bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2604: Constitutive activation of the human aryl hydrocarbon receptor in mice promotes hepatocarcinogenesis independent of its coactivator Gadd45b
Authors: Lu Peipei , Cai Xinran , Fang Zihui , Xie Wen
Published: August 2019

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or dioxin, is a potent liver cancer promoter through its sustained activation of the aryl hydrocarbon receptor (Ahr) in rodents. However, the carcinogenic effect of TCDD and AHR in humans has been controversial. It has been suggested that the inter-species difference in the carcinogenic activity of AhR is largely due to different ligand affinity in that TCDD has a 10-fold lower affinity for the human AHR compared to the mouse Ahr. It remains unclear whether the activation of human AHR is sufficient to promote hepatocellular carcinogenesis. The goal of this study is to clarify whether activation of human AHR can promote hepatocarcinogenesis. Here we reported the oncogenic activity of human AHR in promoting hepatocellular carcinogenesis. Constitutive activation of the human AHR in transgenic mice was as efficient as its mouse counterpart in promoting diethylnitrosamine (DEN)-initiated hepatocellular carcinogenesis. The growth arrest and DNA damage-inducible gene 45 β (Gadd45b), a signaling molecule inducible by external stress and UV irradiation, is highly induced upon AHR activation. Further analysis revealed that Gadd45b is a novel AHR target gene and a transcriptional coactivator of AHR. Interestingly, ablation of Gadd45b in mice did not abolish the tumor promoting effects of the human AHR. Collectively, our findings suggested that constitutive activation of human AHR was sufficient to promote hepatocarcinogenesis.

Note: This abstract was not presented at the meeting.

Citation Format: Peipei Lu, Xinran Cai, Zihui Fang, Wen Xie. Constitutive activation of the human aryl hydrocarbon receptor in mice promotes hepatocarcinogenesis independent of its coactivator Gadd45b [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 2604.
Abstract 2608: EGF signaling activates a TAZ-driven oncogenic program in glioblastoma
Authors: Gao Minling , Fu Yi , John Laterra , Ying Mingyao
Published: August 2019

Glioblastoma multiforme (GBM) is the most aggressive and lethal primary brain tumor in humans. Hyperactivation of the signaling pathway driven by the epidermal growth factor (EGF) and its receptor (EGFR) are commonly occurring in GBM. The oncogenic transcriptional program driven by EGF signaling is still incompletely understood in GBM. Here, we found that EGF induces the upregulation and nuclear translocation of TAZ, an oncogenic transcriptional co-activator. TAZ and its paralog gene YAP are commonly known as downstream mediators of the Hippo signaling pathway. TAZ has been found as a driver of mesenchymal transition in GBM cells and promotes GBM radioresistance. Our results support that EGF induces TAZ transcription and the activation of TAZ gene targets, through the signaling axis including EGFR, STAT3 and ERK. EGFRvIII is the most frequently occurring EGFR mutation in GBM causing constitutively active EGFR. We found that EGFRvIII causes EGF-independent activation of TAZ and its downstream oncogenic targets. Next, we used next-generation sequencing to establish a genome-wide map of TAZ gene targets in GBM cells after EGF stimulation. The results suggest that EGF signaling activates a TAZ-driven oncogenic program including autocrine and paracrine factors that promote GBM growth, angiogenesis, stemness and immunoescape. Moreover, we found that TAZ targeting by shRNAs or the FDA-approved drug Verteporfin effectively inhibits the TAZ-driven oncogenic program activated by EGF stimulation or the EGFRvIII mutant. TAZ targeting also inhibits GBM cell growth and xenograft formation. In contrast, enforced TAZ induces the TAZ-driven oncogenic program and promotes GBM cell growth and xenograft formation. In conclusion, our results reveal a novel TAZ-driven oncogenic program that is activated by EGF signaling in GBM. Our discoveries strongly support TAZ as a therapeutic target for GBM and likely other human cancer with hyperactivation of EGF signaling. Our preclinical studies also provide a clinically translatable strategy for GBM therapy based on using the FDA-approved drug Verteporfin.

Citation Format: Minling Gao, Yi Fu, Laterra John, Mingyao Ying. EGF signaling activates a TAZ-driven oncogenic program in glioblastoma [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 2608.
Abstract 2609: A novel MYB-AR cross-talk promoting castration-resistance in prostate cancer
Authors: Srivastava Sanjeev Kumar , Zubair Haseeb , Patel Girijesh K. , Khan Mohammad Aslam , Deshmukh Sachin Kumar , Sing Seema , Andrews Joel , Wang Bin , Carter James E. , Singh Ajay Pratap
Published: August 2019

Progression of prostate cancer from castration-sensitive to castration-resistant disease is a complex process, which likely involves multiple genetic and epigenetic alterations. Here we report a novel mechanism where MYB acts as a novel binding partner of AR enabling its ligand-independent activation to support castration resistance in prostate cancer. MYB and AR interact and co-localize with each other predominantly in the nuclei. Androgen-depletion or enzalutamide treatment does not interfere with MYB-AR interaction, and MYB-overexpressing prostate cancer cells retain AR in the nucleus even when cultured under androgen-deprived condition. Transcriptional activity of KLK3 (an androgen-responsive gene encoding PSA) promoter is increased in MYB-overexpressing cells, while sustained under androgen-depleted condition. In silico analysis identifies a MYB-binding region in KLK3 promoter in close proximity to the AR binding site, and MYB is shown to cooperatively promote AR binding to the KLK3 promoter. MYB-overexpressing prostate cancer cells exhibit greater tumorigenicity when implanted orthotopically and quickly regain growth following castration leading to the poorer survival of mice, compared to those carrying low MYB-expressing prostate tumors. Together, these findings establish a novel and significant role of MYB-AR cross-talk in prostate cancer, which could be exploited for its therapeutic management.

