bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6310: Ex vivo phosphoflow cytometry to capture fast changes of phospho-proteins at single cell level
Authors: Zhang Qiyao , Wang Jingjing , Ge Yun , Xiong Yajie , Qin Beibei , Gu Qingyang , Ji Qunsheng
Published: September 2020

BACKGROUND: Protein phosphorylation is an important cellular regulatory process and a key indicator of signaling activation or deactivation. However, the fast switch-on/off of the process makes difficult the timely and accurate detection of the phosphorylation status. It is even more challenging to monitor the protein phosphorylation during cancer immune response in vivo, whereas immune responses vary among different cell types and can sometimes only be detected in certain rare populations like Treg (T regulatory) and TCM (T central memory) cells. The ability of Phosphoflow cytometry to accurately and rapidly detect many phospho-protein targets simultaneously at single cell level provides significant advantages over conventional methods. Ex vivo phosphoflow cytometry is increasingly being used as a tool for the discovery of biomarkers used in monitoring of disease and therapy progress.

RESULTS: We established an ex vivo phosphoflow system to evaluate fast changes of pStat5 at single cell level in mouse models. Method validation was carried out step by step to ensure the reproducibility of this assay. The method development included optimizing gating strategy for non-phosphorylated proteins, selecting the reagents and optimizing the procedures of Lyse/Fix, perm, setting up positive controls under in vitro and ex vivo conditions, and optimizing phosphorylated protein staining and detection. Our data showed that the test articles induced differential pStat5 expression in different cell types, and in a dose-dependent manner.

SUMMARY: We have established reliable ex vivo phosphoflow method to detect the fast changes of phospho-proteins at single cell level in animal models. This powerful method will enable us to examine protein post-translational modification in complicated host systems.

Citation Format: Qiyao Zhang, Jingjing Wang, Yun Ge, Yajie Xiong, Beibei Qin, Qingyang Gu, Qunsheng Ji. Ex vivo phosphoflow cytometry to capture fast changes of phospho-proteins at single cell level [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6310.
Abstract 6323: Identification of genomic alterations related to treatment progression in RWD
Authors: Chaudhary Nayan , Luhn Patricia , Jansen Gunther , Metcalfe Ciara , Hafner Marc
Published: September 2020

Real-world data (RWD) are potentially a highly valuable resource to accelerate drug discovery and improve clinical practice. In particular, datasets combining clinical outcomes and comprehensive genomic profiling (CGP) could uncover new mechanisms of resistance and identify new patient populations for targeted therapies. Here, we used a de-identified clinico-genomic database (CGDB) from routine clinical care based on a clinical EHR-derived data from Flatiron Health linked to tumor sequencing results from Foundation Medicine to assess how prevalence of genomic alterations changes following progression on targeted therapies in the metastatic setting.

One of the main challenges we faced is the lack of systematic and longitudinal genomic testing: among the patients receiving a specific treatment, some had a CGP performed prior to treatment, others only around the end of the line of therapy, but only a handful had multiple tests. We therefore established a methodology to assess changes in alteration prevalence and tested how our results are sensitive to cohort building parameters such as timing of testing, duration of therapy, or prior lines of treatment. Our approach successfully recovered three established cases of therapeutic resistance: (1) EGFR T790M in non-small cell lung cancer patients treated with erlotinib or gefitinib; (2) AR alterations following hormonal therapies in prostate cancers; and (3) ESR1 mutations in hormone-receptor positive breast cancer patients progressing on endocrine therapies other than fulvestrant. We delved into the latter case and identified differences in the type of ESR1 mutation that occurred following endocrine therapies if CDK4/6 inhibitors were co-administered or not.

