bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 371: Utilizing metabolic pathways to improve diagnosis and risk stratification of prostate cancer
Authors: Alqazzaz Aymen , Fu Dexue , Karimi Arman , Ferreira Gustavo , McKenna Mary , Siddiqui Mohummad M.
Published: August 2018

Prostate cancer has well-characterized changes that take place metabolically in the citric acid cycle (CAC) such as increased lactate formation, reversal of physiologic buildup of citrate and breakdown into downstream CAC products. Metabolic MR spectroscopy utilizing 13C-labeled pyruvate now allows for clinical characterization of metabolic pathways in tissue. However, the traditionally utilized substrate of [1-13C]pyruvate has limitations because as the pyruvate is broken down, the labeled carbon does not enter the CAC and instead is lost as CO2. We propose that [2-13C]pyruvate, is better to characterize changes in the CAC. Specifically, [2-13]C-pyruvate can be converted to 13C-labeled acetyl CoA and subsequently metabolized in the CAC as 13C-labeled intermediate compounds (such as glutamate which rapidly pools alpha-ketogluterate). Alternatively in altered bioenergetic states, [2-13C]pyruvate is reduced to 13C-labeled lactate (Warburg effect) or transaminated to 13C-labeled alanine. By exposing prostate cancer cells to [2-13C]pyruvate and determining the relative labeling of lactate, glutamate, and alanine, we can elucidate which metabolic pathways (CAC, Warburg, transamination) are utilized in prostate cancer metabolism. The aim of this study was to determine how the metabolism of [2-13C]pyruvate via the CAC, reduction to lactate, and transamination differed in high and low malignancy prostate cancer cells.Methods: Tumorigenic prostate cells LNCaP and PC3 were exposed a medium containing [2-13C]pyruvate for 4 hours. PCA extraction was then performed to obtain the metabolites. 13C labeled compounds formed intracellularly and those released into the medium were analyzed using NMR chemical shifts with focus on peaks associated with pyruvate, lactate, alanine, glutamate, and other compounds related to the CAC. Dioxane was used as an internal standard for NMR characterization. Results: The labeled carbon from the [2-13C]pyruvate can be identified in the downstream metabolites of the citric acid cycle, after reduction to lactate, and/or transamination by determining labeled metabolites by 13C-NMR analysis. This allows for the ability to calculate the relative metabolism of pyruvate via the three pathways; transamination (74%), lactate formation (16%), and CAC (10%).

Conclusions: [2-13C]pyruvate can be successfully utilized as a tool to explore the alterations in pathways of metabolism, including the citric acid cycle, in prostate cancer cells.

Citation Format: Aymen Alqazzaz, Dexue Fu, Arman Karimi, Gustavo Ferreira, Mary McKenna, Mohummad M. Siddiqui. Utilizing metabolic pathways to improve diagnosis and risk stratification of prostate 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 371.
Abstract 3712: Optimizing design parameters of a VLA-4-targeted liposomal nanoparticle in a multiple myeloma disease model
Authors: Bilgicer Basar , Kiziltepe Tanyel , Omstead David , Deak Peter
Published: August 2018

Ligand-targeted liposomes have garnered interest as drug delivery vehicles for cancer therapy, however they have not consistently produced successful outcomes. These inconsistencies likely arise from differences in disease models and target receptors, as well as from nanoparticle design parameters which can significantly influence therapeutic efficacy. We systematically evaluated the role that peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size play on tumor targeting using a multifaceted synthetic strategy to prepare highly controlled and consistent peptide-targeted liposomes. We analyzed these parameters in a VLA-4-expressing multiple myeloma model system where liposomes were functionalized with a VLA-4-antagonist peptide to evaluate in vitro targeting efficiency and in vivo biodistribution and tumor cell uptake. Our studies demonstrated that by increasing the hydrophilicity of the targeting peptide sequence via addition of an oligolysine chain and simultaneously optimizing the EG peptide-linker length, cellular uptake of targeted liposomes was significantly enhanced in vitro. Specifically, the cellular uptake was greatly enhanced for 50-100 nm size liposomes with shorter peptide-linker lengths of EG6 when compared to the industry standard EG45 linker. For in vivo applications, although targeted formulations did not enhance tumor accumulation directly relative to non-targeted formulations, liposomes designed with EG6 linker and an oligolysine chain provided a significant advantage by demonstrating significantly enhanced tumor cell uptake relative to non-targeted liposomes. These results highlighted the importance of creating a comprehensive understanding of the effect of each liposome design parameter on multifactorial biological endpoints in determining the therapeutic potential of peptide-targeted liposomes.

Citation Format: Basar Bilgicer, Tanyel Kiziltepe, David Omstead, Peter Deak. Optimizing design parameters of a VLA-4-targeted liposomal nanoparticle in a multiple myeloma disease model [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 3712.
Abstract 3713: Targeting β-catenin pathway by nanoparticle-mediated delivery of norcantharidin impairs the stemness of triple negative breast cancer cells
Authors: Li Yunfei , Wang Zhishan , Xiao Yajuan , Yang Chengfeng
Published: August 2018

Background Norcantharidin (NCTD) is known as an inhibitor of serine/threonine protein phosphatase types 1 and 2A (PP1/PP2A) and exhibits potent anticancer efficacies. Our preliminary study identified NCTD interfering with the interaction between phospho-β-catenin and PP1/PP2A could downregulate the levels of non-phospho β-catenin in the cytoplasm in Triple Negative Breast Cancer (TNBC) cells. As β-catenin pathway plays an important role in cancer stemness, we hypothesize that NCTD could prevent β-catenin nuclear translocation, impair the stemness and reduce the metastasis of TNBC. However, the inherent features of short half-life and nephrotoxicity hinder NCTD's further in vivo application. The goal of this study was to test the therapeutic effect of targeting β-catenin pathway by nanoparticle-mediated delivery of NCTD against TNBC.

