bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3515: LHX9 is a novel oncogene bypassing KRAS addiction
Authors: Ly Seav Huong , Dai Chao , Hong Andrew L. , Ilic Nina , Krall Elsa B. , Hahn William C.
Published: July 2017

Background Oncogenic mutations of RAS are detected in approximately 30% of human cancers. KRAS is highly mutated in pancreatic, colorectal and lung cancers. Direct therapeutic targeting of Ras and its membrane association has been challenging due to high affinity of GTP to Ras and unexpected mechanism of alternative post-translational modification. While few inhibitors of downstream RAS effectors - MAPK and PI3K pathways - show some clinical efficacy, they are complicated by complex feedback loops, and as single agents are not effective in KRAS-mutant cancers. Combination therapies may be promising yet limited by narrow therapeutic window. Identification of alternative strategies to directly inhibiting RAS and MAPK/PI3K pathways is key to address this unmet need.

Methods To this end, we performed a genome-scale open reading frames screen to identify genes capable of restoring viability to KRAS-dependent cells following KRAS suppression. We tested the top 5-standard-deviation hits in low-throughput manner. One of the top hits included LIM homeobox 9 (LHX9), a homeobox family transcription factor essential for mouse gonad, limb, and brain development. While LHX9 is amplified in several cancers, little is known regarding its roles in cancers. We used hypothesis-driven as well as unbiased approaches including RNA-seq, ChIP-seq, and IP-mass spectrometry to investigate the basis of LHX9-mediated bypass of KRAS dependency.

Results We showed that LHX9 rescued KRAS suppression in multiple cell lines as well as xenograft. LHX9 overexpression substituted for KRAS in KRAS-driven xenograft. Moreover, in activated-MEK-overexpressing HA1E cells, LHX9 formed tumors as robustly as myristoylated AKT. LHX9 rescued KRAS suppression by both restoring KRAS-dependent pathways and activating KRAS-independent pathways. First, LHX9 reactivated the main KRAS effectors - MAPK and PI3K pathways and conferred resistance to MEK inhibitor Trametinib. Second, using ChIP- and RNA-seq, we observed LHX9 binding to promoters/enhancers of genes whose expression was regulated by KRAS, particularly YAP1. Third, LHX9 increased expression and activation of KRAS-independent genes, including STAT3.

Conclusions Here, we identified LHX9 as a novel bypass of KRAS addiction. Multiple unbiased approaches revealed at least three mechanisms by which LHX9 rescued KRAS suppression. Our findings contribute to our understanding of KRAS biology. Further investigation of downstream effectors of LHX9 may highlight potentially novel therapeutic targets for KRAS-dependent cancers.

Citation Format: Seav Huong Ly, Chao Dai, Andrew L. Hong, Nina Ilic, Elsa B. Krall, William C. Hahn. LHX9 is a novel oncogene bypassing KRAS addiction [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 3515. doi:10.1158/1538-7445.AM2017-3515
Abstract 3543: Targeting the metabolic mevalonate pathway with statins as anti-breast cancer agents
Authors: Leeuwen Jenna van , Pandyra Aleksandra , Goard Carolyn , Mullen Peter J. , Yu Rosemary , Penn Linda Z.
Published: July 2017

The statin family of drugs target the mevalonate pathway and have been used for decades in the control of hypercholesterolemia, however recent evidence suggests these approved agents may also be useful as anti-cancer therapeutics (see our recent Nature Review Cancer article1). For example, statins can trigger tumor-specific apoptosis and two independent pre-op clinical trials in breast cancer, evaluating cholesterol-lowering doses of fluvastatin and atorvastatin, resulted in breast tumor shrinkage due to decreased growth and increased apoptosis. Our hypothesis is that statins have utility as anti-breast cancer agents. To maximize efficacy and speak to personalized medicine, our objectives are to develop biomarkers to distinguish which patients will benefit from the addition of statins to their treatment regimen and how best to use statins in combination with other agents to augment anti-tumor efficacy. To identify biomarkers of statin sensitivity we evaluated fluvastatin activity across a panel of BCa cell lines and showed that the basal, estrogen receptor-negative subtype were significantly sensitive to statin-induced apoptosis. As this included the difficult-to-treat triple negative BCa (TNBCa) we have extended this work and further evaluated a panel of TNBCa cell lines for statin sensitivity. From these results we are identifying features associated with robust apoptosis in response to statin exposure. To identify how best to use statins, we conducted two unbiased screens and have shown that blocking the restorative feedback response to statin exposure potentiates statin-induced apoptosis. This is reversible with exogenous mevalonate, reinforcing that this is an on-target effect. We also identified another approved agent, dipyridamole, as able to potentiate the anti-cancer activity of statins. Mechanistically we have shown that dipyridamole blocks the feedback response to statin exposure. We have extended these studies and shown that the combination of statins and dipyridamole is effective against TNBCa both in vitro and in vivo. Thus, we provide essential pre-clinical data to support the further evaluation of statins and dipyridamole in BCa.