On one hand, RWD lack randomization and can thus be subject to confounding variables but on the other hand, RWD have the advantage of including a larger number of patients in a real-world setting compared to clinical trials. Our analysis shows that real-world CGDB can recapitulate the expected emergence of mutations following specific lines of treatment. Our study is a proof of concept of usage of RWD to assess how prevalence of genomic alterations changes following treatment. Our method allowed us to assess differences between patient cohorts and generate novel hypotheses on treatment-related changes in genomic profiles, which can ultimately lead to new drug indications.

Citation Format: Nayan Chaudhary, Patricia Luhn, Gunther Jansen, Ciara Metcalfe, Marc Hafner. Identification of genomic alterations related to treatment progression in RWD [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6323.
Abstract 6327: Artesunate reduces tumor growth and induces different kinds of cell death in docetaxel-resistant prostate carcinoma cells
Authors: Braeunig Vitus , Schupp Patricia , Markowitsch Sascha , Vakhrusheva Olesya , Erb Holger , Efferth Thomas , Haferkamp Axel , Juengel Eva
Published: September 2020

Introduction: Prostate carcinoma (PCa) is the most common cancer in men. The therapeutic effect of approved compounds, such as docetaxel, is limited due to the development of therapy resistance, making new treatment options essential. Artesunate (ART), used in Traditional Chinese Medicine, has shown anti-tumor activity in several tumor types. However, little is known about the efficacy of ART on therapy-resistant PCa. Therefore, the impact of ART on docetaxel-resistant PCa cells was investigated.

Material and Methods: Parental (=sensitive) and docetaxel-resistant PCa cell lines, PC3, DU145, and LNCaP, were exposed to ART [1-100 µM] for 24, 48, or 72 hours. Cells not exposed to ART served as controls. Tumor cell growth, proliferation, cell cycling, and the expression of cell cycle regulating proteins were assessed. Apoptotic, ferroptotic, and necrotic effects were also evaluated.

Results: Exposure to ART significantly reduced tumor cell growth and proliferation of parental and docetaxel-resistant PCa cells in a time- and dose-dependent manner. Cell cycle arrest in the G0/G1 phase after 48h exposure to 37.5 µM ART was observed in parental LNCaP and in all three resistant cell lines. The observed changes in cell growth and cell cycle phases were accompanied by marked modulation of cell cycle regulating proteins. Significant apoptotic effects were observed in parental PC3 and both parental and resistant LNCaP, whereas ferroptosis was apparent in parental and resistant DU145 after exposure to ART. Necrotic events were not detectable in the PCa cells after ART exposure.

Conclusion: ART induced anti-tumor effects, though differing according to the cell line, were apparent in not only parental but also in docetaxel-resistant PCa cells. Thus, adding ART to standard therapies might be a promising treatment strategy for patients with advanced prostate cancer. Further investigations are necessary to verify our findings.

Citation Format: Vitus Braeunig, Patricia Schupp, Sascha Markowitsch, Olesya Vakhrusheva, Holger Erb, Thomas Efferth, Axel Haferkamp, Eva Juengel. Artesunate reduces tumor growth and induces different kinds of cell death in docetaxel-resistant prostate carcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6327.
Abstract 6349: A subgroup of potent CB002 xanthine derivatives define a novel class of anti-cancer drugs that restore the p53 pathway in tumors with mutated p53 and target an S-phase checkpoint
Authors: Borrero Liz Janice Hernandez , Dicker David T. , El-Deiry Wafik S.
Published: September 2020