Citation Format: Sanjeev Kumar Srivastava, Haseeb Zubair, Girijesh K. Patel, Mohammad Aslam Khan, Sachin Kumar Deshmukh, Seema Sing, Joel Andrews, Bin Wang, James E. Carter, Ajay Pratap Singh. A novel MYB-AR cross-talk promoting castration-resistance in prostate 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 2609.
Abstract 2610: The FOXC2 transcription factor promotes growth of melanoma cells in both in vitro and in vivo settings
Authors: Williams Corey J. , Johnson Coleman E. , Bushhouse David Z. , Hargadon Kristian M.
Published: August 2019

Melanoma, a cancer derived from pigment-producing melanocytes of the skin, is a highly aggressive cancer characterized by rapid growth and metastasis. Recent evidence has emerged demonstrating that overexpression of the FOXC2 transcription factor contributes to tumor progression in many cancer types, but the role of FOXC2 in melanoma progression has not previously been studied. Using CRISPR-Cas9 gene editing technology, we have generated and characterized a novel FOXC2 knockout B16-F1 melanoma cell line (B16-F1ΔFOXC2). In comparative in vitro and in vivo studies with wild-type B16-F1 and B16-F1ΔFOXC2 melanomas, we show for the first time that the FOXC2 transcription factor is a critical regulator of melanoma cell growth. The B16-F1ΔFOXC2 melanoma cell line described herein will serve as a useful tool for investigating the function and target genes of FOXC2 in melanoma.

Citation Format: Corey J. Williams, Coleman E. Johnson, David Z. Bushhouse, Kristian M. Hargadon. The FOXC2 transcription factor promotes growth of melanoma cells in both in vitro and in vivo settings [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 2610.
Abstract 2617: FOXC2-associated regulation of melanoma cell adhesion to the extracellular matrix and lymphatic endothelial cells
Authors: Johnson Coleman E. , Williams Corey J. , Hargadon Kristian M.
Published: August 2019

The FOXC2 transcription factor has emerged as a tumor-promoting oncogene in many cancer types. We have recently investigated the impact of Foxc2 gene disruption and FOXC2 overexpression in melanoma on tumor cell adhesion to extracellular matrix (ECM) proteins and lymphatic endothelial cells. We show that melanoma-associated FOXC2 is a critical regulator of tumor cell adhesion to the ECM proteins fibrinogen and fibronectin as well as to lymphatic endothelial cells. These effects on tumor cell adhesion correlate with FOXC2-mediated regulation of the integrins ITGA5 and ITGA9. As changes in cellular adhesion frequently accompany tumor cell transformation and progression, our data suggest that FOXC2 and genes regulated by this transcription factor may play key roles in promoting melanoma invasion and metastasis.

Citation Format: Coleman E. Johnson, Corey J. Williams, Kristian M. Hargadon. FOXC2-associated regulation of melanoma cell adhesion to the extracellular matrix and lymphatic endothelial 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 2617.
Abstract 2639: Insulin receptor alternative splicing during development and cancer
Authors: Khurshid Safiya
Published: August 2019

It has been shown that alternative splicing of some genes changes during development from embryogenesis to adulthood. Intriguingly, transformation of cells is also often accompanied by a reversion to the embryonic spliced isoforms. Insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB isoform and the exon 11-skipped IN-RA isoform. The IN-RB isoform encodes a receptor with high affinity for insulin, while the IN-RA isoform has high affinity for both insulin & IGF2 growth hormones. IN-RAexploits the IGF pathway to accelerate growth, proliferation and angiogenesis. It is known that during embryogenesis the IN-R gets spliced to primarily express the INR-A form. Recently, the A isoform has has also been shown to be expressed in certain cancer types. Because the insulin receptor signaling converges on the mTOR/AKTpathway that is one of the most critical pathways for organismal development as well in cancer, it is important to understand the expression of these isoforms and their role in signaling and cellular fate. However, a tissue and a temporal description of the expression of these two isoforms is lacking. Towards this end we have shown that the mouse insulin receptor gene is spliced similarly to the human gene and have used the mouse model to study IR splicing during development. We harvested tissues from mice at different developmental stages (E10, E18, P1, P7, 3 months and 6 months) and performed RT-PCR for the insulin receptor gene. Our data confirmed that the whole embryos at embryonic day 10 (E10) express mostly IN-RA (90%). However, as the embryo develops and we can obtain discrete tissues we are able to detect tissue-specific profiles of IN-Rsplicing. For example, in bone at E18 as well as postnatal day P1 and 7, IN-RBis highly expressed (65%) and decreases to (40%) as the mouse reaches 6 months of age. Likewise, the liver and pancreas, which are primarily involved with metabolic functions, express mostly IN-RB(90%) from postnatal day P1 and into adult hood (6 months). In contrast, when we analyzed IR-splicing in human cancers, we find that in both liver cancer and cancers of the bone (osteosarcoma and Ewing’s sarcoma) there is a shift toward increased levels of the pro-proliferative isoform, IN-RA. We believe that understanding the molecular underpinnings of the splicing regulation during development will provide us with knowledge that will help us better understand the transformation process and how to control it, possibly through splicing corrective therapy.