Reference: Mullen, P.J. et al. The interplay between cell signalling and the mevalonate pathway in cancer. Nat Rev Cancer. 16, 718-731 (2016).

Citation Format: Jenna van Leeuwen, Aleksandra Pandyra, Carolyn Goard, Peter J. Mullen, Rosemary Yu, Linda Z. Penn. Targeting the metabolic mevalonate pathway with statins as anti-breast cancer agents [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 3543. doi:10.1158/1538-7445.AM2017-3543
Abstract 3545: Glucose-dependent regulation of PXR is modulated by AMPK
Authors: Oladimeji Peter O.
Published: July 2017

The sensitivity of patients to drugs depends on the pharmacokinetics of the drugs, but largely on the bioavailability of the drugs in the body. Orally administered drugs must pass through the intestinal wall and the portal circulation to reach the liver; both are sites of first-pass metabolism. Thus, many drugs may be metabolized before optimal plasma concentrations are reached, or before getting to target tissues, resulting in diminished drug efficacy and acquired drug resistance. Among the several drug-metabolizing enzymes (DME) is cytochrome P450 (CYP) enzyme 3A4, the most abundant in the liver, metabolizes approximately 50 percent of marketed drugs and a main target of pregnane X receptor (PXR) In addition, transporters including MDR1 are also regulated by PXR. There is increasing evidence that drug metabolism can be affected by energy metabolism and vice versa. PXR has been implicated to have a complex role in energy homeostasis, and increased drug clearance was observed in untreated diabetic patients. The elimination of any chemotherapeutic agents in humans is markedly dependent on the relative expression and activity of the various DMEs and drug transpoters. The heterotrimeric AMP-activated protein kinase (AMPK) serves as a monitor of cellular energy status in most eukaryotic cell, and is activated under energy stress conditions. Interestingly, AMPK is also regarded as a tumor suppressor in various cancers because of its inhibitory effects on anabolic processes that are important for tumor development. Despite the knowledge of the cross-regulation between drug metabolism and energy metabolism, the molecular pathways of this interaction remain vague. In our study, we exposed HepG2 cells to varying glucose concentrations and observed that PXR is greatly induced in high glucose conditions. This finding is consistent with clinical observation of increased drug clearance in untreated diabetic patients. We demonstrate that AMPK modulates PXR transcriptional activity. As such, the pharmacological manipulations of AMPK activation status show an inverse relationship with PXR activity. Activation of AMPK was shown to decrease PXR activity as observed by decreased CYP3A4 transcription, and consistent with that a potentiation of response to drugs. Interestingly, we observed that PXR is not a direct target of AMPK: it remains to be determined what the intermediary between AMPK and PXR is. Taken together, our studies identify novel links between the energy status of the cell, the activation of AMPK and down regulation of PXR.

Citation Format: Peter O. Oladimeji. Glucose-dependent regulation of PXR is modulated by AMPK [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 3545. doi:10.1158/1538-7445.AM2017-3545
Abstract 3547: B cell receptor signaling regulates cellular metabolism in Chronic Lymphocytic Leukemia
Authors: Vangapandu Hima V. , Havranek Ondrej , Wierda William G. , Keating Michael J. , Davis Richard Eric , Stellrecht Christine M. , Gandhi Varsha
Published: July 2017

Peripheral blood B cells in chronic lymphocytic leukemia (CLL) are quiescent, yet have an active RNA transcription and protein translation processes, suggesting that these malignant lymphocytes may be metabolically active. However, CLL metabolism and its relationship to disease biology has not been well-explored. In our previous investigation, we reported that compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in quiescent peripheral blood B lymphocytes from CLL patients (Vangapandu, H.V. et al, AACR 2014). In 45 patient samples tested, glycolysis (extracellular acidification rate, ECAR) was consistently low (1 to 15 mpH/min/5 x 105 lymphocytes), whereas, oxygen consumption rate (OCR) varied considerably (5 to 190 pMoles/min/5 x 105 lymphocytes). Among the prognostic factors, high OCR correlated strongly with ZAP 70 positivity, unmutated IGHV status, greater β2M levels and higher Rai stage. In contrast, glycolytic flux (ECAR) from same patient samples did not associate with prognostic factors. Further, OCR did not vary on the basis of frequently occurring cytogenetic abnormalities, 13q14, 17p, and 11q deletions or trisomy 12. Since, ZAP 70 and IGHV unmutated status are associated with augmented B-cell receptor (BCR) pathway signaling, we tested the impact on OCR after genetic ablation of B-cell receptor (CH2 region of IgM). A CRISPR-Cas9-mediated BCR knockout mitigated OCR in a malignant B cell line without impacting the rate of proliferation. A critical node in the BCR pathway is PI3 Kinase and PI3K isoforms, δ and γ are involved in B-cell malignancies. Consistent with BCR ablation results, knocking out PIK3CD (codes for catalytic subunit δ), dramatically reduced OCR and ECAR. Pharmacological inhibitors of the PI3K pathway, duvelisib (a PI3K δ/γ inhibitor in phase III clinical trials) or idelalisib (FDA approved PI3K δ inhibitor) also decreased OCR. Direct inhibition of AKT with MK-2206 showed similar results. Collectively, these data suggest that CLL cellular metabolism is associated with prognostic factors and linked to BCR signaling pathway. Since, the PI3K inhibitors used in our study are being tested clinically for patients with CLL, investigations on the impact on metabolomics during therapy and translation of results to combination strategies need to be explored.