Tumor suppressor TP53 is a transcription factor found to be mutated in over 50% of human tumors. Unique to other tumor suppressors, mutations in TP53 result in a stable protein that acquires a gain-of-function activity leading to an aggressive tumor with more metastatic potential and resistance to chemotherapy. The TP53 gene codes for the p53 protein which controls cell survival fate depending on the type of cellular stress signals. A well-known DNA damage stress response signaling pathway involves the activation of ATR/ATM and phosphorylation of Chk1/Chk2 leading to p53 activation and cell cycle arrest. As of now, there are no FDA approved drugs that restore the p53-pathway in any tumors. We previously discovered the parental compound CB002 along with its analogs that are capable of restoring the p53-pathway in tumors with mutated p53. These compounds are xanthine analogs but unlike known xanthines such as caffeine, pentoxifylline and theophylline, they restore the p53 pathway, including upregulation of Noxa, but do not deregulate the G2 checkpoint. Rather, 24 hr treatment in DLD-1 and SW480 cells, shows that our analogs induce p53 transcriptional targets whereas caffeine, pentoxifylline and theophylline do not. In addition, our cell cycle analysis data in synchronized SW480 cells treated with etoposide suggests that CB002 and its analogs perturb the S-phase of the cell cycle and do not deregulate the G2 checkpoint. This is also evidenced by increased p-RPA/RPA2 and cyclin E, and decreased p-histone 3 protein expression. Our microarray data indicates that our p53-pathway restoring xanthine analogs downregulate essential proteins of the DNA synthesis and repair machinery. We are currently investigating the involvement of key kinases and transcriptional factors regulated in S-phase following treatment with CB002 and its more potent analogs. Our data also suggests the activation of ATR in response to CB002 analog treatment in SW480 cells at 24 hrs. We are evaluating the benefit of synthetic lethality of these analogs in BRCA-deficient cell lines, focusing on target identification, determining the proteomic changes and in vivo efficacy of our p53-pathway restoring CB002 xanthine analogs. Taken together, our data suggests that CB002 and its potent xanthine analogs may provide a unique therapeutic strategy that can be clinically translated.

Citation Format: Liz Janice Hernandez Borrero, David T. Dicker, Wafik S. El-Deiry. A subgroup of potent CB002 xanthine derivatives define a novel class of anti-cancer drugs that restore the p53 pathway in tumors with mutated p53 and target an S-phase checkpoint [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6349.
Abstract 6351: A novel NSC small molecule inhibitor, inhibits proliferation and invasion of triple-negative breast cancer cells
Authors: Yousefi Hassan
Published: September 2020

Treatment of patients with Triple-negative breast cancer (TNBC) has been challenging due to the absence of well-defined molecular targets and high invasive and proliferative capacities of these cells. Current treatments against TNBC have shown minimal activity due to the high recurrence rate in the patients. There is, therefore, a pressing need for novel and efficacious therapies against TNBC. Here, we found a novel small molecule inhibitor with potent anti-tumor activity against TNBC cells. Anti-proliferative effects of NSC small molecule inhibitor were determined using 2D and 3D culture cell proliferation. Propium iodide staining was performed to analyze cell cycle distribution and Annexin/PI assay was carried out to evaluate the induction of apoptosis. Western blot analyses, proteomic and gene array techniques were applied to elucidate the molecular mechanisms underlying the anti-tumor activity of the small molecule inhibitor. Our data show that our NSC small molecule inhibitor, reduced the proliferation of a panel of breast cancer cells, particularly, TNBC cells through the induction of apoptosis but not cell cycle arrest. Proteomic and gene array analysis implicated a myriad of canonical signaling pathways including cell death, and cell survival. Furthermore, we found that treatment with NSC small molecule inhibitor impaired TNBC cell migration and invasion via expression reduction of the epithelial-to-mesenchymal transition (EMT) markers. In addition, we found that NSC small molecule inhibitor decreased TNBC cell proliferation and clonogenic survival and induced apoptotic cell death through inhibition of the anti-apoptotic proteins. Altogether, our data indicate that NSC small molecule inhibitor may exhibit anti-tumor activity in TNBC cells and provide a rationale for further investigation of the potential of NSC small molecule inhibitor as an attractive therapeutic drug for TNBC. We are currently in the process of validating our proteomic data. Further in-depth investigations are required to evaluate the exact targets and mechanism of this potent small molecule inhibitor, and its anti-neoplasm effects on TNBC in vivo.

