bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1218: EMP2 serves as a novel target for immunotoxin mediated ovarian cancer therapy
Authors: Mohamedali Khalid , Kok Su-Yin , Cheung Lawrence H. , Rosenblum Michael G. , Wadehra Madhuri
Published: August 2019

Ovarian cancer (OC) currently accounts for only ~2% of all new cancer cases per year, but once diagnosed, patients only show a 5 year survival of 30%. Subdivided into four subtypes: serous, endometrioid, clear cell, and mucinous carcinoma, high-grade serous ovarian cancer (HGSOC) accounts for 70-80% of the ovarian cancer deaths. Immunoconjugates represent an innovative therapeutic approach to deliver a deadly payload to a tumor cell with more specificity and potentially less avenues for resistance than either can exert individually. However, it has been a challenge to find candidates as well as implement conjugation strategies to achieve these goals for a number of cancers. Epithelial membrane protein-2 (EMP2) is over-expressed in a number of cancers in women including 70% of ovarian tumors, with up to 88% of advanced serous carcinomas showing high expression of the antigen. With an eye towards therapy, we have recently developed an IgG1 targeting the extracellular domain of EMP2 with single digit nM affinity. As the antibody-antigen complex rapidly internalizes, we sought to improve on the efficacy of the naked antibody. Granzyme B (GrB) is a serine protease that plays a critical role in the body’s defense against viral infection and tumor development by initiating the apoptotic cascade via both caspase-dependent and -independent mechanisms. Accordingly, we developed novel fusion proteins composed of the anti-EMP2 backbone as the targeting moiety and granzyme B as the cytotoxic payload. The designs comprised (a) the anti-EMP2 IgG1 with GrB fused to the Fc domain through a cleavable linker, and (b) active GrB fused to the IgG heavy chain (Fc) domain containing 2 single-chain antibodies against EMP2, a format that results in a longer plasma retention time. The constructs were expressed by transient transfection in HEK293E cells and purified to homogeneity. Flow cytometry and ELISA demonstrated binding properties of the fusion protein with high specificity and sensitivity to EMP2 expression. The enzymatic activity of the fusion construct was similar to commercially-available GrB. Internalization of the immunotoxins was confirmed, and they showed high cytotoxicity to EMP2-positive cells compared to EMP2-negative cells. We predict that these designs may improve therapeutic outcomes for EMP2+ tumors.

Citation Format: Khalid Mohamedali, Su-Yin Kok, Lawrence H. Cheung, Michael G. Rosenblum, Madhuri Wadehra. EMP2 serves as a novel target for immunotoxin mediated ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1218.
Abstract 1219: Generation of anti-OX40 antibody with different immunization strategies
Authors: Chai Xiaojuan , Xin Wenfang , Yang Teddy , Duan Qing , Liu Louis
Published: August 2019

Variety of immunogen such as recombinant protein, DNA or cells has been used for animal immunization to develop hybridoma antibody. However, the effectiveness among different types of immunogen in generating functional antibodies especially antibody with agonistic activity has not been systematically investigated.

OX40 (CD134) is a T cell costimulatory receptor that is found primarily on activated CD4+ and CD8+ T cells, regulatory T (Treg) cells and natural killer (NK) cells. Activation of OX40 by its natural ligand or an agonist antibody augments CD4 and CD8 T cell expansion, differentiation, and survival. Agonistic anti-OX40 antibody holds promise as therapeutics for cancer immunotherapy.

In the present study, different immunization strategies (recombinant protein, cell and DNA) have been used to generate anti-OX40 antibody. Antigen specific immune response, number of binders, epitope bins and the functional activities obtained from different immunogens were analyzed and compared. Based on cross reactivity and cell based functional assay results, 25 clones from protein immunization, 22 from DNA immunization and 29 from cell based immunization were expanded and purified. Among the purified antibodies, antibodies obtained from DNA immunization showed stronger agonist activity in both Jurkat OX40-NFAT luciferase reporter activity and primary T cell activation assay. The antibodies obtained from DNA immunization also demonstrated in vivo efficacy and significantly inhibited tumor growth in MC38 syngeneic mouse model using human OX-40 knock-in mice.

In summary, we have evaluated different immunization strategies using OX-40 as a case study and successfully generated high potency high affinity agonistic anti-OX40 antibody that support further development.

Citation Format: Xiaojuan Chai, Wenfang Xin, Teddy Yang, Qing Duan, Louis Liu. Generation of anti-OX40 antibody with different immunization strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1219.
Abstract 1221: Mitomycin C induces programed cell death ligand 1 overexpression but enhances anticancer immunity in none small cell lung cancer
Authors: Luo Min
Published: August 2019