Note: This abstract was not presented at the meeting.

Citation Format: Hima V. Vangapandu, Ondrej Havranek, William G. Wierda, Michael J. Keating, Richard Eric Davis, Christine M. Stellrecht, Varsha Gandhi. B cell receptor signaling regulates cellular metabolism in Chronic Lymphocytic Leukemia [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 3547. doi:10.1158/1538-7445.AM2017-3547
Abstract 3559: Functional characterization of HNF4α in gastric cancer
Authors: Xu Chang , Ooi Wen Fong , Qamra Aditi , Tan Patrick
Published: July 2017

Gastric cancer (GC) is a leading cause of cancer mortality worldwide and particularly common in East Asia. Transcription factors are frequently amplified or overexpressed in GC and might contribute to tumorgenesis, however the underlying mechanisms largely remain unknown. Our previous data indicates that HNF4α is a common target of three amplified transcription factors (GATA4, GATA6 and KLF5) in gastric cancer. HNF4α had also been found to be related to diverse aspects of tumorigenesis. In liver and colon cancers, there are reports indicating that the targets of HNF4α are mainly involved in intermediary metabolism, however, its tissue-specific downstream targets and pathways have not been systemically studied yet. Therefore, a comprehensive functional characterization of HNF4α is required to identify its tissue-specific role in gastric cancer. In this study, we analyzed the expression features of HNF4α in GC, and found the expression level of HNF4α is significantly higher in early stage tumor and in CIN and MSI compared to GS and EBV subtypes of GC according to TCGA classification method. In addition, we performed the HNF4α ChIP in GC cell lines, and the results revealed a number of HNF4α binding sites in GC. Comparison with the published ChIP-seq data of Caco2 and HepG2 cell lines indicated the existence of GC-exclusive targets. Furthermore, functional annotation analysis revealed that the GC-exclusive targets are significantly related to the negative regulation of cellular metabolic process compared to the common targets. These preliminary data suggests that HNF4α might play a tissue specific role in GC, especially in metabolic related processes. Through further target validation and biological function studies, this study hopes to systemically investigate of HNF4α’s role in GC tumorigenesis, especially in metabolism related pathways.

Citation Format: Chang Xu, Wen Fong Ooi, Aditi Qamra, Patrick Tan. Functional characterization of HNF4α in gastric cancer [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 3559. doi:10.1158/1538-7445.AM2017-3559
Abstract 3565: Recurrently detected germline mutation of FLT3 (D358V) associated with prevalence of hematopoietic malignancy and prognosis of AML patients
Authors: SONG SEULKI
Published: July 2017

FLT3 is most frequently mutated gene in acute myeloid leukemia patients. While somatic mutation of FLT3 is well known, germline mutation of FLT3 and predispose to AML remain poorly understood. Here we identify a germline FLT3 p.D358V affected prognosis of AML patients. We performed whole exome sequencing (WES) using 76 patients’ saliva sample to detect FLT3 germline mutation in buccal epithelial cell and demonstrate FLT3 p.D358V mutation for 6 patients among 76 patients. The frequency of FLT3 p.D358V mutation in AML patients is significantly higher in general population (p=9.50E-07), suggesting association of FLT3 mutation with AML predisposition. Asian population especially carried this locus more than other ethic population. When we compared between AML patients and general population of Asian, the chi-square p-value was 0.07 and odd ratio was 2.26. Overall survival was significantly reduced in patients who had FLT3 p.D358V mutation (p=0.03) and disease free survival was show a tendency of reducing in the patients with FLT3 p.D358V mutation.

Citation Format: SEULKI SONG. Recurrently detected germline mutation of FLT3 (D358V) associated with prevalence of hematopoietic malignancy and prognosis of AML patients [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 3565. doi:10.1158/1538-7445.AM2017-3565
Abstract 3585: Determination of gene amplifications with a next-generation sequencing cancer panel
Authors: Deras Ina L. , Wise Aaron , Zhao Chen , Glidewell-Kenney Christine , Le Phillip , Aderhold Elizabeth Upsall , Gutekunst Karen
Published: July 2017

TruSight® Tumor 15* (TST15) uses next-generation sequencing technology to detect somatic point mutations, insertions, and deletions in 15 genes that are commonly mutated in solid tumors. It accurately detects low-frequency mutations with low DNA input and is optimized for formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The test is a multiplexed PCR panel that includes primers to support amplification detection in the EGFR, ERBB2, and MET genes.