Experimental Design NCTD was loaded into an integrin α5 targeting nanoparticle (NP) (RGD-LPH) via complexing with polyethyleneimine. The targeting efficiency of RGD-LPH was tested by the in vivo imaging of RGD-LPH labeled by cyanine 7.5 (cy7.5). The effects of NP-mediated delivery NCTD on TNBC cell malignant behaviors and tumor metastasis were tested in various in vitro and in vivo models.

Results We found that RGD-LPH NP-mediated delivery of NCTD significantly reduced the active and total β-catenin levels as evidenced by Western Blot. The self-renewal capacity was remarkably blocked by RGD-LPH, as examined by sphere formation. RGD-LPH also suppressed features of cancer cells related to proliferation, as examined by MTT, clonogenic assay and soft agar colony formation. In particular, compared to free NCTD, RGD-LPH demonstrated extended-release profile and long-last effect in the

long-term experiments such as clonogenic assay and soft agar colony formation assay. In vivo, systemic delivery of RGD-LPH(cy7.5) indicated a preferential uptake in primary tumor and lung of the tumor-bearing mice. Efficacy studies indicated reduced metastasis of mice from RGD-LPH treatment.

Conclusion: Collectively, our results strongly suggest that NCTD being encapsulated in RGD-LPH, which warrants further development for preclinical and clinical studies as a novel TNBC therapeutic approach, is a robust modulator of Wnt signaling with anti-metastasis activity.

Citation Format: Yunfei Li, Zhishan Wang, Yajuan Xiao, Chengfeng Yang. Targeting β-catenin pathway by nanoparticle-mediated delivery of norcantharidin impairs the stemness of triple negative breast cancer cells [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 3713.
Abstract 3717: α3β1-Integrin targeted DM1 micellar prodrug for targeted therapy against ovarian cancer
Authors: Li Xiaocen , Xiao Wenwu , Wang Yayu , Chen Xiaojia , Liu Ruiwu , Lam Kit S.
Published: August 2018

Ovarian cancer ranks fifth in cancer deaths among women in America. The standard treatment for advanced stages ovarian cancer consists of primary debulking surgery followed by i.v. and i.p. chemotherapy. DM1, or mertansine, is a thiol-containing maytansinoid, that inhibits microtubule assembly and kills various cancer cells at low nM concentration. However, its application in the clinic has been hampered by severe toxic side effects1. Its trastuzumab conjugate (T-DM1), which was approved by the FDA in 2013, is a promising therapeutic agent for patients harboring ovarian cancers with high Her-2 expression2. However, the general limitation for antibody drug conjugates (ADCs) is low drug content, high clearance by the reticuloendothelial, and high manufacturing cost. To overcome this, we designed and synthesized a novel PEG-based, cholic acid/ DM1 hybrid telodendrimer (PEG5k-CA4-DM1) comprised of one DM1 molecule and three molecules of maleimide per telodendrimer, which self-assembled into 25nm nanomicelles for targeted therapy against ovarian cancer. To enhance the stability of this nanoparticle, PEG5k-Cys4-CA8, which contain 4 thiol groups per telodendrimer is formulated together with PEG5k-CA4-DM1 at a ratio of 2:5, to generate thiol/maleimide crosslinked DM1 micellar prodrug (DM1-nano). To further enhance its tumor-targeting ability, the surface of DM1-nano was decorated with LXY30 via a hydrophilic “stealth” linker (EK)3, using copper-free ‘Click' chemistry. LXY30 is a potent and specific ligand against α3β1 integrin which is overexpressed on the surface of many epithelial cancer cells including a panel of ovarian cancer cell lines (e.g. SKOV-3). Compared with free DM1 and non-targeted DM1-nano, LXY30-EK-DM1-nano not only prevented premature drug release, but also further enhanced tumor targeting ability, drug solubility and in vivo circulation time in blood. When loaded with a fluorescent dye (DiD), LXY30-EK-DM1-nano was found to (i) internalize inside SKOV-3 cells in vitro, (ii) significantly accumulated at the tumor site of SKOV-3 xenograft mice, and (iii) exhibit potent anti-cancer activity in vitro (IC50=26nM with SKOV-3 cells) and in vivo (SKOV-3 xenograft model). In addition, LXY30-EK-DM1-nano was found to be significantly more efficacious and less toxic than free DM1 and non-targeted DM1-nano. In conclusion, the LXY30-DM1-nano offers a promising novel nanotherapeutic agent against ovarian cancer.