Blockade of the checkpoint inhibitor programmed death 1 ligand (PDL1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are progressive with low response rate. To enhance the efficacy of PDL1 blockade immunotherapies, we tried to combine chemotherapy capable of enhancing antitumor immunity. Here, we screened that tumor cells with MMC pre-treatment was significantly killed by co-cultured lymphocytes than without ones. Indeed, tumor cells treated with MMC showed the increased release of granzyme B and TNF-α. And tumor lysis was enhanced by combined with PD1 Ab in vitro. Mice treated with both anti-PDL1 and MMC showed an improved overall survival and inhibition within the tumor, indicating that this combination improves the overall quality of the immune response generated. Mechanically, MMC was observed to upregulate PDL1 expression in a concentration and time manner. MMC activated c-JUN to bind with PDL1 promoter and recruited its co-factor, STAT3 to accelerate PDL1 expression. MMC induced an IFN-γ independent signature within the tumor cell, and ERK signaling pathway was activated. A reason for its enhancement in antitumor immunity was the high expression of MHC-I after MMC treatment which attracted more CD8+ T cells. These findings identified a potential mechanism for the observed benefit of combining MMC and PDL1 blockade, in which MMC induces PDL1 upregulation and MCH-I high expression, thereby converting the tumor cell into an immunogenic one, when block PDL1, the antitumor response was enhanced.

Citation Format: Min Luo. Mitomycin C induces programed cell death ligand 1 overexpression but enhances anticancer immunity in none small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1221.
Abstract 1223: Somatic mutations in collagens impact patient outcomes in cancer
Authors: Brodsky Alexander S. , Khurana Jay , Yang Dongfang , Gamsiz Ece , Wong Ian , Resnick Murray
Published: August 2019

Stomach cancer remains among the most lethal diseases around the world demanding improved treatments. The tumor microenvironment (TME) is a critical driver of tumor

progression and response to therapy and mediates many of the cancer hallmarks. Collagens are major components of the TME that controls disease progression and mediate therapeutic response. Collagens have been associated with stiffening of the tumor stroma, malignancy and therapeutic resistance. Collagenopathies are caused by loss of function collagen mutations that disrupt tissue formation and are dominant negative variants. We have found somatic tumor variants in collagens that are similar loss-of-function mutations as the germline variants observed in collagenopathies, providing guidance to interpret these cancer mutations. Indeed, the role of somatic mutations in collagens has not been addressed. Remarkably, we found multiple independent lines of evidence that collagen variants impact stomach adenocarcinoma tumors, consistent with a model that collagen variants disrupt the extracellular matrix (ECM) leading to improved therapeutic responses. 1) We find that loss-of-function collagen missense and nonsense mutations are associated with improved survival and disease-free progression times in gastric tumors in the TCGA dataset. 2) The same type of mutations observed in cancer are known to cause rare human diseases and therefore have the potential to impact cell function. 3) Independent of the patient outcome data, collagen variants were associated with significant changes in expression of tumor environment factors including ECM glycoproteins and EMT expression signatures. 4) Remapping RNA-seq reads reveals that single nucleotide variants of the collagens, matching the exome sequencing data are expressed. We will present computational and functional data supporting the hypothesis that somatic variants impact gastric tumors as well as analysis of the impact of collagens somatic mutations PanCancer. Somatic mutations in collagens represent a new paradigm for how cancer cells mediate the TME providing new biomarker and therapeutic opportunities to disrupt the ECM and improve outcomes for cancer patients.

Citation Format: Alexander S. Brodsky, Jay Khurana, Dongfang Yang, Ece Gamsiz, Ian Wong, Murray Resnick. Somatic mutations in collagens impact patient outcomes in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1223.
Abstract 1224: Correlation of tumor angiogenesis with peripheral ring-enhancement on CT: Its predictive value of chemotherapy responses of colorectal metastatic hepatic tumor
Authors: Okamoto Hirotaka , Shiba Shugo , Iino Hiroshi , Sudoh Makoto , Wakana Hiroyuki , Maruyama Suguru , Kawashima Kenji , Fukasawa Toshio , Ichikawa Daisuke , Fujii Hideki
Published: August 2019

[Background]The treatment of colorectal cancer (CRC) with metastasis is now widely accepted to be systemic chemotherapy of 5-FU base chemotherapy (FOLFOX or FOLFILI) in combination with molecular antibody. The objective of this study was to determine the significance of tumor angiogenesis in metastatic hepatic tumors in relation to the response of systemic chemotherapy.

[Methods]A total of 48 consecutive CRC patients with hepatic metastasis who had been treated with or without hepatic resection were retrospectively reviewed, and factors, such as metastatic tumor angiogenesis and chemotherapy response, were analyzed. Tumor angiogenesis in association with ring-enhancement (RE) on computed tomography (CT) was also microscopically evaluated by microvessel counting (MVD) in sections stained immunochemically with CD34.

[Results]The overall response rate after 6 courses of first-line chemotherapy for the liver metastasis with RE on CT was 64% (23/36), whereas the response rate without RE was 25% (3/12), which was significantly different. The survival rate of patients with or without liver resection of all RE-positive tumors was not different, but the survival rate of patients with liver resection was longer than that of patients without liver resection for all RE-negative tumors. Microscopic examination revealed that peripheral RE on CT of the metastatic tumor was associated with tumor angiogenesis by MVD.

[Conclusions]The peripheral RE on CT of the metastatic hepatic tumor was associated with tumor angiogenesis and may predict chemotherapy response. A combination of liver resection with chemotherapy improves the survival of patients who have multiple hepatic metastasis.