Citation Format: Safiya Khurshid. Insulin receptor alternative splicing during development and 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 2639.
Abstract 2641: Transactivation of EGFR and mTOR pathway by H2Sin cancer cells
Authors: Higgins Jillian L. , Kraus Aileen K. , Arruda Dominic T. , Guo Hao , Lautato Mattew , Lautato Christopher , DaCunha Eliana , Chen Ashley , Yao Ricky , Yang Dongqin , Wan Yinsheng
Published: August 2019

Hydrogen sulfide regulates a variety of physiological and pathological processes. Previous studies have suggested that H2S plays a significant role as a secondary messenger in cellular signaling. Interestingly, it has been found that this unique gaseous molecule, or gasotransmitter, is capable of producing both pro- and anti- apoptotic activity in cultured cells. It has been speculated that these gasotransmitters are endogenously generated in mammalian systems through enzymatic catalysis. Our pursuits focus on comparing the effects of H2S on various lines of cancer cells (CaOV3-ovarian cancer cells, A375-melanoma cells, HeLa-cervical cancer cells, and WM 266-4-melanoma cells). Using a water-soluble, slow-releasing H2S donor, GYY4137 (200 μM), we studied how both concentration and time affect cell viability and investigated further whether H2S causes pro- or anti- apoptotic activity. Existing data have indicated that GYY4137 may activate molecular targets, including EGFR, ERK and JNK. We hypothesize that GYY4137 activates mTOR (mammalian target of rapamycin) with result of phosphorylation of S6, leading to either cell death or cell proliferation. By monitoring various proteins through Western blot analysis and confocal microscopy we studied the activation of EGFR/mTOR pathway in response to GYY4137 treatment. Our data showed that the longer the treatment the more prevalent phosphor S6 and phosphor-EGFR are. Noticeably, we observed a significant difference between 15 minutes and 30 minutes, suggesting that GYY4137 has the largest effect after 30 minutes of treatment. We further studied cell growth and viability using MTT assay to investigate the effects of various concentrations of GYY4137 on the cells. Our data indicated that the stronger the concentration is, the lower the cell viability. Similarly, we used Mitotracker ® Red dye to stain for mitochondrial activity in CaOV3 and WM 266-4 cells treated at various time points. Overall, mitochondrial activity increased when cells were exposed to GYY4137, suggesting that H2S may cause anti-apoptotic activity, and instead favor cell growth at the concentration we used. Collectively, our data suggests that the H2S donor, GYY4137 has a significant effect on cell proliferation and survival in several cancerous cells likely mediated by transactivation of EGFR and mTOR pathways.

Note: This abstract was not presented at the meeting.

Citation Format: Jillian L. Higgins, Aileen K. Kraus, Dominic T. Arruda, Hao Guo, Mattew Lautato, Christopher Lautato, Eliana DaCunha, Ashley Chen, Ricky Yao, Dongqin Yang, Yinsheng Wan. Transactivation of EGFR and mTOR pathway by H2Sin cancer 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 2641.
Abstract 2642: Antisense oligonucleotide-based induction of dominant-negative variants of STAT3 and EGFR in breast and lung cancer cells
Authors: Subramaniam Prasad , Cartegni Luca
Published: August 2019

The signal transducer and activator of transcription 3 (STAT3) is linked to multiple cancers, including breast and lung adenocarcinomas, where it is known to activate multiple oncogenic pathways and serve as a bypass mechanism in drug resistance. For example, STAT3 is known to be a driver in some basal-like Triple-negative breast cancers (TNBC) and a resistance mechanism to EGFR inhibitors in non-small cell lung cancer (NSCLC). Thus STAT3 has become an attractive target in cancer therapeutics. A naturally occurring alternative splicing variant, STAT3-beta, is truncated and lacks the C-terminal trans-activation domain. Stat3-beta can act in a dominant negative fashion. We recently demonstrated that splice-switching oligonucleotides (SSOs) can be used to induce a shift in endogenous expression from the abundant, oncogenic STAT3-alpha to the truncated dominant-negative STAT3-beta isoform. This leads to decreased cell viability in triple-negative breast cancer cells and to tumor regression in mice, as compared to full STAT3 knock-down or controls. Our aim was to test a panel of next-generation SSOs in order to evaluate their efficacy in basal-like breast cancer cells, which are typically refractory to tyrosine kinase inhibitors. Based on our previous studies, we generated a panel of optimized SSOs targeting STAT3, and assayed the extent of the STAT3 alpha-to-beta switch, at the RNA and protein level, and at their efficacy at eliciting cell apoptosis in MDA-MB-468 TNBC cells, and identified one which show a 5-10 fold improvement of activity in vitro, compared to our previous leading published compound. The optimized STAT3 SSO sequence was also tested in RTK inhibitor-resistant H1975 non-small cell lung cancer cells and was found to be effective in killing the cancer cells as compared to Stat3 knockdown. We also used a similar antisense approach to induce the expression of a dominant-negative variant of the EGFR and tested in combination with our STAT3 SSO. While both the SSO-induced dominant negative variants of EGFR or STAT3 were able to effectively kill lung cancer cells individually, we observed a marked synergistic increase in inhibition when the two compounds where used in combination.