Citation Format: Xiaocen Li, Wenwu Xiao, Yayu Wang, Xiaojia Chen, Ruiwu Liu, Kit S. Lam. α3β1-Integrin targeted DM1 micellar prodrug for targeted therapy against ovarian 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 3717.
Abstract 3728: α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in KC and KPC mice
Authors: Verma Raj K. , Yu Wei , Shrivastava Anju , Srivastava Rakesh K. , Shankar Sharmila
Published: August 2018

Activation of sonic Hedgehog (Shh) in cancer stem cell (CSC) has been demonstrated with aggressiveness of pancreatic cancer. In order to enhance the biologic activity of α-mangostin, we formulated mangostin-encapsulated PLGA nanoparticles (Mang-NPs) and examined the molecular mechanisms by which they inhibit human and KC mice (PdxCre;LSL-KrasG12D) pancreatic CSC characteristics in vitro, and pancreatic carcinogenesis in KPC (PdxCre;LSLKrasG12D;LSL-Trp53R172H) mice. Mang-NPs inhibited human and KrasG12D mice pancreatic CSC characteristics in vitro. Mang-NPs also inhibited EMT by upregulating E-cadherin and inhibiting N-cadherin and transcription factors Slug, and pluripotency maintaining factors Nanog, c-Myc, and Oct4. Furthermore, Mang-NPs inhibited the components of Shh pathway and Gli targets. In vivo, Mang-NPs inhibited the progression of pancreatic intraneoplasia to pancreatic ductal adenocarcinoma and liver metastasis in KPC mice. The inhibitory effects of Mang-NPs on carcinogenesis in KPC mice were associated with downregulation of pluripotency-maintaining factors (c-Myc, Nanog and Oct4), stem cell markers (CD24 and CD133), components of Shh pathway (Gli1, Gli2, Patched1/2, and Smoothened), Gli targets (Bcl-2, XIAP and Cyclin D1), and EMT markers and transcription factors (N-cadherin, Slug, Snail and Zeb1), and upregulation of E-cadherin. Overall, our data suggest that Mang-NPs can inhibit pancreatic cancer growth, development and metastasis by targeting Shh pathway.

Citation Format: Raj K. Verma, Wei Yu, Anju Shrivastava, Rakesh K. Srivastava, Sharmila Shankar. α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in KC and KPC mice [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 3728.
Abstract 3735: Somatostatin receptor-based imaging and treatment of murine pancreatic neuroendocrine tumors induced by MEN1-loss
Authors: Li Janet W. , Zhang Hanwen , Carlin Sean D. , Raj Nitya , Klimstra David S. , Libutti Steven K. , Weber Wolfgang A. , Reidy-Lagunes Diane , Untch Brian R.
Published: August 2018

Background: Somatostatin receptor ligands are used for the detection and treatment of neuroendocrine tumors. Mutations of MEN1 are frequently observed in human pancreatic neuroendocrine tumors (PanNETs). We utilized a previously described murine model of PanNET that harbors a floxed Men1 allele to study the feasibility of somatostatin receptor type 2 (SSTR2) imaging and therapy in mice. Methods: Mice with pancreatic-specific expression of Cre recombinase and floxed Men1 (Pdx1-cre, Men1f/f) were aged to 9-12 months to allow for the development of PanNETs. Mice were injected with either the SSTR2 agonist 68Ga-DOTA-TATE or antagonist 177Lu-DOTA-JR11 via tail vein. Imaging with 68Ga-DOTA-TATE was performed using PET/CT. Percent uptake of injected activity was calculated for visualized tumors. 177Lu-DOTA-JR11 binding was visualized with autoradiography. Immunofluorescence (IF) detected SSTR2 expression and a marker of DNA damage (γ-H2AX). Results: IF staining for SSTR2 showed membranous localization of the receptor limited to areas of neoplasia with high and low levels of SSTR2 expression variable by individual animal and tumor. Tumors were successfully detected in mice injected with 68Ga-DOTA-TATE using PET/CT. The mean maximum percent uptake of injected activity for visualized tumors was 2.8% +/- 0.34. Injection of 177Lu-DOTA-JR11 and subsequent autoradiography confirmed localization of radionuclide to areas of histology-confirmed neoplasia. Treatment effect (DNA damage) was observed with co-localization of γ-H2AX and SSTR2 staining in neoplastic areas as compared to hyperplastic islets and normal pancreatic parenchyma that were both SSTR2 and γ-H2AX negative. Conclusion: PanNETs from Pdx1-cre, Men1f/f mice can be successfully imaged and treated using somatostatin-based radionuclides. This genetically accurate model can be employed to study peptide receptor radionuclide therapy efficacy and toxicity either alone or in combination with other treatment strategies.

Citation Format: Janet W. Li, Hanwen Zhang, Sean D. Carlin, Nitya Raj, David S. Klimstra, Steven K. Libutti, Wolfgang A. Weber, Diane Reidy-Lagunes, Brian R. Untch. Somatostatin receptor-based imaging and treatment of murine pancreatic neuroendocrine tumors induced by MEN1-loss [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 3735.
Abstract 3741: Deciphering the regulome of androgen receptor variants in prostate cancer
Authors: Daniel Mark L. , Hickey Theresa E. , Carroll Jason S. , Tilley Wayne D. , Selth Luke A. , Dehm Scott M.
Published: August 2018

Prostate cancer is an androgen-dependent disease. Androgen function is controlled by the androgen receptor (AR), and treatments targeting AR are effective at preventing disease progression. Despite this, resistance and emergence of castration resistant prostate cancer (CRPC) is responsible for virtually all prostate cancer-related death. Constitutively active AR variants (AR-Vs) have been implicated as drivers of CRPC. AR-Vs consist of the unstructured AR NH2-terminal domain (NTD), and the AR DNA binding domain, but lack the ligand binding domain (LBD), which is targeted by all current therapies. While our understanding of proteins that regulate the AR LBD is mature, there is less known about the molecular regulation of the NTD. This represents a gap in knowledge given that the AR NTD is essential for AR/AR-V transcriptional activity. To address this, we used a technique termed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) with a pair of isogenic prostate cancer cell lines that were manipulated via genome engineering to express either full-length AR or AR-V. RIME identified several candidate proteins that interact with the AR NTD including BCL9, a β-catenin transcriptional coactivator. Interrogation of public databases revealed that BCL9 mRNA is overexpressed in metastatic prostate cancer compared with localized disease. Additionally, knockdown of BCL9 in cell lines expressing full-length AR inhibited expression of AR target genes as well as β-catenin target genes. Interestingly, knockdown of BCL9 did not affect these same genes in cell lines expressing AR-Vs, indicating that AR truncation could represent a mechanism of escape from BCL9 regulation. Collectively, these findings identify a new mechanism of AR regulation by BCL9 in prostate cancer.