Citation Format: Hirotaka Okamoto, Shugo Shiba, Hiroshi Iino, Makoto Sudoh, Hiroyuki Wakana, Suguru Maruyama, Kenji Kawashima, Toshio Fukasawa, Daisuke Ichikawa, Hideki Fujii. Correlation of tumor angiogenesis with peripheral ring-enhancement on CT: Its predictive value of chemotherapy responses of colorectal metastatic hepatic tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1224.
Abstract 1229: Neoadjuvant therapy alters collagen architecture of pancreatic cancer
Authors: Nakajima Kosei , Ino Yoshinori , Nara Sotoshi , Iwasaki Toshimitsu , Hiraoka Nobuyoshi
Published: August 2019

Treatment of pancreatic cancer remains clinically challenging and requires the novel therapeutic interventions to improve patient outcome. Neoadjuvant therapy i.e. preoperative treatments comprising chemotherapeutic agents with/without radiation offers down-staging and improvement of surgical resectability. Here, we report that neoadjuvant therapy alters collagen architecture of pancreatic cancer. Transcriptomic profiles of type 1 to type 28 collagens depicted changes of collagen composition due to neoadjuvant therapy. Immunohistochemically, the expressed area of collagen type 1, 3, 4, and 5 were significantly reduced post treatment. The bioinformatic approaches provided a comprehensive insight into treatment-induced matrix remodeling, which showed that Ephrin-A signaling and Ephrin-A5 appeared as a highly possible pathway and a crucial ligand. Ephrin-A5 co-localized with alpha-SMA(+) cancer-associated fibroblasts, whereby Ephrin-A5 have been implicated to synthesize collagens. The Ephrin-A5 positive cells significantly reduced after neoadjuvant therapy, also inversely correlated with tumor shrinkage rate. Using primary culture cells of cancer-associated fibroblasts, experimental exposure of radiation and chemotherapeutic agents suppressed the proliferation of cancer-associated fibroblasts, collagen synthesis and Ephrin-A5 expression. Thus, our studies demonstrate that neoadjuvant therapy down-regulates Ephrin-A5 expression, leading to a change of extracellular matrix structure. It should be a clinical concern of surgical handling when following resection.

Note: This abstract was not presented at the meeting.

Citation Format: Kosei Nakajima, Yoshinori Ino, Sotoshi Nara, Toshimitsu Iwasaki, Nobuyoshi Hiraoka. Neoadjuvant therapy alters collagen architecture of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1229.
Abstract 1233: Myeloperoxidase (MPO) activity in myeloid-derived suppressor cells (MDSCs): effects on anticancer drug activity
Authors: Yalowich Jack C. , Kanagasabai Ragu , Netherby Colleen S. , Messmer Michelle N. , Abrams Scott I.
Published: August 2019

In cancer, suppression of host immunity is related, in part, to immature myeloid-derived suppressor cells (MDSCs) expanded in response to various tumor- or stroma-derived factors. Here, the expression and activity of myeloperoxidase (MPO) was examined in both granulocytic (PMN) and monocytic (M) MDSC subsets recovered from the spleens of 4T1 tumor-bearing mice, as well as interferon regulatory factor-8 (IRF8) knockout mice (IRF8-/-) which develop high levels of MDSCs due to IRF8-deficiency. Phenotypically identical cells from non-tumor-bearing mice served as controls. In addition, the role of MPO in PMN-MDSCs as a determinant of DNA damage induced by the anticancer agents etoposide (VP-16), camptothecin (CPT) and parthenolide (PTL), was evaluated for potential immunomodulatory effects. MPO protein content was ~4-fold greater in PMN-MDSCs from tumor-bearing compared to non-tumor-bearing mice, while MPO specific activity was equivalent in these two populations. MPO protein content in PMN-MDSCs from IRF8-/- mice was at least comparable to levels found in tumor-bearing hosts. In both IRF8-/- and tumor-bearing mice, PMN-MDSCs contained 5-10-fold greater MPO protein and overall enzyme activity compared to isolated M-MDSCs. VP-16 (5 µM)- and PTL (100 µM)-induced DNA strand breaks were 4-fold greater in PMN- compared to M-MDSCs from tumor-bearing mice. In addition, the heme-synthesis inhibitor, succinylacetone (SA) reduced MPO activity 2-3-fold in PMN-MDSCs from tumor-bearing mice with a corresponding decrease in drug-induced DNA damage for both VP-16 and PTL with no effect on CPT-induced DNA damage. The antioxidant dehydroascorbate decreased VP-16-induced DNA damage in PMN-MDSCs containing MPO activity, but not in those cells pre-incubated with SA to deplete MPO. These results indicate that MPO-dependent activation of VP-16 and PTL enhance DNA damage. Antioxidant protection against VP-16-induced DNA damage is consistent with MPO-mediated oxidative activation of VP-16 to a redox-cycling phenoxyl radical (Mol. Pharmacol. 79: 479-487, 2011). Overall, these results suggest that: 1) PTL may be useful to eliminate PMN-MDSCs; 2) MPO activation of VP-16 in PMN-MDSCs may be deleterious via oxidative DNA damage resulting in mixed-lineage leukemia (MLL) gene rearrangements known to be causal for therapy-related myeloid leukemias. Support: CA172105; CA090787; CA140622