Here we describe development of a bioinformatics analysis method to enable detection of these gene amplifications. DNA extracted from 31 FFPE samples with amplifications in EGFR, ERBB2, or MET—some with dual amplifications—were diluted with DNA extracted from 18 normal FFPE samples to create a total of 67 unique samples at low-level amplification. Undiluted and diluted samples were assessed for quality based on amplifiability (ΔCq or library yield) and tested with TST15. For comparison, the samples were also tested with droplet digital PCR (ddPCR) to determine the copy ratio of target to wild type. For amplifiable samples, the TST15 algorithm was able to detect amplifications in EGFR and ERBB2 at ddPCR ratios of 1.4 and 1.6, respectively. ddPCR was unable to accurately quantify low MET amplifications; therefore, expected ddPCR ratios were used for comparison. The TST15 algorithm was able to detect amplifications in MET samples at an expected ddPCR ratio of 1.3. Poor sample quality (i.e., high ΔCq or low library yield) impaired amplification detection. In some samples, the presence of multiple amplifications also impaired detection.

The addition of this algorithm to the TST15 workflow will allow researchers to more accurately characterize tumor samples by detecting somatic mutations and amplifications simultaneously.

*For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Ina L. Deras, Aaron Wise, Chen Zhao, Christine Glidewell-Kenney, Phillip Le, Elizabeth Upsall Aderhold, Karen Gutekunst. Determination of gene amplifications with a next-generation sequencing cancer panel [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 3585. doi:10.1158/1538-7445.AM2017-3585
Abstract 3611: Proteasome subunit PSMD1 participates in p53 degradation and regulates proliferation of breast cancer cells
Authors: Okumura Toshiyuki , Ikeda Kazuhiro , Sato Wataru , Horie-Inoue Kuniko , Inoue Satoshi , Takeda Satoru , Okamoto Koji
Published: July 2017

Breast cancer is the most common cancer among women in developed countries. Generally, 70% of primary breast cancer patients express estrogen receptor, thus endocrine therapy is often efficient for the disease using antiestrogen such as tamoxifen as well as aromatase inhibitor. During long-term therapy, however, a substantial proportion of women recurrent and acquire resistance to endocrine therapy. Understanding of the mechanism underlying acquired endocrine resistance would be a first step toward overcoming hormone-refractory cancer. In this study, we identified candidate genes that contributed to tamoxifen resistance in MCF-7 breast cancer cells by functional screening based on short hairpin RNA (shRNA) library. We found that several candidate genes were overexpressed in breast cancer patients and their abundance was positively associated with poor prognosis of cancer patients in clinical database. Moreover, the genes associated with poor prognosis also showed positive association with shorter survival for patients treated with tamoxifen. Expression levels of these candidate genes were upregulated in hydroxytamoxifen (OHT)-resistant MCF-7 cells (OHTR) compared with parental cells. Proliferation of OHTR cells was repressed by siRNAs targeting 26S subunits, non-ATPase 1 (PSMD1) and tetraspanin12 (TSPAN12). Furthermore, knockdown of PSMD1 inhibited cell cycle progression and p53 protein degradation. p21 and 14-3-3σ, target genes of p53, were upregulated by PSMD1 knockdown. These findings would be used for the development of alternative diagnostic and therapeutic options for endocrine-resistant breast cancers.

Citation Format: Toshiyuki Okumura, Kazuhiro Ikeda, Wataru Sato, Kuniko Horie-Inoue, Satoshi Inoue, Satoru Takeda, Koji Okamoto. Proteasome subunit PSMD1 participates in p53 degradation and regulates proliferation of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3611. doi:10.1158/1538-7445.AM2017-3611
Abstract 3615: Combined targeting of estrogen receptor alpha and nuclear transport pathways remodel metabolic pathways to induce apoptosis and overcome tamoxifen resistance
Authors: Kulkoyluoglu-Cotul Eylem , Madak-Erdogan Zeynep , Landesman Yosef , Wrobel Kinga
Published: July 2017

There is a critical need for novel therapeutic approaches to re-sensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and XPO1, a nuclear transport protein in overcoming endocrine resistance. Using Cignal Finder pathway analysis, Seahorse metabolic profiling and GC/MS whole metabolite profiling, we found that combination of 4-OH-Tam and Selinexor (SXR), being evaluated in multiple later stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies, inhibited Akt phosphorylation by changing the localization of the kinase. Since we observed dramatic changes in Akt activity we hypothesized that glucose utilization pathways and metabolic profile of breast cancer cells would change in the presence of 4-OH-Tam and SXR. These cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine as the mitochondrial fuel source. We showed that combined targeting of XPO1 and ERα rewires metabolic pathways, increase demand on mitochondria and causes increased production of ROS that would eventually lead to apoptosis. Re-modelling metabolic pathways to regenerate new vulnerabilities in endocrine-resistant tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα (+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences.