Citation Format: Mark L. Daniel, Theresa E. Hickey, Jason S. Carroll, Wayne D. Tilley, Luke A. Selth, Scott M. Dehm. Deciphering the regulome of androgen receptor variants in prostate 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 3741.
Abstract 3750: Xenoestrogens cause estrogen receptor-dependent R-loop formation and DNA damage
Authors: Dunphy Karen A. , Majhi Prabin Dhangada , Sharma Aman , Roberts Amy L. , Daniele Elizabeth A. , Schneider Sallie S. , Jerry D Joseph
Published: August 2018

Background: The hormone 17β-estradiol (E2) plays an important role in breast cell proliferation and development but prolonged exposures and higher levels of E2 have been linked to genomic instability and breast cancer. Recent studies show E2 induced transactivations lead to DNA damage. A variety of environmental chemicals mimic the activities of estrogen, referred to as xenoestrogens. Thus, our hypothesis is to determine whether xenoestrogens stimulate transcriptional response and induce DNA damage similarly like E2.

Methods: The T-47D breast cancer cell line is used as a model because it expresses both estrogen receptors (ERα & ERβ) as found in normal breast tissue. Cells were exposed a physiologic range of E2 (17β-estradiol) [0.5-100nM] and the xenoestrogens, e.g., benzophenone-3 (BP-3) [0.5-50µM] and propyl paraben (PP) [0.5-10µM] with or without ER-antagonist ICI (1µM) for 24 hours. Transcriptional responses were quantified using an integrated ERE-luciferase reporter and qPCR for endogenous genes. Proliferation was monitored using the Alamar Blue assay. DNA damage was determined using γ-H2AX. R-loops were detected using the S9.6 antibody against DNA-RNA hybrids in the genome.

Results: Exposure of T-47D cells to physiologic levels of E2, PP and BP3 causes significant increase in DNA double-strand breaks (DSBs) as determined by γ-H2AX staining. We demonstrate a concomitant increase in R-loop formation with increase in γH2AX intensity. Treatment with RNaseH depletes R-loop intensity, corroborating the result. E2 and PP induce transactivation as determined by Luciferase reporter assay as well as expression of ER-response genes (PGR and AREG), whereas BP3 has modest effect on transactivation of PGR. ER transactivation as well as R-loop formation by E2, PP and BP3 were abrogated with ICI, demonstrating ER-dependency of R-loop formation. Consistent with the transcriptional response, proliferation studies determined that E2 showed highest proliferation followed by PP. However, BP3 showed no increase in proliferation.

Conclusions: Our results show that xenoestrogens have strikingly different activities in transactivation and proliferation. However, both xenoestrogens appear to stimulate R-loop accumulation that leads to DSB in an ER-dependent manner.

Citation Format: Karen A. Dunphy, Prabin Dhangada Majhi, Aman Sharma, Amy L. Roberts, Elizabeth A. Daniele, Sallie S. Schneider, D Joseph Jerry. Xenoestrogens cause estrogen receptor-dependent R-loop formation and DNA damage [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 3750.
Abstract 3764: Porcupine inhibitor RXC004 enhances immune response in pre-clinical models of cancer
Authors: Bhamra Inder , Armer Richard , Bingham Matilda , Eagle Catherine , Cook Alicia Edmenson , Phillips Caroline , Woodcock Simon
Published: August 2018

Background: RXC004, a potent and selective porcupine (PORCN) inhibitor, is being investigated in a safety and tolerability study in cancer patients with solid tumours (CT 2017-000720-98). In addition to the tumour targeting role of RXC004 and other Wnt pathway inhibitors, we present pre-clinical data which suggests further potential for RXC004 in modulating the immune system of the tumour microenvironment. Materials and Methods: To evaluate the potential of a novel porcupine inhibitor, RXC004, as an immunomodulatory anti-cancer agent, sub cutaneous B16F10 melanoma (C57BL/6 mice) and CT26 colorectal (BALB/c mice) murine tumour models were utilised. Mice in both models were treated with RXC004 alone or in combination with mouse anti-PD-1 antibody. Flow cytometry analysis was utilised to measure key immune cell populations in the tumour microenvironment. To probe the underlying mechanism of immune modulation in these models and to provide a link to the emerging clinical data suggesting a role for Wnt pathway activation in immune escape, human monocytic cells were isolated from PBMCs and human dendritic cells were derived in vitro. The Wnt pathway was induced in derived Dendritic cells and expression of IDO was measured. Results: In the murine CT26 model, RXC004 treatment reduced tumour size when dosed in combination with anti-PD-1 antibody, causing regression and cures in some animals. Furthermore, flow cytometry showed RXC004 in combination with anti-PD-1 antibody increased the proportion of CD8+ cytotoxic T cells as well as decreasing FoxP3+ regulatory T cells when compared to the monotherapy anti-PD-1 arm. In a syngeneic murine melanoma B16F10 model, RXC004 monotherapy at a dose of 5mg/kg QD orally significantly inhibited tumour growth, as did RXC004 combined with anti-PD-1. RXC004 had no effect on the proliferation of B16F10 cells in vitro, suggesting this was not caused by the compound directly affecting B16 cell proliferation. Flow cytometry analysis of the B16 tumours showed significant immune modulatory effects in the tumour microenvironment. In addition to mouse model data, Wnt pathway activation in human dendritic cells was shown to increase IDO expression. Conclusion: Taken together, data from murine syngeneic mouse models corroborate literature data suggesting that inhibiting the Wnt pathway may promote the immune response against human cancers.