Citation Format: Jack C. Yalowich, Ragu Kanagasabai, Colleen S. Netherby, Michelle N. Messmer, Scott I. Abrams. Myeloperoxidase (MPO) activity in myeloid-derived suppressor cells (MDSCs): effects on anticancer drug activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1233.
Abstract 1235: Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis
Authors: Wu Xiaoqing , Gardashova Gulhumay , Lan Lan , Han Shuang , Zhong Cuncong , Gowthaman Ragul , Karanicolas John , Dixon Dan A. , Welch Danny R. , Li Ling , Ji Min , Aubé Jeffrey , Xu Liang
Published: August 2019

The majority of breast cancer-related mortality is due to metastasis. Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is overexpressed in breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors and cytokines that implicate major tumor hallmarks including invasion and metastasis. Knocking out HuR by CRISPR/CAS9 technology inhibits cell invasion in breast cancer cells. Therefore, HuR is an emerging target for breast cancer therapy, especially the lethal metastatic breast cancer. RNA-binding proteins had previously been considered “undruggable” due to lack of a well-defined binding pocket for target RNAs. Using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that inhibit HuR-mRNA interaction at nM to sub-µM potency. Our lead compound, KH-3, potently inhibits breast cancer cell growth and decreases cell invasion in vitro similar to HuR knockout, as well as increasing the expression of epithelial marker E-cadherin. In the study of mechanism of action, a transcription factor, FOXQ1, which is recently revealed to implicate in breast cancer invasion and metastasis processes, is found for the first time to be a direct mRNA target of HuR and one of the top genes that are reduced by KH-3 treatment. Exogenous introduction of FOXQ1 can rescue cell invasive capability impaired by HuR knockout and abolish the effect of KH-3 on inhibiting cell invasion in breast cancer cells. Moreover, KH-3 disrupts HuR-FOXQ1 interaction in RNP-IP, RNA pull down and FOXQ1 3′-UTR luciferase reporter assays. In vivo efficacy studies show that KH-3 not only exhibits potent antitumor efficacy in an orthotopic xenograft model of breast cancer, but also efficiently inhibits lung metastasis and improves mouse survival in an experimental metastasis model. Our data provide a proof-of-principle that HuR inhibition by KH-3 may be developed as a promising molecular therapy for inhibiting progression and metastasis of breast cancer with high HuR.

Citation Format: Xiaoqing Wu, Gulhumay Gardashova, Lan Lan, Shuang Han, Cuncong Zhong, Ragul Gowthaman, John Karanicolas, Dan A. Dixon, Danny R. Welch, Ling Li, Min Ji, Jeffrey Aubé, Liang Xu. Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1235.
Abstract 1236: Subclinical doses and choice of replication stress inducer in combination with CHK1 inhibitors for optimal tumor control and immune responses in vitro and in vivo
Authors: Oo Zay Yar , Proctor Martina , Stevenson Alex , Nazareth Deborah , Larsen Jill , Haass Nikolas , Gabrielli Brian G.
Published: August 2019

Background: Lung cancer and melanoma are responsible for almost 25% of cancer deaths in Australia. Despite recent progress with targeted and immuno-therapies, the 5 year survival rate in late stage patients is still<20%. CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress, primarily Gemcitiabine. Clinical trials of this combination have shown some good responses but high levels of normal tissue toxicity.

Materials and methods: Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as combinations with subclinical doses of hydroxyurea (HU) in melanoma and non-small cell lung cancer (NSCLC) tumor spheres and cell lines in 2% O2 conditions and xenograft models in immune compromised mice and with humanized immune systems.

Results: We report that low dose HU increased the sensitivity of >70% of both melanoma and NSCLC cell lines to CHK1 inhibitor (GDC-0575) triggered apoptosis, with complete loss of viability found with clinically achievable doses of this combination. Similar sensitivity was observed in xenograft models of both melanoma and NSCLC. We also demonstrate that a low dose of Gemcitabine combination with CHK1 inhibitor results in complete loss of proliferative potential in normal tissue, whereas normal tissue retaining proliferative potential after treatment with even high doses of hydroxyurea in combination with CHK1 inhibitor. In vivo, this translates to minimal effect on lymphocyte populations in the blood. The combination also triggers an inflammatory response involving the recruitment of macrophages, associated with increased HMGB1 nuclear staining. Immune responses were also assessed in humanized mouse models.

Discussion: These data indicate that the combination of low dose HU and CHK1 inhibitor have strong anti-cancer activity in the setting of melanoma and NSCLC cancer, and triggers an inflammatory response, and is likely to be better tolerated than current combinations with Gemcitabine. Our data suggest a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination, and this appears to include an immune response to the tumor.