Citation Format: Hassan Yousefi. A novel NSC small molecule inhibitor, inhibits proliferation and invasion of triple-negative breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6351.
Abstract 6361: Identification of mitochondrial RNA polymerase inhibitors via deep convolutional neural networks: A therapeutic strategy for cancer metabolism
Authors: Khalid Noor-Ul-Ain , Warrington Jeffrey M. , Laposa Rebecca Rachelle
Published: September 2020

Dysregulated metabolism is a hallmark of cancer and a substantial subset of many cancers rely on oxidative phosphorylation (OXPHOS) for growth and viability, including AML, breast cancer, lung cancer, glioblastoma, ovarian and pancreatic cancer. The electron transport chain (ETC) in mitochondria contains both nuclear DNA- and mitochondrial DNA-encoded subunits. Mitochondrial DNA-encoded subunits are essential for ETC function and are transcribed by the mitochondrial RNA polymerase POLRMT. We have previously validated POLRMT as an anticancer target in acute myeloid leukemia models. The challenge of inhibiting POLRMT with small molecules will arise from both a) selectivity to off targets and b) transport of the drug to the mitochondria. There exist several small nucleoside / nucleotide analogues that are known to inhibit RNA polymerase. However, this target space is rife with the opportunity for off-target activities and toxicity. Therefore, we targeted a novel pocket with the aim of finding novel chemical matter. Using AtomNet, an artificial intelligence platform that utilizes deep convolutional neural networks to predict small molecule recognition, a virtual library of 8 million compounds was screened against POLRMT. The top 72 hit compounds were tested in OCI-AML2 human acute myeloid leukemia cells for a potential decrease of mitochondrial RNA levels (MT-ND1) using reverse transcriptase polymerase chain reaction (RT-PCR). OCI-AML2 cells were treated with compounds at 0.1, 1 or 10 micromolar for 72 hours, 3 days or 6 days or with the positive control POLRMT ribonucleoside analogue inhibitor, 4'-azidocytidine. Gene expression was normalized to 18S. Twelve compounds demonstrated a time-dependent decrease in mitochondrial gene expression. In summary, these data indicate that functional inhibitors of human POLRMT can be identified rapidly via deep convolutional neural networks and machine learning techniques. Financial relationships: RL received an Artificial Intelligence Molecular Screening Award from Atomwise, and funding from the Centre for Collaborative Drug Research, Toronto, Canada. JW is an employee of Atomwise.

Citation Format: Noor-Ul-Ain Khalid, Jeffrey M. Warrington, Rebecca Rachelle Laposa. Identification of mitochondrial RNA polymerase inhibitors via deep convolutional neural networks: A therapeutic strategy for cancer metabolism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6361.
Abstract 6377: Curcumin analog GO-Y030 diminishes cancer stem cell population by inhibiting interaction of HSP70 with its substrates
Authors: Suzuki Maya , Yamamoto Yohei , Nishijima Aki , Shibata Hiroyuki , Omori Yasufumi
Published: September 2020