Citation Format: Inder Bhamra, Richard Armer, Matilda Bingham, Catherine Eagle, Alicia Edmenson Cook, Caroline Phillips, Simon Woodcock. Porcupine inhibitor RXC004 enhances immune response in pre-clinical models of 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 3764.
Abstract 3771: Mechanistic and pharmacodynamic characterization of the immuno-oncological activity of RXDX-106, a novel TYRO3, AXL, and MER (TAM) inhibitor in clinical development
Authors: Albert Amanda , Lew Erin D. , Smith Kristen , Tindall Elizabeth A. , Yokoyama Yumi , Diliberto Amy , Ely Heather , Lee Jack , Nevarez Robin , Oh Joanne , Walsh Colin , Christiansen Jason , Li Gary , Shoemaker Robert
Published: August 2018

The TAM family of receptor tyrosine kinases (RTKs) includes TYRO3, AXL, and MER, and is a unique set of molecular targets for cancer immunotherapy. TAM RTKs play a key role in the tumor microenvironment (TME) as negative regulators of innate immune responses and contribute to the evasion of cancer cells from immunosurveillance. RXDX-106 is a novel small molecule TAM inhibitor with single-digit nanomolar kinase inhibition and slow dissociation binding kinetics resulting in potent and durable target inhibition. In preclinical studies, RXDX-106 has demonstrated immune-mediated single agent in vivo activity in multiple syngeneic tumor models and potentiated anti-tumor activity in combination with immune checkpoint inhibitors. To characterize the mechanism of action of and pharmacodynamic responses to RXDX-106 treatment, an integrated approach was employed to explore RXDX-106-mediated immune modulatory effects at the cellular, protein, and transcriptional levels. In syngeneic tumor models, RXDX-106 treatment resulted in the recruitment of both innate and adaptive immune cell subtypes to the TME. Specifically, RXDX-106 treatment resulted in increased immune cell infiltration, macrophage polarization towards an antitumor M1 phenotype, activation of T cell and NK cell function, and modulation of immune markers at both the protein and RNA level, indicative of a pro-inflammatory and anti-tumorigenic effect. Importantly, the immune modulatory effects of RXDX-106 were concordant across platforms. Immune modulation was further characterized in a dose-dependent manner, with increasing immune cell infiltration observed with escalating doses of RXDX-106. In summary, RXDX-106 has the potential to restore and enhance immune function by modulating the local immunosuppressive TME. The unique mechanism of activating both innate and adaptive immunity, and regulating cross-talk between immune cells and tumor cells, supports clinical evaluation of RXDX-106 as an immunomodulatory agent for the treatment of a variety of cancers.

Citation Format: Amanda Albert, Erin D. Lew, Kristen Smith, Elizabeth A. Tindall, Yumi Yokoyama, Amy Diliberto, Heather Ely, Jack Lee, Robin Nevarez, Joanne Oh, Colin Walsh, Jason Christiansen, Gary Li, Robert Shoemaker. Mechanistic and pharmacodynamic characterization of the immuno-oncological activity of RXDX-106, a novel TYRO3, AXL, and MER (TAM) inhibitor in clinical development [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 3771.
Abstract 3772: Preclinical study of a novel TLR8 selective agonist for cancer immunotherapy
Authors: Wang Yuxun , Yang Heping , Zhang Xingzhong , Ren Shuwen , Li Huanping , Zhao Shuda , Gu Longjun , Zhang Yin , Zeng Yikun , Wang Longsheng , Fu Guangliang , Bao Fang , Liu Fang , Wu Zhiheng , Zhu Panhu , Xu Hui , Gao Yaqiao , Zhang Jian , Wang Pei , Chen Shoujun , Gao Daxin
Published: August 2018

The innate immune system has emerged as one of the major pathways for drug discovery focusing on cancer immunotherapy. TLR8 plays a crucial role in activating innate immunity and facilitates adaptive immunity thereby exerting potent immune-mediated anticancer activity. We previously presented preliminary characterization of a novel TLR8 agonist DN-A1 (Abstract 2995, AACR Annual Meeting 2017). In the present study, we carried out further comprehensive comparative preclinical study of DN-A1 and motolimod, the only TLR8 selective drug candidate in clinical trials. The result indicated that DN-A1 clearly differentiated from motolimod. DN-A1 was highly selective for TLR8 while motolimod exhibited moderate activity over TLR7 in addition to its primary agonistic activity over TLR8. DN-A1 had superior in vitro ADMET and in vivo PK profiles. Moreover, DN-A1 displayed more potent in vitro and in vivo biological activity and produced stronger anticancer efficacy than motolimod. These findings were further supported by DN-A1's stronger effect than motolimod in activating innate immunity in both monkey in vivo and human PBMC ex vivo systems. Furthermore, DN-A1 significantly suppressed tumor growth as a single agent and combination with the chemotherapeutic agent enhanced efficacy of either agent alone in leukemia tumor model. GLP toxicity study in rats and preliminary toxicity study in monkeys revealed acceptable safety profile of DN-A1. Importantly, DN-A1 consistently caused skin injection site reaction (ISR) when administered subcutaneously in both rats and monkeys. In contrast, motolimod failed to induce ISR under the same condition where DN-A1 caused ISR. Recent reports of motolimod's clinical data indicated that ISR was strongly correlated with substantial survival benefit in cancer patients whereas there was no survival benefit when motolimod failed to induce ISR. Therefore, DN-A1 holds great potential as the best-in-class TLR8 selective immunotherapeutic drug candidate poised for human clinical trials.