Citation Format: Prasad Subramaniam, Luca Cartegni. Antisense oligonucleotide-based induction of dominant-negative variants of STAT3 and EGFR in breast and lung cancer 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 2642.
Abstract 265: YWHAZ amplification/overexpression defines aggressive bladder cancer and contributes to chemo-/radio-resistance via deregulation of caspase-mediated apoptosis
Authors: Sheu Jim Jinn-Chyuan , Yu Chia-Cheng , Chen I-Hsuan , Korla Praveen , Ko Grace , Chai Chee-Yin , Li Chien-Fong
Published: August 2019

The objective of this study is to characterize the oncogenic roles of a recently identified cancer-associated gene called YWHAZ (also known as 14-3-3 α/ζ ) in urothelial carcinomas of urinary bladder (UCUB). Based on a genomewide study, YWHAZ was found located at the 8q22.3 amplicon and its genetic amplification was predominantly detected in tumors at more advanced stages, muscle-invasive bladder cancer (MIBC). IHC staining confirmed the association of YWHAZ overexpression with higher tumor stages, lymph node/vascular invasion, and mitotic activity. Univariate and multivariate analyses further indicated the prognostic potential of YWHAZ for more aggressive cancer types. Both gene set enrichment analysis (GSEA) and STRING network studies suggest the involvement of YWHAZ in regulating caspase-mediated apoptosis. Ectopic expression of YWHAZ in bladder cells with low YWHAZ levels boosted cells becoming more tolerated toward chemical and ionizing radiation treatments. Gene knockdown by specific shRNA in cells with high YWHAZ levels attenuated the survival activity, resulting in cell growth suppression and cell death. Our data confirmed an essential role of YWHAZ in providing survival signals to sustain cell proliferation during chemo- or radio-therapy. Treatments based on anti-YWHAZ strategies may be beneficial for patients with YWHAZ overexpression.

Citation Format: Jim Jinn-Chyuan Sheu, Chia-Cheng Yu, I-Hsuan Chen, Praveen Korla, Grace Ko, Chee-Yin Chai, Chien-Fong Li. YWHAZ amplification/overexpression defines aggressive bladder cancer and contributes to chemo-/radio-resistance via deregulation of caspase-mediated apoptosis [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 265.
Abstract 2654: Synergistic inhibition of cancer cell viability and suppression of HIF1α by CDK4 inhibitors and HSP90 inhibitors across cancer types
Authors: Zhao Shuai , Zhou Lanlan , Dicker David T. , El-Deiry Wafik S.
Published: August 2019

Hypoxia is common and hypoxia-inducible factor 1α (HIF1) promotes various aspects of cancer, such as angiogenesis, or metastasis, and confers therapy resistance. Targeting HIF’s is a promising cancer treatment strategy. We previously showed that cyclin-dependent kinase 1 and 4 (CDK1, CDK4) stabilize HIF1α protein in cancer cells (Warfel et al., Cell Cycle, 2013). We also found that CDK activity facilitates HSP90 stabilization of HIF1α and that targeting CDK1 or CDK4/6 and HSP90 simultaneously reduces HIF1α levels and inhibits colorectal cancer (CRC) cell viability. The CDK4/6i palbociclib plus HSP90i ganetespib synergistically inhibit cell viability in HCT116 CRC cells under normoxia and hypoxia, show anti-tumor efficacy in vivo, and reduce both HIF1 and HIF2 levels. To investigate the translational potential of the combination strategy, we explored use of a HSP90i in clinical trials as no HSP90i has been FDA-approved for cancer treatment. Onalespib is a HSP90i undergoing phase I trials for solid tumors and lymphoma. and XL888 is being tested in GI cancer and melanoma. A third HSP90i, TAS116, is under investigation in solid tumors. We found that all these HSP90 inhibitors, in combination with palbociclib, reduce HIF1α levels in CRC cells. Cell viability was inhibited by the combination treatment in a synergistic manner. Similar effects were observed in pancreatic cancer, breast cancer and other tumor types upon treatment with TAS116 and palbociclib. As palbociclib is not the only FDA-approved CDK4/6 inhibitor, we examined the anti-tumor effects of HIF targeting effect using an alternative FDA-approved CDK4/6 inhibitor, abemaciclib. Abemaciclib, in combination with TAS116, decreased HIF1α levels and inhibited cancer cell viability. Our findings demonstrate a class effect towards HIF1α inhibition and cancer cell viability across multiple tumor types from the combination of a CDK4/6i and HSP90i. Ongoing experiments are examining the role of HIF in the sensitivity to the drug combination and the impact of the combined inhibitor therapy on cancer cell migration and invasion. Further clinical translation of this concept is warranted for cancer therapy.

Citation Format: Shuai Zhao, Lanlan Zhou, David T. Dicker, Wafik S. El-Deiry. Synergistic inhibition of cancer cell viability and suppression of HIF1α by CDK4 inhibitors and HSP90 inhibitors across cancer types [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 2654.
Abstract 2656: Regulation of HIF-1a by MDM2 in prostate cancer cells
Authors: Alobid Saad Ebrahim , Rathinavelu Appu
Published: August 2019