It has been established that a cancer tissue contains, in general, a small subpopulation of cancer stem cells (CSCs) having tumor initiation capacity. Because CSCs are resistant to chemoradiotherapy and play important roles in cancer relapse and metastasis, strategies targeting CSCs are crucial for effective anticancer therapies. Curcumin (diferuloylmethane) isolated from the rhizome of the plant turmeric (Curcuma longa) is one of the most widely characterized phytochemicals and well known to suppress cancer cell proliferation, invasion, angiogenesis, and metastasis by regulating multiple cell signaling pathways. However, its bioavailability is limited due to poor absorption. To improve this disadvantage, Ohori et al. (Mol Cancer Ther 2006;5:2563-71.) synthesized a new curcumin analog GO-Y030 (1,5-bis(3,5-bis(methoxymethoxy)phenyl)-1,4-pentadiene-3-one), which has an about 30-fold greater growth-inhibitory effect than curcumin. In the current study, we verified the potency of GO-Y030 to act against CSC population. We first discovered that GO-Y030 could dose-dependently suppress a sphere-forming ability in human HuH7 hepatocellular carcinoma and human PC3 prostatic carcinoma cell lines. Interestingly, the specific inhibitor of the stress-inducible heat shock protein (HSP) 70 (also called HSP72, HSP70-1, or HSPA1A) exhibited effects similar to GO-Y030, i.e., inhibition of CSC sphere formation and upregulation of HSP70 and HSP40 protein expression. As this result suggested that target molecules of GO-Y030 might be HSP70 and its co-chaperones such as HSP40, we next performed in vitro HSP70/HSP40-mediated refolding activity assay and revealed that GO-Y030 efficiently inhibited HSP70/HSP40 chaperone activity. Finally, performing the substrate-binding assay, we have proven that GO-Y030 reduces the substrate-recognition and binding activity of HSP70. HSPs are molecular chaperones that function to prevent denaturation or unfolding of client proteins when they are threatened by stress or high temperature. Since CSCs by nature adapt to oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress by enhancing synthesis of protein-folding chaperones, the function of HSP70/HSP40 is important for the maintenance of CSC population. Our data suggest that GO-Y030 should impair stress tolerance in CSCs by inhibiting the interaction of HSP70 with substrate proteins and disrupting the function of HSP70/HSP40 and thus should finally contribute to reduction of CSC population.

Citation Format: Maya Suzuki, Yohei Yamamoto, Aki Nishijima, Hiroyuki Shibata, Yasufumi Omori. Curcumin analog GO-Y030 diminishes cancer stem cell population by inhibiting interaction of HSP70 with its substrates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6377.
Abstract 6383: HTBPI inhibits liver tumorigenesis by combined inhibition of Akt
Authors: Yu Haiyang , Liu Hongwei , Chen Qian , Pang Xu , Yin Shuangshuang , Wang Kailong , Wang Rui , Wang Tao , Qiu Yuling
Published: September 2020

Akt, a crucial protein involved in a variety of signaling pathways in cancer, acts as an important regulator of cell survival in hepatocellular carcinoma (HCC), and provides curative option for the related drug development. We have found an active phenanthroindolizidine alkaloid, (13aR,14R)-9,11,12,13,13a,14-hexahydro-3,6,7- trimethoxydibenzo[f,h]pyrrolo[1,2-b]isoquinolin-14-ol (HTBPI), which is a promising Akt inhibitor effective in the suppression of HCC cells proliferation through stimulating apoptotic and autophagic capability in vivo and in vitro. Treatment of HTBPI combined with a classical autophagy-lysosomal inhibitor (bafilomycin A1), could enhance stimulation effects of apoptosis on HCC cell lines. In addition, we confirmed that HTBPI, targeting Akt, occupied the kinase binding domain (Thr 308) of Akt to inactivate its function by CETSA and DARTS assay. Moreover, HTBPI showed a potential therapeutic effect in patient-derived xenograft (PDX) tumors without observed toxicity. Furthermore, high p-Akt (Thr 308) expression was collated with liver tumor formation and poor survival in HCC patients. In conclusions, HTBPI impeded HCC progress through regulation of apoptosis and autophagy machinery via interaction with p-Akt (Thr 308). This may provide potential molecular candidate by inhibiting Akt for the therapy of HCC patients.

Citation Format: Haiyang Yu, Hongwei Liu, Qian Chen, Xu Pang, Shuangshuang Yin, Kailong Wang, Rui Wang, Tao Wang, Yuling Qiu. HTBPI inhibits liver tumorigenesis by combined inhibition of Akt [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6383.
Abstract 6386: Combined PDE4 and VEGFR2 inhibition as a new preclinical rationale in clear cell renal cell carcinoma
Authors: Cao Minghua , Nawalaniec Karol , Luo Yueming , Moench Romana , Hsiao Li-Li , Waaga-Gasser Ana Maria
Published: September 2020