Citation Format: Zay Yar Oo, Martina Proctor, Alex Stevenson, Deborah Nazareth, Jill Larsen, Nikolas Haass, Brian G. Gabrielli. Subclinical doses and choice of replication stress inducer in combination with CHK1 inhibitors for optimal tumor control and immune responses in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1236.
Abstract 123A: The critical role of interleukin-8 chemokine axis in the development of benign prostatic hyperplasia (BPH)
Authors: Smith Diandra K. , Venugopal Natasha , Terris Martha K. , Lokeshwar Bal L.
Published: August 2019

Benign prostatic hyperplasia (BPH), a non-malignant proliferative disease of the prostate, has a global incidence of over 210 million men aged 50 years or older. BPH is essentially a non-cancerous (non-malignant tumor) with significant morbidity. The severity varies from mild urinary retention to severe lower urinary tract symptoms (LUTS) accounting for greater than 50% of hospital visits for difficulty with urination, retention, and pain, and close to 40% of the prostate surgeries. BPH strongly associates with prostate volume that begins to increase in men aged 40 or higher. We investigated the role of chemokine and chemokine receptors in three immortalized cell lines and primary cultures derived from fresh BPH tissue following transurethral resection of the prostate. We analyzed the levels of chemokines and chemokine receptors secreted in the culture medium and in the mRNA from cells by qPCR, western blotting and Cytokine arrays. The epithelial identity of primary cultures was established by analyzing cytokeratin expression. Two natural products isolated from Allspice were identified that inhibit the growth of BPH cultures. A cell line identified as the BPH progenitor, a BPH intermediary cell line and a cell line with frank BPH features secreted a large excess of chemokine, predominantly CXCL-VIII or Interleukin-8 (IL-8). Unusually high amount of IL-8 was secreted by BPH progenitor cells and intermediate cells as compared to a stable normal prostate cell line (RWPE1) derived from the peripheral zone of the prostate, which did not secrete any IL-8 or express its receptor. The investigation found that expression of CXC receptors CXCR1, CXCR2 and an IL-8 inducible CXCR (CXCR7) was absent in progenitor cells and in intermediate cells, but was highly expressed in the cell line derived from BPH tissue and more significantly, in primary cultures of BPH tissue. Further, primary cultures and progenitor cells expressed high levels of immune stimulatory factors indicating PMN and monocyte infiltration leading to potential chronic inflammation of the transitional zone of the prostate. The natural products from Allspice inhibited all members of the IL-8 axis and proliferation, suggesting their potential preventative potential for BPH. These results demonstrate how a novel chemokine axis is likely to enhance pathogenesis of BPH and suggest several avenues to control the abnormal growth of prostate in aging male.

Note: This abstract was not presented at the meeting.

Citation Format: Diandra K. Smith, Natasha Venugopal, Martha K. Terris, Bal L. Lokeshwar. The critical role of interleukin-8 chemokine axis in the development of benign prostatic hyperplasia (BPH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 123A.
Abstract 1241: Characterization of a newly developed anti-TIGIT monoclonal antibody by double staining of T cells and NK cells by IHC and FACS
Authors: Qi Weimin , Chu Joseph , Luo Lily , Scoggin Tatiana , Dickinson Kim
Published: August 2019

T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory domain (TIGIT) is a newly identified surface protein expressed in regulatory, memory, naturalkiller (NK), and activated T cells. Several studies indicate that mouse TIGIT is a vital immunomodulator that can control the activities of both NK and T cells and plays an important role in transplantation tolerance and tumor immune surveillance. In this study, we generated a mouse monoclonal antibody, clone: BC41, by immunizing a recombinant human TIGIT, corresponding to an extra cellular domain. The protein was injected into BALB/c mice for three times and then the splenocytes were electro fused with mouse myeloma cells. The TIGIT-specific hybridoma cells were screened and a TIGIT-specific clone, named as BC41, was selected. The BC41 antibody was characterized by Western blot and immunohistochemistry. In immunoblots, TIGIT was able to detect a 26 kda protein corresponding to human TIGIT on over-expressed cells and native protein in human spleen lysates. Moreover, the BC41 was able to bind mouse TIGIT. FACS result showed that the BC41 antibody bond specifically to CD3/CD28+ T cells. We further analyzed the antibody binding profile by Immunohistochemistry on human T cells and NK cells by double staining. BC41 was able to bond portion of CD8 T cells (EOMES+), Treg cells (FOXP3+), PD1+ T cells and NK (CD57+) cells. Our results demonstrated that the antibody is TIGIT-specific and can be applied for detecting and analyzing tumor-infiltrating cells profile in transplantation and tumor immunotherapy.