Citation Format: Eylem Kulkoyluoglu-Cotul, Zeynep Madak-Erdogan, Yosef Landesman, Kinga Wrobel. Combined targeting of estrogen receptor alpha and nuclear transport pathways remodel metabolic pathways to induce apoptosis and overcome tamoxifen resistance [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 3615. doi:10.1158/1538-7445.AM2017-3615
Abstract 3616: Potential therapeutic use of Estrogen Receptor β agonist in the prevention and progression of breast cancer using HER-2/neu mouse model
Authors: Ramasamy Kumaraguruparan , Samayoa Cathy , Krishnegowda Naveen K. , Vadlamudi Ratna K. , Tekmal Rajeshwar R.
Published: July 2017

Breast cancer is the most common cancer among women worldwide. Hormone-mediated therapy to treat estrogen receptor alpha (ERα) positive breast cancers include the use of ER antagonist, Tamoxifen, aromatase inhibitors and other compounds that degrade ERα. Unlike ERα, ERβ has been shown to have tumor-suppressive function in various cancers, including breast cancer. Recent studies have identified, synthesized, and tested the clinical safety of ERβ-selective agonists. Given the tumor-suppressive properties of ERβ, it may be possible to use these compounds to induce or activate the ERβ and test their role in the chemoprevention and blocking the progression of breast cancer. We have investigated the therapeutic utility of ERβ agonists in the prevention and progression of breast cancer using MMTV-HER2/neu mice transgenic mouse model. MMTV-HER2/neu mice develop premalignant lesions at 4-5 months, and tumors starting at month 7 due to overexpression of the HER2/neu proto-oncogene. MMTV-HER2/neu mice were treated with an ERβ agonist, LY500307 examined the prevention and progression of mammary cancers in these mice. When compared to controls, ERβ agonist-treated mice exhibited a significant decrease in the development of preneoplastic changes. Differential gene expression analysis revealed a significant change in the expression of a number of genes in response to LY500307 treatment. Pathway analysis identified an enrichment for chemokines signaling pathways, particularly TNF, in blocking the development of preneoplastic changes resulting from treatment with ERβ agonists. Our studies also show a decrease in the formation of mammary tumors in HER-2/neu mice with preexisting preneoplastic changes when treated with LY500307. This study suggests that ERβ agonist treatment may be a valuable therapeutic option for the prevention in women at increased risk of breast cancer and in blocking the progression of hormone receptor positive breast tumors.

Citation Format: Kumaraguruparan Ramasamy, Cathy Samayoa, Naveen K. Krishnegowda, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. Potential therapeutic use of Estrogen Receptor β agonist in the prevention and progression of breast cancer using HER-2/neu mouse model [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 3616. doi:10.1158/1538-7445.AM2017-3616
Abstract 3671: Anti-PD-1 treatment may potentiate the radiation-induced lung injury
Authors: Xue Jianxin , Du Shisuo , Lu You , Dicker Adam , Lu Bo
Published: July 2017

Purpose: Combination of radiation therapy and anti-PD1 immunotherapy has been investigated both in the lab and in the clinic. Pneumonitis is a rare but potentially fatal toxicity of ant-programmed death-1 (PD-1) monoclonal antibodies (mAbs). The purpose of our study is to address whether anti-PD-1 mAbs will potentiate radiation-induced lung toxicity and mortality in a murine model using Small Animal Radiation Research Platform (SARRP) for lung-targeting irradiation (IR).

Methods: Both lungs of male C57bl/6 mice were targeted for 20Gy using the SARRP. Mice were stratified into 4 treatment groups receiving IgG, anti-PD1, IR + IgG, or IR + anti-PD1. IgG or anti-PD-1 mAbs administrated via i.p. injection, with a dosage of 10mg/kg, twice per week for five doses. Acute lung injury was assessed by H&E staining and flow cytometry to measure CD4 or CD8 positive T lymphocytes. A duplicate study (n=10) was performed to determine long-term survival following lung irradiation.

Results: 30 days following lung irradiation, lung tissues exhibited abnormal alveoli, with exudates and inflammatory cells in the alveolar septa (H&E staining). The extent of these changes was more prominent in IR+anti-PD-1 group. Moreover, there were significantly (2.1 fold increase; p<0.05) more CD8+ cytotoxic T lymphocytes, rather than CD4+ cells lymphocytes, in the irradiated lung tissues in the group of IR+anti-PD1 compared to that of IR+IgG. Up to 120 days post IR, 90% mice survived in the group of IR+IgG while 70% mice survived in the IR+anti-PD-1 group (p=0.657).

Conclusions: Anti-PD-1 mAbs potentiates the radiation-induced pneumonitis, likely mediated by increased CD8+ cytotoxic T lymphocytes. Anti-PD-1 mAbs may increase the radiation-related mortality although it was not statistically significance at the day 120 following IR. Care should be taken for excessive lung toxicities in ongoing clinical trials of combining thoracic RT and anti-PD1 therapy. We will collaborate with NRG to further investigate clinical lung toxicities from combining thoracic radiotherapy with Nivo in the ongoing RTOG 3505 though analyzing the collected biospecimens.

Note: This abstract was not presented at the meeting.