MDM2 is an oncogene amplified in approximately 40% of all human prostate carcinomas, and such amplification is correlated very well with aggressive tumor phenotype, distant metastasis, poor treatment outcomes and overall increase in mortality. Interestingly, in cancer cells, hypoxia inducible factor-1alpha (HIF-1α) is activated to facilitate adaptation to the non-conducive tumor microenvironment, through the regulation of downstream genes to ensure tumor survival and progression through modification of protective biological processes. Our previous investigations have outlined a significant role for MDM2 overexpression in regulating HIF-1α under both normoxic and hypoxic conditions in MDM2 expressing LNCaP and MDM2 transfected LNCaP-MST cells, leading to angiogenesis and other detrimental effects. Therefore, our study was aimed to identify how HIF-1α level is regulated in these prostate cancer cells, as a result of MDM2 overexpression, under both normoxic and hypoxic conditions. Analysis of mRNA levels exhibited a significant transcriptional upregulation of HIF-1α in LNCaP-MST compared to LNCaP cells under normoxic condition. Interestingly, targeting the transcriptional machinery with Actinomycin D resulted in a significant downregulation of HIF-1α protein levels also under normoxic conditions. On the other hand, inhibition of proteasomal degradation with MG-132 resulted in further elevation of HIF-1α protein levels in normoxic condition. However, under hypoxic condition, the levels of HIF-1α mRNA seem to be unchanged in both cell lines. In support of these findings, LNCaP-MST cells exhibited a significant downregulation of VHL (Von Hippel-Lindau) levels compared to LNCaP cells under hypoxic condition. The VHL protein typically facilitates polyubiquitination of HIF1α that are marked for proteasomal degradation subsequent to prolyl hydroxylation. So far, our results have confirmed a positive correlation between MDM2 expression and HIF-1α levels under normoxic conditions, while protein degradation may play a significant role during hypoxic conditions. In conclusion, our study provides new evidences that are linking MDM2 overexpression to HIF-1α protein levels in prostate cancer cells under both normoxic and hypoxic conditions, which may be important while considering targeted therapies. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

Citation Format: Saad Ebrahim Alobid, Appu Rathinavelu. Regulation of HIF-1a by MDM2 in prostate cancer 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 2656.
Abstract 2661: Targeting endoplasmic reticulum-resident proteins for the treatment of B cell cancer
Authors: Tang Chih-Hang Anthony , Valle Juan R. Del , Hu Chih-Chi Andrew
Published: August 2019

IRE-1 splices XBP-1 mRNAs, leading to activation of the functional XBP-1 transcription factor. We showed that genetic deletion of XBP-1 in chronic lymphocytic leukemia (CLL) cells decelerated malignant progression of CLL in mice. We synthesized and characterized a specific inhibitor, B-I09, which could block the RNase activity of IRE-1 with high potency and efficacy. B-I09 clearly suppressed activation of the IRE-1/XBP-1 pathway, as evidenced by the decreased mRNA and protein levels of XBP-1 in intact cells. B-I09 specifically targeted mouse CLL cells in vivo by inducing apoptosis. Because XBP-1 deficiency could compromise the BCR signaling, a crucial survival signal for CLL, we tested whether pharmacological inhibition of XBP-1 could enhance the effect of inhibitors of the BCR signaling by combining B-I09 with ibrutinib (a Bruton’s tyrosine kinase inhibitor) to treat human CLL cells. A strong pharmacological synergism was determined using the Chou-Talalay combination index method, suggesting that B-I09 could help ibrutinib to achieve higher cytotoxicity at a lower dose, addressing ibrutinib’s toxicity issue. Our studies also led us to discover that IRE-1 interacted with STING, an ER-resident protein critical for cytoplasmic DNA sensing and interferon production. We showed that the IRE-1/XBP-1 pathway was required for the interferon-producing function of STING, and that agonists of STING selectively triggered mitochondria-mediated apoptosis in malignant B cells. Upon stimulation, STING was degraded inefficiently in malignant B cells, implying that prolonged activation of STING could lead to apoptosis. In CLL-bearing mice, injection of the STING agonist, 3'3'-cGAMP, induced apoptosis and regression of leukemia. Similarly efficacious effects were elicited by 3'3'-cGAMP injection in syngeneic or immunodeficient NSG mice grafted with malignant B cells. These data suggested that STING agonists could directly eradicate CLL and other B cell malignancies in vivo. IRE-1 and STING are thus useful ER-resident protein targets for the treatment of B cell cancer.

Citation Format: Chih-Hang Anthony Tang, Juan R. Del Valle, Chih-Chi Andrew Hu. Targeting endoplasmic reticulum-resident proteins for the treatment of B cell 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 2661.
Abstract 2666: ARD1-mediated Hsp70 acetylation protects cancer cells against the cellular stress
Authors: Seo Ji Hae , Shin So-Jin , Kim Jin Young , Lee Seungmee , Chung Hyewon , Cho Chi-Heum
Published: August 2019

The 70 kDa heat shock proteins (Hsp70s) are a family of ubiquitously expressed intracellular proteins that are required for the maintenance of protein homeostasis. Hsp70 is highly induced in response to cellular stressors, including oxidative stress, hyperthermia, hypoxia and changes in pH, contributing to cellular resistance to stress-induced cell death. Most tumor cells, which live under continuous stress conditions, express elevated levels of Hsp70 to combat these harsh conditions and suppress apoptosis. During cellular stress, Hsp70 maintains protein homeostasis through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. In addition, this switch is essential for the maintenance of protein homeostasis and ultimately rescues cancer cells from stress-induced cell death. Therefore, ARD1-mediated Hsp70 acetylation is a key regulatory mechanism that enables Hsp70-expressing cancer cells to be more resistant to proteotoxic stress, and regulation of Hsp70 K77 acetylation might be helpful to cancer treatment.

Note: This abstract was not presented at the meeting.

