human in-vivo models and the inadequate recapitulation of physiological conditions in-vitro are intrinsic weaknesses. Here we show that perfusion is a vital factor for engineered human tissues to recapitulate key aspects of the tumour microenvironment. Organotypic culture and human tumour explants were allowed to grow long-term (14–35 days) and phenotypic features of perfused microtumours compared with those in the static culture. Differentiation status and therapeutic responses were significantly different under perfusion, indicating a distinct biological response of cultures grown under static conditions. Furthermore, heterogeneous co-culture of tumour and endothelial cells demonstrated selective cell-killing under therapeutic perfusion versus episodic delivery. We present a perfused 3D microtumour culture platform that sustains a more physiological tissue state and increased viability for long-term analyses. This system has the potential to tackle the disadvantages inherit of conventional pharmaceutical models and is suitable for precision medicine screening of tumour explants, particularly in hard-to-treat cancer types such as brain cancer which suffer from a lack of clinical samples.

Abstract 1138: The protein disulfide isomerase inhibitor XCE853 inhibits in vitro, ex-vivo and in vivo growth of human tumors

Abstract Protein disulfide isomerase (PDI) is a chaperone protein that regulates oxidative protein folding as well as cell viability. Increased PDI levels have been documented in a variety of human cancers associated with a poor overall survival, including ovarian, prostate, brain and lung cancers. Inhibition of PDI activity leads to apoptosis in cancer, suggesting that PDI is a promising druggable target. XCE853 is a synthetic small molecule displaying an excellent docking with the catalytic domain of the human PDI. XCE853 inhibits in vitro recombinant PDI enzymatic activity. In addition, the proliferation of a large panel of human tumor cells is blocked by XCE853 with IC50s in the nanomolar range through an irreversible cytolysis leading to a tumor cell death by autophagy and particles release (vesicles or protein aggregates). XCE853 is also active on a large panel of drug resistant human cancer cells. XCE853 induced an irreversible cytolysis of human tumor cells after a short in vitro exposure independently of efflux pumps. In addition, the ex-vivo approach using fresh human tumor explants cultivated in 3 dimensions with low concentrations of XCE853 has shown a strong decrease of the proliferation (KI-67 labeling) in several tumor types. Finally, XCE853 displayed excellent oral bioavailability in mice and was able to block the growth of several human cancers using in vivo xenograft models leading to a complete tumor growth arrest even after the cessation of the treatment. Altogether, these data strongly support further efforts to move this drug candidate to the preclinical studies to access advanced cancer patients. Citation Format: Gregoire Pierre Prevost, Anne Chauchereau, Patrick Ladam, Renaud Seigneuric, Denis Carniato, Marc-Henry Pitty, Paul Foster. The protein disulfide isomerase inhibitor XCE853 inhibits in vitro, ex-vivo and in vivo growth of human tumors [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 1138. doi:10.1158/1538-7445.AM2017-1138

Posttranslationally modified progesterone receptors direct ligand-specific expression of breast cancer stem cell-associated gene programs

BackgroundEstrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities.MethodsTo better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology.ResultsPhospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate.ConclusionsWe conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.

Abstract P3-03-05: PI3K/PDK1 mediates resistance to CDK4/6 inhibitors through dysregulation of S-phase cyclins/cyclin dependent kinases (CDKs)