Citation Format: Jianxin Xue, Shisuo Du, You Lu, Adam Dicker, Bo Lu. Anti-PD-1 treatment may potentiate the radiation-induced lung injury [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 3671. doi:10.1158/1538-7445.AM2017-3671
Abstract 3674: Local irradiation promotes antitumor immunity and abscopal effect of antigen-specific immunotherapy by enhancing dendritic cell maturation
Authors: Chang Ming-Cheng , Cheng Jason Chia-Hsien , Chiang Ying-Cheng
Published: July 2017

Background: Ionizing radiation therapy (RT) is a well-established method to eradicate locally advanced tumors. Whether or no local RT can enhance the potency of an antigen-specific DNA vaccine and the possible underlying mechanism were evaluated.

Materials and Methods: The number of E7-specific CD8+ cytotoxic T cell precursors and anti-E7 Abs from Tumor-bearing mice treated with local RT (6Gy twice per week) and the CTGF/E7 DNA vaccine were evaluated. Abscopal effects were also monitored. HMGB-1and DC maturation from local irradiation were analyzed by flow cytometry. The ratio of effector-to-suppressor cells in the tumor microenvironment was also investigated.

Results: Tumor-bearing mice treated with local RT (6Gy twice per week) and the CTGF/E7 DNA vaccine exhibited a dramatic increase in the number of E7-specific CD8+ cytotoxic T cell precursors, higher titers of anti-E7 Abs, a significant reduction of tumor size, and longer survival. The combination of local RT and the CTGF/E7 vaccine also elicited abscopal effects on non-irradiated local subcutaneous and distant pulmonary metastatic tumors. Local irradiation can induce the expression of HMGB-1 in apoptotic tumor cells and stimulate DC maturation, which results in inducing antigen-specific immune responses. In addition, local irradiation, although inducing Treg cells, can eventually increase a higher ratio of effector-to-suppressor cells in the tumor microenvironment.

Conclusions: Local irradiation can enhance the antigen-specific immunity and antitumor effects on local and distant metastatic tumors generated by an antigen-specific DNA vaccine. Irradiation combined with antigen-specific immunotherapy is a potential new strategy for cancer therapy.

Citation Format: Ming-Cheng Chang, Jason Chia-Hsien Cheng, Ying-Cheng Chiang. Local irradiation promotes antitumor immunity and abscopal effect of antigen-specific immunotherapy by enhancing dendritic cell maturation [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 3674. doi:10.1158/1538-7445.AM2017-3674
Abstract 3703: A genomic characterization of testicular germ cell tumor immune microenvironment
Authors: Hollern Daniel P. , Hoadley Katie , Vincent Benjamin , Perou Charles M. , Group TCGA Testicular Germ Cell Tumor Analysis Working
Published: July 2017

Testicular germ cell tumors (TGCT) are a histologically diverse set of tumors and the most common cancer type in young men. As part of the TCGA effort, we analyzed tumors from 133 patients including 4 patients with asynchronous double primary TGCTs (137 primary tumors) by histopathologic, genomic, epigenomic, transcriptomic, and proteomic methods. We noted molecular signatures of various immune cells were greater in seminomas than other histologies. Specifically, we find seminomas show elevation of expression signatures for B-Cells, Cytotoxic T-Cells, Th17 Cells, and T-regulatory cells. This increase in immune cells were accompanied by high expression of specific cytokines and key immune checkpoint molecules such as CTLA4, LAG3, and PD-L1. Strong positive correlations were identified between immune cell signatures and increases in T-Cell and B-Cell receptor diversity measures. Interestingly, increases in receptor diversity also showed strong positive associations to gene expression signatures for anti-tumor immune responses. This indicates that anti-tumor immune responses in seminomas may be polyclonal. We found no evidence for relationships between immune cells and mutation load, neoantigen load, or detected viruses. We do observe that seminomas have significantly high expression of the testis specific antigens, yet the contribution of these antigens to the immune response did not show strong or uniform statistical indications. Instead, the strongest association between expanded immune cells populations in seminomas were the presence of activating mutations in KIT accompanied by elevation of gene expression of signatures associated with KIT and expression of MHC class I and II genes. Together, these data describe the immune microenvironment of seminomas and suggest alterations and events in the kit pathway as being critical to the nature of the immune microenvironment in seminomas.

Citation Format: Daniel P. Hollern, Katie Hoadley, Benjamin Vincent, Charles M. Perou, TCGA Testicular Germ Cell Tumor Analysis Working Group. A genomic characterization of testicular germ cell tumor immune microenvironment [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 3703. doi:10.1158/1538-7445.AM2017-3703
Abstract 3708: RAS-MAPK signal is required for enhanced PD-L1 expression in human lung cancers
Authors: Sumimoto Hidetoshi , Takano Atsushi , Teramoto Koji , Daigo Yataro
Published: July 2017

Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Since the precise mechanisms responsible for ectopic PD-L1 expression remains obscure, we analyzed the molecular mechanisms of ectopic PD-L1 expression in human lung cancers, focusing on the MAPK signal. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50-60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. Post-transcriptional mechanism by miR-200s appears not to be in general under the downstream of MAPK in the PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10-32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression at a transcriptional level, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers.