Citation Format: Ji Hae Seo, So-Jin Shin, Jin Young Kim, Seungmee Lee, Hyewon Chung, Chi-Heum Cho. ARD1-mediated Hsp70 acetylation protects cancer cells against the cellular stress [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 2666.
Abstract 2669: A role for HOXA5 in the transcriptional response of ovarian and fallopian tube surface epithelial cells to norepinephrine
Authors: Dash Sweta , Gjyshi Anxhela , Cen Ling , Cheng Chia-Ho , Zhang Chaomei , Yoder Sean J. , Teer Jamie K. , Armaiz-Pena Guillermo N. , Monteiro Alvaro N.
Published: August 2019

Chronic stress has been suggested to play a role in tumor initiation, progression and patient survival. Studies have shown that activation of the sympathetic nervous system and increased levels of stress-associated hormones such as norepinephrine can enhance migration, invasion and growth of ovarian cancer cells. However, the extent to which stress contributes to tumor initiation remains unknown. The purpose of this study was to explore the effects of norepinephrine on the transcriptional program of immortalized normal cell lines presumed to be the origin of epithelial ovarian cancers. We treated normal immortalized ovarian (iOSE) and fallopian tube (iFTE) surface epithelial cells with 10uM norepinephrine for 15 min, 1h and 4h and generated global transcriptomic profile of treated vs untreated cells by RNA-Seq. The most significant gene expression changes (P(adj) < 0.05) were observed at the 1h time point; with an overlap of 45 differentially expressed genes in both iOSE and iFTE cells. Genes in immune-related and developmental pathways were found to be highly enriched in the norepinephrine treated samples set. By the 4h time point, the number of differentially expressed genes had increased to 313 genes from 234 at 1h in iFTE cells, while it reduced to 34 genes from 53 in iOSE cells. Additionally, the number of differentially expressed genes common to iOSE and iFTE cells had reduced to 14. Gene ontology analysis of the differentially expressed genes revealed ‘transcription factor’ to be the most significantly overrepresented class (based on FDR value) at both 1h and 4h time-points. Promoter enrichment analysis identified HOXA5, GFI1 and FOXI1 transcription factor binding sites to be highly enriched (based on Z-score and Fisher-Score) at both time points. HOXA5 ranked consistently at the top in both cell lines and at both 1h and 4h time points. These observations suggest that HOXA5 may play a central role in regulating the early response to norepinephrine in normal ovarian and fallopian tube cells. We will further assess the function of HOXA5 through ChIP-Seq and shRNA- mediated silencing.

Citation Format: Sweta Dash, Anxhela Gjyshi, Ling Cen, Chia-Ho Cheng, Chaomei Zhang, Sean J. Yoder, Jamie K. Teer, Guillermo N. Armaiz-Pena, Alvaro N. Monteiro. A role for HOXA5 in the transcriptional response of ovarian and fallopian tube surface epithelial cells to norepinephrine [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 2669.
Abstract 2678: A clonal expression biomarker improves prognostic accuracy: TRACERx lung
Authors: Biswas Dhruva , Birkbak Nicolai J. , Rosenthal Rachel , Hiley Crispin T. , Lim Emilia L. , Papp Krisztian , Krzystanek Marcin , Djureinovic Dijana , Wu Yin , Moore David A. , Skrypski Marcin , Abbosh Christopher , Bakir Maise Al , Watkins Thomas B. , Veeriah Selvaraju , Wilson Gareth A. , Jamal-Hanjani Mariam , Chinnaiyan Arul M. , Micke Patrick , Bartek Jiri , Csabai Istvan , Szallasi Zoltan , Herrero Javier , McGranahan Nicholas , Swanton Charles
Published: August 2019

At the point of cancer diagnosis, molecular biomarkers aim to stratify patients into precise disease subtypes predictive of outcome independent of standard clinical parameters such as tumour stage. Although prognostic gene expression signatures have been derived for many cancer types, seldom have they been shown to improve therapeutic decision making, limiting their clinical use. While intra-tumour transcriptomic heterogeneity (RNA-ITH) has been shown to bias existing biomarkers, efforts to control for this biological parameter have not been considered in biomarker development. Here, we analyse multi-region RNA-seq and whole-exome data for 156 tumour regions from 48 TRACERx patients to explore RNA-ITH in NSCLC. We show that chromosomal instability is a major driver of RNA-ITH, through the generation of heterogeneous copy number events within tumours, and that existing prognostic gene expression signatures are vulnerable to sampling bias. To address this issue, we develop the Outcome Risk Associated Clonal Lung Expression (ORACLE) assay, comprised of genes expressed homogeneously within individual tumours but heterogeneously between patients. These genes are enriched in modules associated with cell proliferation, such as mitosis and nucleosome assembly, that are often selected for through copy number gain events occurring early in tumour evolution. Our approach to identify “clonal” transcriptomic biomarkers in NSCLC overcomes tumour sampling bias, improves survival risk forecasting over current clinicopathological risk factors, and may be generalised to other cancer types, whilst revealing the early evolutionary selection of high risk DNA copy number events driving poor clinical outcome.