Citation Format: Weimin Qi, Joseph Chu, Lily Luo, Tatiana Scoggin, Kim Dickinson. Characterization of a newly developed anti-TIGIT monoclonal antibody by double staining of T cells and NK cells by IHC and FACS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1241.
Abstract 1242: Examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype
Authors: Dey Sanchareeka , Kalainayakan Sarada Preeta , Ghosh Poorva
Published: August 2019

Currently there are few successful targeted therapies for non-small cell lung cancer (NSCLC). Targeting mutations in EGFR, BRAF, ALK etc. provide treatment options only for a defined subset of adenocarcinoma patients leaving majority of adenocarcinoma and squamous carcinoma patients without any options. Activating KRAS mutations are found in 15-30% of all NSCLC patients; however, existing treatment options for KRAS-mutant cancer are not effective in the subset with concomitant loss of LKB1 (kinase that phosphorylates AMPK). Several studies demonstrate the importance of mitochondrial metabolism and oxidative phosphorylation (OXPHOS) in lung tumorigenesis. Components of OXPHOS complexes and markers of mitochondrial biogenesis are highly predictive of reduced overall survival in NSCLC patients. A study using genetically engineered mouse models (GEMMs) for lung cancer (KrasLSLG12D/+Trp53-/- and KrasLSL-G12D/+Lkb1-/-) showed that along with glucose, lactate contributes to the TCA cycle. This highlights the importance of OXPHOS in these models. Also, heme is a prosthetic group for several of these OXPHOS proteins and is involved in oxygen transport and utilization. Hence, we aim to investigate unique therapeutic targets by examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype. We used LSL-KrasG12D LKB1-/- (LSL-KRASG12D; LKBloxP/loxP; LSL-Luciferase (KLLuc)) GEMM model for our study. Briefly, the mice were infected with AdenoCre virus, and sacrificed at different intervals in the progression of the tumors. For histological and immunohistochemical analysis, lung tissues were formalin fixed, paraffin embedded and sectioned (5µm thick). H&E staining showed consistent progression in tumor size over time. We will examine the time-course expression of OXPHOS, heme pathway proteins, and hemoproteins to determine the extent to which this lung tumor subtype depends on heme and respiratory proteins for its progression.

Citation Format: Sanchareeka Dey, Sarada Preeta Kalainayakan, Poorva Ghosh. Examining the role of heme and respiratory proteins in the progression of KRAS/LKB1 mutant subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1242.
Abstract 1250: Radotinib induces cell death of multiple myeloma cells
Authors: Jo Jae-Cheol , Heo Sook-Kyoung , Noh Eui-Kyu , Kim Jeong Yi , Sung Jun Young , Yu Ho-Min , Jeong Yoo Kyung , Ju Lan Jeong , Choi Yunsuk
Published: August 2019

Background: Radotinib is a medicine for the treatment of some types of cancer. It is approved in South Korea for use as a second-line treatment of chronic myeloid leukemia (CML). Its mechanism of action involves inhibition of the tyrosine kinase Bcr-Abl and of platelet-derived growth factor receptor (PDGFR). It has been little known the effects of radotinib on multiple myeloma (MM) cells.

Methods: First, we examined cytotoxicity of radotinib on MM cell lines, RPMI-8226 and MM.1S. Annexin V positive cell, caspas-3 and -9 activities, cell cycle distribution and mitochondrial membrane potential (MMP, ΔΨm) were observed by analyzed with flow cytometric analysis. And diverse signaling pathways were investigated by Western blotting in MM cells.

Results: Interestingly, radotinib caused cell death of MM cells. Radotinib induced Annexin V positive cells, and caspase pathway activation including caspase-3, -7 and -9. And its treatment remarkably decreased MMP in MM cells. As well as we observed that cytochrome c accumulated dose dependently in the cytosol of radotinib-treated RPMI-8226 and MM.1S cells. Moreover, radotinib decreased the expression of Bcl-xL and Bcl-2, and increased the expression of Bax and Bak in MM cells. Moreover, radotinib significantly suppressed MM cell growth in a xenograft animal model using RPMI-8226 cells.

Conclusion: Radotinib may play an important role as a candidate or chemosensitizer for treatment agent in MM. These data indicate that radotinib has a potential for anti-cancer therapy in MM.

Figure 1. Radotinib significantly suppressed MM cell growth in a xenograft animal model using RPMI-8226 cells.

Citation Format: Jae-Cheol Jo, Sook-Kyoung Heo, Eui-Kyu Noh, Jeong Yi Kim, Jun Young Sung, Ho-Min Yu, Yoo Kyung Jeong, Lan Jeong Ju, Yunsuk Choi. Radotinib induces cell death of multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1250.
Abstract 1252: Intracellular iron chelation by a novel compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle via the ERK-autophagy axis in EBV-positive epithelial cancers
Authors: Chiang Alan K S , Yiu Stephanie PT , Hui Kwai F. , Choi Chung K. , Kao Richard YT , Yang Dan
Published: August 2019