Citation Format: Yuxun Wang, Heping Yang, Xingzhong Zhang, Shuwen Ren, Huanping Li, Shuda Zhao, Longjun Gu, Yin Zhang, Yikun Zeng, Longsheng Wang, Guangliang Fu, Fang Bao, Fang Liu, Zhiheng Wu, Panhu Zhu, Hui Xu, Yaqiao Gao, Jian Zhang, Pei Wang, Shoujun Chen, Daxin Gao. Preclinical study of a novel TLR8 selective agonist for cancer immunotherapy [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 3772.
Abstract 3775: Use of RLI-15 a clinical grade fusion protein with IL-15 superagonistic activity for the activation of anti-tumor immune response
Authors: Adkins Irena , Sadilkova Lenka , Hradilova Nada , Tomala Jakub , Tomalova Barbora , Kovar Marek , Mikyskova Romana , Reinis Milan , Martynoff Guy de , Bechard David , Moebius Ulrich , Spisek Radek
Published: August 2018

RLI-15, a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain represents a promising candidate for the induction of anti-tumor immunity. RLI-15 was designed to bypass the need of endogenous IL-15Rα, thereby leveraging the activity of IL-15 in vivo on target immune cells. RLI-15 stimulates the proliferation and the cytotoxic activity of natural killer (NK) cells and memory CD8+ T cells with no significant expansion and activation of regulatory T cell compartment. RLI-15 was previously shown to exhibit a potent anti-metastatic activity in B16F10 melanoma and Renca renal cell carcinoma mouse models. RLI-15 also significantly delayed tumor growth and prolonged survival when combined with anti-PD1 therapy in CT26 and MC38 colon carcinoma models. Here, we report that the combination treatment with clinical-grade RLI-15 and an anti-PD1 antibody leads to a significant anti-tumor efficacy in a TRAMP-C2 prostate cancer mouse model with 70 % of mice remaining tumor free after the treatment. We evaluated the optimal schedule of such combination therapy to set the basis for the design of upcoming clinical trials. We further tested how the administration schedule affects the pharmacodynamics properties of clinical-grade RLI-15 and translates into the anti-tumor efficacy in metastatic Renca and CT26 mouse models. In cynomolgous monkeys, various schedules of administration of RLI-15 showed a dose-dependent expansion of peripheral blood lymphocytes, predominantly of NK cell and memory CD8+ T cell compartments. The toxicity in mice and cynomolgous monkeys was evaluated to determine the maximal tolerated dose of RLI-15. Furthermore, the activity of clinical-grade RLI-15 was tested in vitro on human PBMCs and the superiority over IL-2 and IL-15 stimulatory capacity has been confirmed. The complex analysis of RLI-15 behavior and of the induced anti-tumor immune response will be explored in the design of a planned Phase I clinical study in patients with both solid tumors and hematological malignancies.

Citation Format: Irena Adkins, Lenka Sadilkova, Nada Hradilova, Jakub Tomala, Barbora Tomalova, Marek Kovar, Romana Mikyskova, Milan Reinis, Guy de Martynoff, David Bechard, Ulrich Moebius, Radek Spisek. Use of RLI-15 a clinical grade fusion protein with IL-15 superagonistic activity for the activation of anti-tumor immune response [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 3775.
Abstract 3777: RORγ agonist enhances antitumor effects of immune checkpoint anti-PD1
Authors: Gaur Pankaj , Verma Vivek , Nandre Rahul , Vir Pooja , Wang Hua , Kang Baolin , Carter Laura , Hu Xiao , Liu Xikui , Gupta Seema , Khleif Samir
Published: August 2018

Anti-PD1/PDL1 therapies have demonstrated great clinical benefit in some cancers, albeit in a limited percentage of patients. RORγt is a master transcription factor, which promotes Type 17 T cell differentiation and is known to enhance the stemness and plasticity of effector cells and to modulate PD1 expression. Since one of the potential failure mechanism of anti-PD1 is the lack of sustained CD8 infiltration within the TME, we assessed whether combining a synthetic, small-molecule RORγ agonist could enhance the antitumor effects of anti-PD1 treatment. To assess this combination treatment, we tested the effects of anti-PD1 antibody (1 mg/kg twice weekly) combined with a RORγ-agonist (LYC-54143; 100 mg/kg PO, BID given continuously till the end of study) in tumor-bearing murine mode lthat utilizes a vaccine for proper priming. We found that RORγ agonist combined with PD1 antibody significantly enhanced the anti-PD1 antitumor effect. This result was manifested by further delaying the tumor growth and prolonging mice survival. At day 35 after tumor implantation, 66% of mice survived in the group treated with anti-PD1 and RORγ agonist and vaccine when compared to 0% survival following anti-PD1 + vaccine or vaccine- alone treatments. This enhanced antitumor activity was observed/confirmed in several tumor models with the combination of RORγ agonist + anti-PD1 treatment. These results show that RORγ agonists may enhance the effectiveness of anti-PD1 therapy and therefore this combination is a promising therapeutic strategy for cancer.