Citation Format: Dhruva Biswas, Nicolai J. Birkbak, Rachel Rosenthal, Crispin T. Hiley, Emilia L. Lim, Krisztian Papp, Marcin Krzystanek, Dijana Djureinovic, Yin Wu, David A. Moore, Marcin Skrypski, Christopher Abbosh, Maise Al Bakir, Thomas B. Watkins, Selvaraju Veeriah, Gareth A. Wilson, Mariam Jamal-Hanjani, Arul M. Chinnaiyan, Patrick Micke, Jiri Bartek, Istvan Csabai, Zoltan Szallasi, Javier Herrero, Nicholas McGranahan, Charles Swanton. A clonal expression biomarker improves prognostic accuracy: TRACERx lung [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 2678.
Abstract 268: Engineering of transgenic mice expressing human mesothelin for investigation of mesothelin-targeted therapeutics
Authors: Zhang Xianyu , Hagerty Brendan , Guerin Theresa , O'Sullivan Norene , Kozlov Serguei , Alewine Christine
Published: August 2019

Most monoclonal antibody-based therapeutics are specific for the human isoform of their targets and do not cross-react with orthologous mouse isoforms. This frequently limits pre-clinical modeling to human-derived tumors growing in immune compromised mice and precludes combination studies with immune modulating agents which rely on an active immune system for activity. Although introduction of human cDNA into mouse tumor cells will make these cells susceptible to targeting, a syngeneic mouse may reject or partially reject cells bearing the foreign transgene, impacting anti-tumor efficacy studies. Mesothelin (MSLN) is the target of many cell- and antibody-based therapies that have reached clinical trials due to its lack of expression in critical normal tissues and robust expression in many solid tumors. We have generated two distinct transgenic C57/Bl6 mouse models with compartment-limited expression of human MSLN (hMSLN) for use in pre-clinical testing of MSLN-targeted therapeutics: 1) TPO/hMSLN mice express full-length hMSLN from an insulated rat thyroid peroxidase gene promoter. Analysis by RT-PCR, immunoblot and IHC demonstrate strong thyroid-specific expression hMSLN. 2) In Nor/hMSLN mice, the hMSLN coding region was inserted as a full-length cDNA into the native mMSLN locus via a homologous recombination knock-in technique such that hMSLN is expressed in place of the mouse isoform under the control of endogenous murine MSLN transcription regulatory elements. IHC studies show expected expression of hMSLN in mouse pleura, pericardium and peritoneum. Shed hMSLN can be readily detected in the serum of both transgenic lines using ELISA, similar to human patients. Treatment of either model with MSLN-targeted immunotoxin LMB-100 at the maximum dose tolerated by non-transgenic C57/Bl6 mice resulted in no gross toxicity. Histologic analysis revealed subclinical pericarditis in Nor/hMSLN mice. No post-treatment thyroid damage was observed in TPO/hMSLN mice even upon detailed histologic examination of thyroid tissue. Syngeneic mouse pancreatic cancer cells expressing hMSLN transgene implanted orthotopically into TPO/hMSLN or Nor/hMSLN grew as well or better as those grown in non-transgenic C57/Bl6 mice. We have established two immunologically proficient transgenic mouse models expressing hMSLN that can be used for evaluating immune effect of hMSLN-targeted therapeutics as well as their on-target/off-tumor toxicities.

Citation Format: Xianyu Zhang, Brendan Hagerty, Theresa Guerin, Norene O'Sullivan, Serguei Kozlov, Christine Alewine. Engineering of transgenic mice expressing human mesothelin for investigation of mesothelin-targeted therapeutics [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 268.
Abstract 2687: Dolasynthen–a novel, homogeneous Auristatin F hydroxypropyl amide antibody-drug conjugate platform
Authors: Toader Dorin , Damelin Marc , Dirksen Anouk , Fesler Shawn P. , Collins Scott D. , Nehilla Barrett J. , Xu Jian , Xu Ling , Cattcott Kalli C. , Uttard Alex , Lee Winnie , Clardy Susan , Stevenson Cheri A. , Qin LiuLiang , Conlon Patrick R. , Kozytska Mariya V. , Chin Chen-Ni , Lee David H. , Lowinger Timothy B.
Published: August 2019

Dolasynthen is a novel, fully synthetic, structurally homogeneous platform that enables the construction of ADCs with tunable drug-to-antibody ratios (DAR), from a low of 2 to a high of 24. The resulting ADCs exhibit excellent physicochemical properties and fully homogeneous conjugates can be created through a variety of bioconjugation chemistries. Analogous to our first platform, Dolaflexin®, Dolasynthen is loaded with the proprietary payload Auristatin F hydroxypropylamide (AF-HPA) with precisely defined numbers of the cytotoxin per Dolasynthen scaffold.Studies that evaluate the tolerability and efficacy of Dolasynthen in preclinical models are described herein. ADCs containing a range of DAR values were generated following conjugation of Dolasynthen to two different monoclonal antibodies. The DAR of the ADCs was achieved by controlled reduction of native disulfide bonds in IgG1 antibodies, chromatographic fractionation, or through use of site-specific conjugation technologies. ADCs with both DAR6 and DAR12 were evaluated in vitro and also in vivo in the mouse, rat and monkey, for efficacy, tolerability and PK. Dolasynthen conjugates had excellent physicochemical properties and displayed the expected cell binding and in vitro cytotoxicities. In vivo pharmacology of Dolasynthen ADCs in in vivo xenograft models showed dose-dependent tumor growth inhibition at low mg/kg mAb doses. Tolerability in the rat at multiple doses was determined, including histopathological evaluation. Dolasynthen ADCs showed excellent PK characteristics in mouse, rat and NHP. Overall, the Dolasynthen platform appears to offer significant potential for clinical application.