Pharmaceutical reactivation of lytic cycle of Epstein-Barr virus (EBV) represents a potential therapeutic strategy against EBV-associated epithelial malignancies e.g. gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC). A novel lytic inducing compound, C7, which exhibits structural similarity to Dp44mT, a known chelator of intracellular iron, is found to reactivate EBV lytic cycle in GC and NPC. This study aims to investigate the role of intracellular iron chelation by C7 and other iron chelators in lytic reactivation of EBV in GC and NPC. Testing of 6 structural analogues of C7 revealed only those which had high affinity towards transition metals could induce EBV lytic cycle. Pre-complexing C7 and iron chelators to iron prior to treatment of the cells abolished EBV lytic reactivation. Though hypoxia signaling pathway was activated, it was not the only pathway associated with EBV reactivation. Specifically, C7 and iron chelators initiated autophagy by activating extracellular signal-regulated kinase (ERK1/2) to reactivate EBV lytic cycle since autophagy and EBV lytic reactivation were abolished in cells treated with ERK1/2 blockers whilst autophagy inhibition by 3-MA and ATG5 knockdown significantly abolished EBV lytic reactivation. In summary, we discover a novel mechanism of reactivation of EBV lytic cycle through intracellular iron chelation and induction of ERK-autophagy axis in EBV-positive epithelial malignancies, raising the question whether clinically available iron chelators can be combined with existing therapeutic modalities to treat these cancers.

Note: This abstract was not presented at the meeting.

Citation Format: Alan K S Chiang, Stephanie PT Yiu, Kwai F. Hui, Chung K. Choi, Richard YT Kao, Dan Yang. Intracellular iron chelation by a novel compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle via the ERK-autophagy axis in EBV-positive epithelial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1252.
Abstract 1253: MDM2 inhibitor APG-115 synergizes with CDK4/6 inhibitors in a patient-derived xenograft model of dedifferentiated liposarcoma
Authors: Fang Douglas D. , Zhai Guoqin , Xu Chunhua , Gu Qingyang , Wang Jingwen , Zhu Saijie , Yang Dajun , Zhai Yifan
Published: August 2019

Liposarcoma is a rare but highly recurrent soft tissue neoplasm. Combination of surgery and radiotherapy is the main treatment for liposarcoma patients while chemotherapy remains experimental and limited depends on disease subtypes. Well-differentiated and dedifferentiated liposarcoma (WDLPS/DDLPS) represents ~50% of all diagnosed liposarcoma and their benefit from chemotherapy has been documented to be minimal. Concomitant amplifications of MDM2 and CDK4 are frequently found in WDLPS/DDLPS patients, suggesting that the combination with two targeted agents may worth further development for the treatment. APG-115 is a potent, selective, orally bioavailable small molecule inhibitor of MDM2-p53 protein-protein interaction and thus activates tumor suppression activity of p53 in p53 wild-type tumors. Currently, APG-115 is in clinical development as a single agent or in combination with PD-1 blockade immunotherapy for solid tumors, including sarcoma. To further explore the sensitive patient population and predictive biomarkers for APG-115, in this study, we evaluated the effect of the combined MDM2/CDK4 inhibition in p53 wild-type, MDM2 and CDK4 amplified liposarcoma using a preclinical xenograft tumor model derived from a dedifferentiated liposarcoma patient (PDX). Tumor-bearing mice were treated with MDM2 inhibitor APG-115, CDK4 inhibitors palbociclib/ribociclib, or their combinations. In the first experiment, APG-115 administered at 40 mg/kg as a single agent demonstrated antitumor activity in the DDLPS PDX model. In comparison with the single agents, the combination treatment with APG-115 and palbociclib or APG-115 and ribociclib exerted synergistic antitumor effects, resulting in T/C values of 16.9% and 27.1%, respectively (T/C value, average tumor volumes in treatment group vs. the vehicle control group). Partial tumor regression (PR) was achieved in 1/5 animals in both combination treatment groups but not in single arms. Another independent experiment with a higher dose of APG-115 (80 mg/kg) combined with palbociclib showed further improved antitumor activity with T/C value of 7.0% (2/5 PR). Collectively, our results suggest that the combination therapy of APG-115 and a CDK4 inhibitor may benefit WDLPS/DDLPS patients with p53 wild-type, and MDM2 and CDK4 amplifications clinically.

Citation Format: Douglas D. Fang, Guoqin Zhai, Chunhua Xu, Qingyang Gu, Jingwen Wang, Saijie Zhu, Dajun Yang, Yifan Zhai. MDM2 inhibitor APG-115 synergizes with CDK4/6 inhibitors in a patient-derived xenograft model of dedifferentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1253.
Abstract 1259: HYAL4: The first study on a potential molecular driver and biomarker of invasive bladder cancer
Authors: Morera Daley S. , Lahorewala Sarrah S. , Lokeshwar Soum D. , Jordan Andre R. , Lokeshwar Vinata B.
Published: August 2019

INTRODUCTION AND OBJECTIVE: The majority of morbidity and mortality in bladder cancer (BCa) patients is due to muscle invasive bladder cancer (MIBC). The prognosis of patients with MIBC could be improved by identification of novel biomarkers and therapeutic agents. Chondroitin sulfate proteoglycans are known to promote tumor growth and metastasis. However, a Chondroitinase (Chase) that degrades chondroitin sulfate has not been identified. HYAL4, a member of the glycosaminoglycan (GAG) degrading enzyme-family, potentially has Chase activity; however, HYAL4 (HY4) has not be studied in any biological system, normal or disease. In this study, we evaluated the expression of all six members of the GAG-degrading enzyme family in BCa specimens and evaluated HY4 functions in preclinical models of BCa.