Citation Format: Pankaj Gaur, Vivek Verma, Rahul Nandre, Pooja Vir, Hua Wang, Baolin Kang, Laura Carter, Xiao Hu, Xikui Liu, Seema Gupta, Samir Khleif. RORγ agonist enhances antitumor effects of immune checkpoint anti-PD1 [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 3777.
Abstract 378: Stat3 promotes ovarian cancer by modulating the energy metabolism and develops drug resistance in patients
Authors: Raja Vaishnavi , Giri Shailendra , Sakr Sharif , Hijaz Miriana , Quiles Raymond , Munkarah Adnan R. , Rattan Ramandeep
Published: August 2018

Signal Transducers and Activators of Transcription (STAT) are a group of transcription factors that are known to play a major role in cancer progression. In ovarian cancer, increased STAT3 leads to cancer proliferation in response to cytokines and confers resistance to chemotherapy-induced apoptosis in epithelial malignancies. STAT3 is constitutively activated in patient derived ovarian cancer cells, and increased STAT3 expression is a predictor of poor prognosis. Apart from its function as a transcription factor, recently STAT3 has been shown to modulate mitochondrial function to promote carcinogenesis. The aim of our study was to investigate if STAT3 activation can modulate cellular metabolism of ovarian cancer cells. Stable clones expressing STAT3 were generated in A2780 ovarian cancer cells, along with empty vector clones. Ectopic expression of STAT3 in A2780 ovarian cancer cell line resulted in increased proliferation (p<0.01) and colony formation ability (p<0.001) in vitro and led to large ovarian tumors (p<0.01) compared to parental and vector controls in xenograft mouse model. Bioenergetics profiling showed higher mitochondrial respiration (OCR) and glycolysis (ECAR) in STAT3 clones compared to parental and vector clones. Ratio of ECAR/OCR in the STAT3 overexpressing cells placed them in the ‘metabolically active' phenotype, while parental A2780 and vector clones were in ‘metabolically less active' phenotype. A selective inhibitor of STAT3, STATTIC, inhibited the STAT3 mediated growth of A2780 cells both in vitro (p<0.01) and in vivo (p<0.01). In addition, STATTIC treatments reversed the ‘metabolically active' state of STAT3 overexpressing clones to a ‘lower metabolic state', placing them in the same category as the control cells. In addition, STATTIC inhibited the cell proliferation and modulated bioenergetic phenotype of other ovarian cancer cells lines (PEO4, C200 and OVCAR3) that display a ‘metabolically active' phenotype. Overall, STAT3 can induce metabolic changes in ovarian cancer cells, maybe as survival mechanism and enhances the cellular fitness of the ovarian cancer cell resulting in increased oncogenic abilities.

Citation Format: Vaishnavi Raja, Shailendra Giri, Sharif Sakr, Miriana Hijaz, Raymond Quiles, Adnan R. Munkarah, Ramandeep Rattan. Stat3 promotes ovarian cancer by modulating the energy metabolism and develops drug resistance in patients [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 378.
Abstract 3782: Transcriptional regulation of MHC Class II in tumor associated plasmacytoid dendritic cells in glioblastoma
Authors: Chintala Sreenivasulu , Tewari Brij N. , Henriquez Mario , Fetcko Kaleigh , Filley Anna , Dey Mahua
Published: August 2018

Background: Glioblastoma (GBM), an aggressive grade IV astrocytoma, is the most common primary brain tumor in adults and carry very poor prognosis. One of the hallmark features of GBM is tumor induced dense immunosuppression that renders immunotherapeutic strategies against GBM ineffective. In order to develop successful immunotherapeutic strategies for GBM, it is critical to understand the mechanism of immunosuppression in the tumor microenvironment (TME). In our previous publication, we have shown that plasmacytoid dendritic cells (pDCs) accumulate in TME and preferentially up-regulate MHC-II expression in comparison to myeloid DCs (mDCs). GBM infiltrating pDCs are immunosuppressive in nature and lack the ability to secrete IFN-alpha, and MHC-II upregulation by immunosuppressive pDCs lead to development of tumor tolerance. MHC-II molecules are transcriptionally regulated by the multiprotein complex enhanceosome consisting of a catalytic Class II transactivator (C2ta) in a cell type-dependent mechanism. There are no studies clearly showing differential enhanceosome and C2ta regulation in GBM associated pDC and mDC. In the present study, we investigated the mechanism of differential expression of enhanceosome multiprotein complex in GBM associated pDC and mDC and identify the upstream regulators and downstream effectors. Methods: GL261 cells were intracranially injected (i.c.) in C57BL6 mice and allowed to establish tumors. Tumor and normal brain (n=5), along with cervical lymph nodes (cLN), were collected and processed for isolation of pDC and mDC using flow sorting. Quantitative RT-PCR analysis was used to determine the enhanceosome components. IHC/IF and Western blot analysis were performed to confirm differential expression of enhanceosome components in mouse and human GBM specimens. Cytokine/chemokine protein array and ELISA methods were used to determine the differential expression of cytokines/chemokines in pDC and mDC. Results: We found differential expression of enhanceosome components in tumor associated pDC compared to mDC. Additionally, we found that GBM derived cytokines regulate differential expression of enhanceosome in tumor associated pDC and mDCs. Conclusions: In conclusion results from our studies show that GBM microenvironment regulates differential expression of enhanceosome transcription factors in pDC compared to mDC in a cytokine dependent process. This in turn helps to maintain an immunosuppressive microenvironment for continued tumor growth.