Abstract Background: CDK4/6 inhibitors in combination with antiestrogens are approved for the treatment of ER+ advanced breast cancer. However, not all patients benefit from CDK4/6 inhibitors, underscoring the need to develop therapeutic strategies to circumvent de novo and acquired drug resistance. Methods: ER+ breast cancer cells (MCF-7, T47D, HCC1428, and HCC1500) were made resistant to increasing doses to the CDK4/6 inhibitor ribociclib (LEE011; Novartis). LEE011-resistant cells were characterized by 2D/3D growth, cell cycle, and immunoblot analyses. GSK2334470 (PDK1 inhibitor) and dinaciclib (CDK2 inhibitor) were used to modify resistance to ribociclib. PDK1 and pS6 immunohistochemistry (IHC) were performed on primary human tumor explants treated ex vivo with palbociclib. Results: Resistant cell lines (MCF-7/LR, T47D/LR, HCC1428/LR, and HCC1500/LR) exhibited an IC50 at least 20-fold higher than that of their parental cells. They displayed cross-resistance to the CDK4/6 inhibitors palbociclib and abemaciclib. Immunoblot analysis of ribociclib-resistant cells showed increased levels of 3-phosphoinositide dependent protein kinase 1 (PDK1), S227 pRSK2 (target of PDK1), T308 pAKT (target of PDK1), and pS6 (downstream effector of the PDK1 target p70S6K), compared to parental drug sensitive cells. PDK1 is a master kinase that functions downstream of phosphoinositide 3-kinase (PI3K) and is crucial for the activation of AKT and many other AGC kinases including PKC, S6K, SGK, and RSK. Primary tumor explants treated ex vivo with palbociclib for 96 h also exhibited upregulation of PDK1 and pS6 by IHC. Cell cycle analysis revealed that CDK4/6 inhibition failed to induce G1 arrest, a reduction in S phase, and senescence in MCF-7/LR and T47D/LR compared to parental cells. Progression into S phase in the presence of ribociclib suggested upregulation of S-phase cyclins/CDKs. Indeed, the resistant cells exhibited significantly higher levels of pCDK2, cyclin A, cyclin E and S477/T479 pAKT, a CDK2-dependent phosphorylation of AKT required for full kinase activity and limited to the S-phase of the cell cycle. Pharmacological inhibition of PDK1 (with GSK2334470) or CDK2 (with dinaciclib) re-sensitized the ribociclib-resistant cells to CDK4/6 inhibitors. However, ribociclib/GSK2334470 inhibited MCF-7/LR and T47D/LR cell proliferation better than ribociclib/dinaciclib. Further, ribociclib/GSK2334470 but not ribociclib/dinaciclib completely abrogated pRb, pS6, pRSK2, pCDK2, cyclin A, and cyclin E, suggesting the PI3K/PDK1 pathway mediates acquired resistance to CDK4/6 inhibitors through dysregulation of the cell cycle. Consistent with these data, ribociclib/GSK2334470 inhibited growth of established MCF-7 xenografts in nude mice, significantly more potently than each drug alone. Conclusions: These data support a critical role for PI3K/PDK1 in acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells. Co-targeting of PI3K/PDK1 and CDK4/6 may overcome resistance to CDK4/6 inhibitors and is worthy of further translational and clinical investigation in patients with ER+ breast cancer. Citation Format: Jansen VM, Formisano L, Witkiewicz A, Estrada MV, Sanchez V, Dugger TC, Knudsen ES, Arteaga CL. PI3K/PDK1 mediates resistance to CDK4/6 inhibitors through dysregulation of S-phase cyclins/cyclin dependent kinases (CDKs) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-05.

Abstract 3058: Phenotype-based discovery and development of novel therapies for the treatment of colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world. The treatment of late stage CRC using Bevacizumab®, a targeted anti-VEGF therapy, produces insufficient response rates among patients and inevitable acquired tumour resistance. There is a clinical need for improved treatment options. Methods: Previously, anti-angiogenic screens in Tg[fli1:EGFP] zebrafish identified quininib, a small molecule drug subsequently shown to be as effective as bevacizumab® at reducing tumour volume in vivo and which reduces tumour CD31 expression in xenografted mice. Here, structural analogues of quininib were synthesised to identify novel chemical entities with higher anti-angiogenic and anti-inflammatory efficacy. Analogues were examined for anti-angiogenic effects in the Tg[fli1:EGFP] zebrafish intersegmental-vessel assay. Hits were then progressed for safety analyses in cytotoxic assays, anti-angiogenic efficacy in aortic explants, and effects on cell proliferation and tubule formation using HT29_Luc2 and HMEC cells. Leads were then tested in ex vivo human CRC tumour explants to determine effects on angiogenic and inflammatory factor secretion. Following determination of the maximum tolerated dose (MTD) of the highest ranking analogues, CRC-specific murine xenograft models created by subcutaneous injection of HT29_Luc2 cells were assessed for anti-tumorigenic analysis. Results: Five out of 12 drug candidates (CC16_HCl, CC12_HCl, Z_HCl, CC8_HCl & OS_1) reduced developmental angiogenesis in Tg[fli1:EGFP] zebrafish significantly more than quininib (***P<0.01). These analogues were well tolerated in human endothelial and CRC cell cytotoxic assays and were more effective than quininib at reducing tubule formation and angiogenic factor secretion in HMEC cells. Two analogues reduced aortic ring vessel sprouting equally or more effectively than quininib. Some analogues can reduce inflammatory and angiogenic factor secretion from ex vivo human CRC explants more significantly than quininib. The analogue with the highest efficacy ranking was identified and its maximum tolerated dose study (MTD) determined using balb/c wild type mice. The anti-tumorigenic effects of the highest ranking analogue is being assessed by i/p injection in an optimised model of HT29_Luc2-CRC tumourigenesis in Balb/c nude mice. Conclusions: 5 analogues significantly more effective than quininib at reducing inflammation and angiogenesis were identified. The highest ranking analogue is tolerated by i/p injection in mice up to 50 mg/kg with no obvious adverse effects. The highest ranking analogue is currently being tested in murine xenografts with preliminary data suggests it is more effective than quininib at reducing tumour growth. Citation Format: Clare T. Butler, Breandán Kennedy, William M. Gallagher, Jacintha O’Sullivan. Phenotype-based discovery and development of novel therapies for the treatment of colorectal cancer. [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 3058.