METHODS: Cohort 1: 79 bladder tissues (normal (NBL) = 31; tumor (TBL) = 52); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment for metastatic disease with chemotherapy. Gene expression was measured by q-PCR in both cohorts. HY4 was either overexpressed (HY4) or knocked-down in immortalized normal urothelial (Urotsa) and 3 BCa cell lines. Transfectants were assayed for Chase activity, anchorage independent growth, motility, invasion, molecular signaling. Tumor growth and metastasis in intravesical examined in HT1376 bladder orthotopic model.

RESULTS: In cohort 1 HYAL1, HYAL4 mRNA levels were significantly (6-13-fold) elevated in TBL tissues when compared to NBL tissues (P<0.001). HY4 levels were 7-fold elevated in MIBC. HY4 levels were an independent predictor of metastasis and death due to BCa (chi-sq: 6.9; P=0.0087; chi-sq: 7.59; P=0.006). In cohort 2, high HY4 levels significantly correlated with G+C treatment failure (chi-sq: 8.7; P=0.003) with > 80% accuracy. HY4-expressing transfectants secreted HY4 and Chase activity in their conditioned media. Overexpression of HY4 in normal urothelial and BCa cells significantly increased, while its knockdown abrogated, anchorage-independent growth, invasion, chemotactic motility (> 3-fold). HY4 expression induced Gemcitabine resistance (IC50: vector: 3.1 nM; HY4: 126 nM). HY4 expression upregulated a stem cell signature. Urothelial cells expressing HY4 formed tumors in NOD/SCID mice. HY4-expressing BCa cells metastasized to lung and spleen in an orthotopic BCa model.

Citation Format: Daley S. Morera, Sarrah S. Lahorewala, Soum D. Lokeshwar, Andre R. Jordan, Vinata B. Lokeshwar. HYAL4: The first study on a potential molecular driver and biomarker of invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1259.
Abstract 126: Multidimensional gene expression models for characterizing response and metastasis in solid tumor samples
Authors: Carney Walter P. , Bhagat Milan , LaFranzo Natalie
Published: August 2019

Predicting response to standard of care therapies and new immunotherapies is enabled by tools that quantitatively analyze the complex heterogeneity of the tumor microenvironment. Current methods to do so primarily rely either on single gene or protein markers, and often report solely on one feature of the multifaceted immune cycle. Unlike other molecular approaches, the ImmunoPrismTM Immune Profiling Assay has been built using multidimensional gene expression models validated to accurately identify immune cells in a complex mixture with high correlation to flow cytometry measurements, but accessible for formalin-fixed paraffin-embedded (FFPE) tissues. We will report results showing the assay is highly sensitive, capable of detecting immune cells present to as little as 2% of the specimen, important for critical immune cell types such as M1 and M2 macrophages and Tregs. Further, the assay requires as few as two sections of FFPE solid tumor tissue or as little as 20 ng of total RNA which enables the analysis of rare clinical archives. In this study, we apply the ImmunoPrism assay to cohorts of patients with indications including non-small cell lung carcinoma (NSCLC), triple-negative breast cancer (TNBC) and pancreatic adenocarcinoma (ADC) treated with standard of care therapies. Immune profile changes for pre vs. post treatment and primary vs. metastatic tissue are reported in the context of clinical data, such as therapy response. Resulting biomarkers, including a novel machine-learning based multidimensional marker are also reported with predictive accuracy, PPV, NPV, and selection thresholds.

Citation Format: Walter P. Carney, Milan Bhagat, Natalie LaFranzo. Multidimensional gene expression models for characterizing response and metastasis in solid tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 126.
Abstract 1261: Targeting Wee1 in laryngeal squamous cell carcinoma
Authors: Yuan Meng-Ling , Jiang Qi-Wei , Yang Yang , Huang Jia-Rong , Shi Zhi
Published: August 2019

WEE1 is a tyrosine kinase that regulates G2/M cell cycle checkpoint and frequently overexpressed in various tumors. However, the expression and clinical significance of WEE1 in human laryngeal squamous cell carcinoma (LSCC) are still unknown. In this study, we found that WEE1 was highly expressed in LSCC tissues compared with adjacent normal tissues. Importantly, overexpression of WEE1 was correlated with T stages, lymph node metastasis, clinical stages and poor prognosis of LSCC patients. Furthermore, inhibition of WEE1 by MK-1775 induced cell growth inhibition, G2/M cell cycle arrest and apoptosis with the increased intracellular reactive oxygen species (ROS) levels in LSCC cells. Pretreatment with ROS scavenger N-acetyl-L-cysteine could reverse MK-1775-induced ROS accumulation and cell apoptosis in LSCC cells. MK-1775 also inhibited the growth of LSCC xenografts in nude mice. Altogether, these findings suggest that WEE1 is a potential therapeutic target in LSCC, and inhibition of WEE1 is the prospective strategy for LSCC therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Meng-Ling Yuan, Qi-Wei Jiang, Yang Yang, Jia-Rong Huang, Zhi Shi. Targeting Wee1 in laryngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1261.