Citation Format: Sreenivasulu Chintala, Brij N. Tewari, Mario Henriquez, Kaleigh Fetcko, Anna Filley, Mahua Dey. Transcriptional regulation of MHC Class II in tumor associated plasmacytoid dendritic cells in glioblastoma [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 3782.
Abstract 38: Mitophagy deficiency promotes osteolytic bone metastasis in breast cancer
Authors: Deng Rong , Zhang Hai-Liang , Cai Rui-Zhao , Tang Jun , Zhu Xiao-Feng
Published: August 2018

Introduction: Metastasis of breast cancer to the bone is a common complication and occurs in 65-80% of late-stage patients. Specifically, the development of osteolytic bone lesions increases patient morbidity and mortality with debilitating skeletal complications. Mitophagy is a selective form of autophagy that removes damaged or excessive mitochondria in response to various metabolic stresses. However, the role of mitophagy in development of cancer remains largely unclear. The Janus-faced role of mitophagy during tumorigenesis leads to a novel challenge when targeting it for cancer therapies. This study focuses on the role of mitophagy deficiency in breast cancer osteolytic bone metastasis, and explores the potential of rescuing mitophagy deficiency as possible therapeutic targets for metastatic disease.

Methods: Specific single-guide RNAs (sgRNAs) were used to deplete ULK1 expression in breast cancer cell lines. Mitophagy was monitored by detecting Tom20 and Hsp60. In addition, mitochondrial DNA (mtDNA) was used to further confirm the reduction of mitochondrial mass. For bone metastasis studies, luciferase-tagged tumor cells were injected into the left cardiac ventricle of anesthetized female nude mice. Metastatic progression was monitored by bioluminescence imaging (BLI). Micro-CT imaging was used to detect bone volume, cortical wall thickness, trabecular thickness and number in mice of tibia. TRAP assay was used to detect osteoclast differentiation assays.

Results: We found that the core Atg protein ULK1 was not only expressed at low level in breast cancer, but also required for hypoxia-induced mitophagy. ULK1-related mitophagy deficiency promoted the formation of metastatic osteolytic lesions in experimental bone metastasis mouse model with MDA-MB-231 cells. TRAP staining revealed that mature osteoclasts significantly increased in RAW264.7 cells treated with CM from ULK1-knockout breast cancer cells. In addition, MEK inhibitor trametinib was sufficient to upregulate the expression of ULK1 and reduced the release of cytokines, which is key to osteoclast differentiation via triggering mitophagy, thereby effectively blocking breast cancer osteolytic bone metastasis.

Conclusion: In conclusion, we demonstrate the functional importance of mitophagy defect in breast cancer for promoting osteolytic bone metastasis. We also provide strong preclinical evidence for using MEK-ERK pathway inhibitors to disrupt ERK kinase-involved ULK1 degradation and induce mitophagy as a breast cancer bone metastasis therapy.

Citation Format: Rong Deng, Hai-Liang Zhang, Rui-Zhao Cai, Jun Tang, Xiao-Feng Zhu. Mitophagy deficiency promotes osteolytic bone metastasis in 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 38.
Abstract 3809: T-cell activation through the inhibition of tumor-expressed IDO1 activity in tryptophan metabolism pathway
Authors: Dong Chunlan , Wang Ying , Gong Kefeng , Xing Frank , Shi Qian
Published: August 2018

Indoleamine 2,3-dioxygenase-1 (IDO1) has been described as a major mechanism of immunosuppression in tumors. Despite histologic evidence showing that most human tumor tissues have extensive infiltration by various pro-inflammatory and immune cells, expression of IDO1 by tumor cells results in aggressive tumor growth and resistance to T cell-targeting immunotherapies. To de-suppress T-cell function through inhibition of IDO1-mediated tryptophan metabolism pathway, we established an in vitro assay system allowing robust detection of production level of tryptophan metabolism product N-formyl-kynurenine (Kyn) in culture medium, plasma and tumor tissue through ELISA and LC-MS/MS. We screened a panel of human and murine tumor cell lines for IDO1 expression with and without INFg induction. Our data suggest that a few of these models constitutively express IDO1; about half of the tumor cell models in the panel express IDO1 in an IFNg-dependent manner while the others do not respond to the cytokine stimulation at all. The mechanism underlying the lack of response to cytokine induction on IDO1 production is under investigation. We next tested the reduction of kynurenine in culture medium through inhibition of IDO1 enzymatic activity. After 24h stimulation by IFNg, a murine cell model was treated by INCB024360 (Incyte) for 48h and 72h. Cell-free supernatants were then collected, followed by Kyn detection by LC-MS/MS. The results showed that longer compound treatment (72h) offered a more robust detection window (1,500 ng/mL Kyn down to 2,00 ng/mL), compared to 48h results (350 ng/mL Kyn down to 100 ng/mL). This reproducible assay offers a reliable platform for initial IDO compound screening. To correlate effective reduction of kynurenine production to reactivation of T-cell activity, a co-culture system was established in that T-cell proliferation and cytokine production (IL2) were examined upon IDO1 inhibition in a time course. To achieve maximal therapeutic effect, a combination of INCB024360 and aPD1 antibody was applied in a T cell-mediated tumor cell killing assay. Detailed data will be discussed.

Citation Format: Chunlan Dong, Ying Wang, Kefeng Gong, Frank Xing, Qian Shi. T-cell activation through the inhibition of tumor-expressed IDO1 activity in tryptophan metabolism pathway [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 3809.