Citation Format: Hidetoshi Sumimoto, Atsushi Takano, Koji Teramoto, Yataro Daigo. RAS-MAPK signal is required for enhanced PD-L1 expression in human lung cancers [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 3708. doi:10.1158/1538-7445.AM2017-3708
Abstract 371: Developing small molecule therapeutics to target AKT signaling in non-small cell lung cancer
Authors: Chandrasekaran Balaji , Pal Deeksha , Kolluru Venkatesh , Ramisetti Srinivasa R. , Sharma Arun K. , Ankem Murali , Damodaran Chendil
Published: July 2017

Lung cancer remains a leading public health problem which is evidenced by its increasing death rate. Platinum-based chemotherapy is the first-line of treatment for patients in advanced stages of non-small cell lung cancer (NSCLC), however the success rates are not quite impressive. Hence, developing individualized treatment strategies for metastatic lung cancer gains momentum, such as Tyrosine kinase inhibitors erlotinib and gefitinib, or ALK inhibitors ceritinib and alectinib are commonly used in the clinic. Recent reports suggest that pAKT(ser473) is highly expressed in NSCLC and higher nuclear expression of pAKT correlated with poor prognosis and an independent prognostic marker for survival. Our lab is interested to develop novel small molecules which specifically inhibit AKT signaling in NSCLC. Structure-activity relationship (SAR) studies in our laboratory have recently ideintifed one such compound, AKS-407, that effectively inhibited cell growth at nanomolar concentration in NSCLC cell lines (A549 and H460; 250nM). Molecular studies revealed AKS-407 inhibited AKT signaling by down regulating pAKT(ser473) expression and downstream events including NFκB activation, BCl-2 expression in both the cell lines. As signaling through AKT regulates epithelial-mesenchymal transition (EMT) in NSCLC, we determined the effect of AKS-407 on EMT phenotype on NSCLC cells. Treatment of AKS-407 inhibited the mesenchymal markers includes snail, MMP9, N-cadherin, β-catenin and vimentin expression that resulted in blocking invasion and migration of A549 and H-460 cells. These results suggest AKS-407 to be a promising small molecule targeting AKT signaling pathway which remains an important target for the development of effective treatment of metastatic NSCLC. Validating in-vivo efficacy of this potential drug candidate would further support our overall goal of the study.

Citation Format: Balaji Chandrasekaran, Deeksha Pal, Venkatesh Kolluru, Srinivasa R. Ramisetti, Arun K. Sharma, Murali Ankem, Chendil Damodaran. Developing small molecule therapeutics to target AKT signaling in non-small cell lung cancer [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 371. doi:10.1158/1538-7445.AM2017-371
Abstract 3742: Exposure burden for patients during an experimental study using a dual-energy contrast-enhanced spectral mammography
Authors: Rawojc Kamila , Kisielewicz Kamil , Dziecichowicz Anna , Tomaszuk Monika , Cepiga Anna , Anna Jankowska , Izabela Cieśla , Dominika Najberg , Elzbieta Luczynska , Kieltyka Bartosz
Published: July 2017

Mammography provides early micro-calcification recognition, that is important for further breast cancer (BC) diagnosis. Imaging choice for BC detection is X-ray mammography (MG) with the use of high-resolution digital modality combined with iodinated contrast agents. However, a conventional MG has some sensitivity limitations, especially in patients with densely treated breasts. Moreover, MG contributes radiation risk for BC that is correlated with an exposure to ionizing radiation. Patients, for whom MG study does not give a clear interpretation are often further diagnosed by contrast-enhanced breast MRI, the most sensitive BC detection technique. Alternatively, it is limited by higher costs, lower availability and increased false positives. Dual-energy contrast-enhanced spectral mammography (CESM) is a new method applied in BC that enables accurate detection of malignant breast lesions similar to breast MRI. The aim of this study was to compare the usefulness of CESM versus MG in BC detection. Additionally, doses given to the BC patients during imaging were compared. In this study, 250 consecutive women (144 MG vs. 105 CESM), that were screened using mammography between 2011 and 2014 were retrospectively enrolled in the study. The comparison of entrance surface air kerma and mean glandular dose values for both options were recorded, respectively and analyzed using different statistical and retrospective approach. Results of presented study showed that CESM might be a new diagnostic tool allowing an accurate detection of BC lesions, giving results similar to those received from magnetic resonance imaging. A good agreement found between sensitivity of the method and tumour diameters measurements based on CESM, supports this tool as a potential replacement for breast MRI in the preoperative evaluation of BC patients. However, a radiation burden for patients might be significantly higher than that for a standard full-field digital mammography procedure. In summary, each method has its own benefits with respect to specific applications that are further discussed in the study.