Citation Format: Dorin Toader, Marc Damelin, Anouk Dirksen, Shawn P. Fesler, Scott D. Collins, Barrett J. Nehilla, Jian Xu, Ling Xu, Kalli C. Cattcott, Alex Uttard, Winnie Lee, Susan Clardy, Cheri A. Stevenson, LiuLiang Qin, Patrick R. Conlon, Mariya V. Kozytska, Chen-Ni Chin, David H. Lee, Timothy B. Lowinger. Dolasynthen–a novel, homogeneous Auristatin F hydroxypropyl amide antibody-drug conjugate platform [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 2687.
Abstract 269: DNA-PK inhibitor, M3814, as a new combination partner of Mylotarg in the treatment of acute myeloid leukemia
Authors: Zimmermann Astrid , Carr Michael , Zenke Frank T. , Blaukat Andree , Vassilev Lyubomir T.
Published: August 2019

Despite significant advances in the development of new treatments for acute myeloid leukemia (AML) the therapeutic success remains relatively poor. There remains an urgent need for improved therapies with increased tumor potency and selectivity. Mylotarg is the first AML drug from a new generation of antibody drug conjugate (ADC) therapies targeting the acute leukemia cell compartment with increased specificity. This agent targets leukemia cells for apoptosis with a cytotoxic warhead, calicheamicin, carried by a CD33-specific antibody. Calicheamicin induces DNA double strand breaks (DSB) which, if left unrepaired, lead to cell cycle arrest and apoptosis in cancer cells. However, repair of DSBs by the non-homologous end joining pathway driven by DNA-PK (DNA-dependent protein kinase) can reduce the efficacy of calicheamicin. M3814 is a novel, potent and selective inhibitor of DNA-PK protein kinase. This compound effectively blocks DSB repair, strongly potentiates the antitumor activity of ionizing radiation and DSB-inducing chemotherapeutics and is currently under clinical investigation. Suppressing DSB repair with M3814 synergistically enhanced the apoptotic activity of calicheamicin in cultured AML cells. Combination of M3814 with Mylotarg in two AML xenograft models, MV4-11 and HL-60, demonstrated increased efficacy and significantly improved survival benefit without elevated body weight loss. Our results support a new application for pharmacological DNA-PK inhibitors as enhancers of Mylotarg efficacy and a potential new combination treatment option for AML patients.

Citation Format: Astrid Zimmermann, Michael Carr, Frank T. Zenke, Andree Blaukat, Lyubomir T. Vassilev. DNA-PK inhibitor, M3814, as a new combination partner of Mylotarg in the treatment of acute myeloid leukemia [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 269.
Abstract 2692: Preclinical development of a novel, highly selective PI3Kδ inhibitor, IOA-244, for the treatment of solid malignancies
Authors: Ewings Katherine , MacQueen Amy , Shah Pritom , Tsapara Anna , Papakonstanti Evangelia , Veen Lars van der , Lahn Michael , Johnson Zoe
Published: August 2019

The Class I Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) family is an attractive target class for the treatment of hematological and solid malignancies. In addition to the direct effects of PI3Kδ inhibition on B cell malignancies, inhibiting PI3Kδ preferentially targets regulatory T cells and myeloid derived suppressor cells, thus breaking tumor-induced immune tolerance and restoring anti-tumor immunity. To date, the development of selective inhibitors of the Class I PI3K family has been hampered by the lack of isoform specificity as well as safety issues. IOA-244 is a novel, highly selective, orally bioavailable PI3Kδ inhibitor, with the unique property of being ATP non-competitive; these characteristics make IOA-244 the ideal drug to explore the hypothesis that PI3Kδ inhibition can modulate anti-tumor immunity as a monotherapy and in combination in clinical trials.

In mouse models, IOA-244 inhibited tumor growth when combined with either anti-PD-1 or anti-PD-L1. In the CT26 model the composition of the tumour infiltrating lymphocytes showed a marked decrease in the suppressor cell populations, i.e. Tregs and MDSCs, as well as tumor associated macrophages (TAMs). Conversely a concomitant increase in natural killer cells as well as the ratio of cytotoxic CD8+ T cells: Treg cells was observed. Furthermore, in vitro studies using primary human T cells demonstrated a selective and concentration-dependent suppression of Treg cells by IOA-244 whilst leaving CD8+ T cell proliferation intact.

In addition to effects on the immune system, we are exploring the potential of IOA-244 to mediate direct anti-cancer effects on solid tumors with a high expression of PIK3CD. IOA-244 induced a concentration-dependent inhibition of p-AKT in MDA-MB-231 cells. Furthermore, we demonstrate strong anti-proliferative activity by IOA-244 on hepatocellular tumor lines in vitro. Activity in these and other cell and patient derived lines is being studied, both in xenograft in vivo studies and in vitro models.

Based on GLP toxicology studies, IOA-244 has the potential to be a best-in-class PI3Kδ inhibitor with a safety and pharmacokinetic profile that is amenable for use alone and in combination with immunotherapies for the treatment of solid malignancies. IOA-244 is poised to enter phase I clinical testing in selected cancer indications in the first half of 2019.

Citation Format: Katherine Ewings, Amy MacQueen, Pritom Shah, Anna Tsapara, Evangelia Papakonstanti, Lars van der Veen, Michael Lahn, Zoe Johnson. Preclinical development of a novel, highly selective PI3Kδ inhibitor, IOA-244, for the treatment of solid malignancies [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 2692.