Citation Format: Kamila Rawojc, Kamil Kisielewicz, Anna Dziecichowicz, Monika Tomaszuk, Anna Cepiga, Jankowska Anna, Cieśla Izabela, Najberg Dominika, Luczynska Elzbieta, Bartosz Kieltyka. Exposure burden for patients during an experimental study using a dual-energy contrast-enhanced spectral mammography [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 3742. doi:10.1158/1538-7445.AM2017-3742
Abstract 3761: Specific and dynamic tumor antigen-specific immune responses were elicited in patients with hepatocellular carcinoma after cell-based immunotherapy
Authors: Han Yanyan , Wu Ye , Yang Chou , Huang Jing , Guo Yabing , Liu Li , Chen Ping , Wu Dongyun , Liu Junyun , Li Jin , Zhou Xiangjun , Hou Jinlin
Published: July 2017

Hepatocellular carcinoma (HCC) is one of the most common cancers in China and frequently occurs with chronic hepatitis B virus (HBV) infection. To investigate whether cell-based cancer immunotherapy induces tumor specific immune responses in patients with HCC and provides clinical benefits, as well as to elucidate the most immunogenic tumor associated antigens (TAAs), multiple antigen stimulating cellular therapy (MASCT) was applied in addition to standard of care. Mature dendritic cells (DCs) and activated T cells prepared for MASCT were generated from autologous peripheral blood mononuclear cells (PBMCs). DCs were loaded with a peptide pool of multiple HCC-related tumor antigens, and T cells were stimulated by these DCs. Thirteen patients with HCC received repeated MASCT after tumor resection during which their immune responses were examined. After three courses of MASCT, the frequency of regulatory T cells in the patients’ PBMCs significantly decreased (p < 0.001), while the antigen peptide pool-triggered T cell proliferation (p < 0.001) and IFNγ production (p = 0.001) were significantly enhanced. The specific T cell responses against each antigen in the pool were detected in eleven patients, but with individualized distinct patterns. The most immunogenic TAAs for HCC are survivin, CCND1, and RGS5. Moreover, the antigen-specific immune responses observed in tumor-free patients’ PBMCs were significantly stronger than that in the patients with recurrence (p = 0.037).

Conclusion: Our study demonstrates that MASCT is well-tolerated by patients with HCC and elicits strong and dynamic immune responses specifically against multiple tumor associated antigens, which may correlate with clinical outcomes.

Citation Format: Yanyan Han, Ye Wu, Chou Yang, Jing Huang, Yabing Guo, Li Liu, Ping Chen, Dongyun Wu, Junyun Liu, Jin Li, Xiangjun Zhou, Jinlin Hou. Specific and dynamic tumor antigen-specific immune responses were elicited in patients with hepatocellular carcinoma after cell-based immunotherapy [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 3761. doi:10.1158/1538-7445.AM2017-3761
Abstract 3784: Expression of estrogen receptor specific signaling transcriptome in epithelial cell adhesion molecule (EpCAM) captured circulating tumor cells from patients with breast cancer
Authors: Singh Ashok , Sperger Jamie M. , Schehr Jennifer , Witkowski Tessa , Weaver Beth A. , Burkard Mark E. , Lang Joshua M.
Published: July 2017

Background: Breast cancer (BC) is the second leading cause of cancer related death in American women. There is a critical need for new biomarkers to evaluate treatment response and emerging mechanisms of resistance. The identification and characterization of new biomarkers such as Circulating Tumor Cells (CTCs) may be helpful tools for these purposes as well as understanding the biology of metastatic BC.

Methods: Blood samples were processed with a Ficoll gradient to isolate nucleated cells. CTCs were isolated from these samples using an exclusion-based sample preparation technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. EpCAM positive CTCs were captured in the VERSA followed by mRNA extraction for gene expression profiling for the estrogen receptor (ER) and other breast cancer relevant targets. CTCs were defined as Hoechst positive, CD45/CD11b/CD14 negative, and cytokeratin positive. Captured CTCs were evaluated for ER protein staining intensity and localization.

Results: The VERSA platform was validated to capture and enumerate CTCs from patients with breast cancer using EpCAM capture and immunofluorescent staining for intact nucleus, ER, and cytokeratin while negative for multiple immune markers (CD45, CD11b, CD14). The VERSA can capture cells with >90% capture efficiency with EpCAM antibody in MCF7 and T47D cell lines. ER specific transcriptome analysis in BC patient CTCs and cell lines was performed. We evaluated expression of ER signaling pathway, prognostic, metastatic/EMT; stemness; epithelial markers with concordant expression of RPLPO and HPRT1. We identified expression of ER in 6 out of 7 BC samples. Expression of PR and HER2 in a subset of samples confirms capture of breast cancer cells for further molecular interrogation.

Conclusion: In conclusion, we report the expression profiles of ER signaling genes in EpCAM captured CTCs from BC patients. These tools will be used to compare subpopulations of CTCs from patients with BC as predictive and pharmacodynamic biomarkers of response to targeted therapies or chemotherapies.

Citation Format: Ashok Singh, Jamie M. Sperger, Jennifer Schehr, Tessa Witkowski, Beth A. Weaver, Mark E. Burkard, Joshua M. Lang. Expression of estrogen receptor specific signaling transcriptome in epithelial cell adhesion molecule (EpCAM) captured circulating tumor cells from patients with breast cancer [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 3784. doi:10.1158/1538-7445.AM2017-3784