Clear cell renal cell carcinoma (ccRCC) is the most lethal form of kidney cancer. Small molecule VEGFR inhibitors are widely used but are not curative and various resistance mechanisms have been described. Previous studies have indicated cross-talk between the cAMP pathway and the MAPK pathway, and the elevation of cAMP levels by inhibition of Phosphodiesterase-4 (PDE4) activities can result in growth arrest and/or cell death. In this study we focused on the effects of PDE4 in ccRCC. We first analyzed the expression of PDE4 in human RCC cell lines and found PDE4D to be dominantly expressed. PDE4D CRISPR Knockout (KO) was then performed on Caki-1 RCC cells and normal renal proximal tubular epithelial cells as control. Cell proliferation, cell cycle cAMP, MAPK plus PI3K/AKT signaling pathways were additionally analyzed to define the effects of PDE4D inhibition. PDE4D KO Caki-1 cells showed decreased cell viability and a higher apoptosis rate compared with wild type controls. Increased intracellular cAMP levels in PDE4D KO resulted in downregulated MAPK but not PI3K/AKT signaling in Caki-1 tumor cells. In addition, PDE4D KO Caki-1 cells demonstrated higher sensitivity to VEGFR inhibitors. To conclude, we provide a new preclinical rationale for dual PDE4/VEGFR2 inhibition in patients with ccRCC. While the MAPK signaling pathway is activated in ccRCC, dual inhibitors may improve the anti-tumor effects in a patient subset with evidence of MAPK involvement. Future work involving in vivo models will be useful to better define efficacy of this dual strategy of anti-tumor activity.

Citation Format: Minghua Cao, Karol Nawalaniec, Yueming Luo, Romana Moench, Li-Li Hsiao, Ana Maria Waaga-Gasser. Combined PDE4 and VEGFR2 inhibition as a new preclinical rationale in clear cell renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6386.
Abstract 639: Acute promyelocytic leukemia
Authors: Chale Ravinder Singh , Jimenez Joaquin J.
Published: September 2020

Introduction: Acute Promyelocytic Leukemia (APL) accounts for 10%-15% of all acute myeloid leukemia's. The use of All-Trans Retinoic Acid/ Arsenic trioxide (ATRA/ATO) in the treatment regimens of low to intermediate-risk patients has led to remarkable cure rates (~80%). However, treatment guidelines of high-risk patients (WBC count≥ 10 x 109/L) remain unclear and these patients are at an increased risk of relapse.

Design: Due to ambiguous treatment guidelines for high-risk APL patients as well as limited options for patients with arsenic resistant disease we sought to analyze clinical outcomes in this subset of patients. We conducted a literature search through Pubmed, EBSCO, and JSTOR to identify original articles that evaluated treatment protocols as well as outcomes for high-risk/refractory APL patients. We also evaluated the literature for clinical data involving patients who had arsenic resistant APL. Only relevant articles from 2000 to 2019 were included. Clinical trials where patients did not receive ATRA/ATO treatment were excluded.

High risk patients have an increased mortality and decreased overall survival compared to intermediate and low risk patients. Although early death resulting from bleeding complications remains an important clinical hindrance, cases of arsenic resistant disease are becoming more prevalent. Resistant/refractory APL is a rare occurrence; however, no effective treatment exists to overcome resistance in this subset of patients. Of particular interest is the identification of variant translocations in APL; those that differ from the classical PML-RARA (~95%). 12 non-characteristic rearrangements have so far been described; of these, the ZBTB16-RARA and STAT5b-RARA are the most common variants. Both ZBTB16-RARA & STAT5b-RARA are insensitive to ATRA/ATO combination therapy, leaving these patients with limited options. Clinical outcomes have recently been described in a subset of 13 patients with arsenic resistant disease. Of these 13 patients with arsenic resistant disease, 11 eventually died. Mutations in a particular region of the PML protein have been shown to mediate arsenic resistant disease in both in vitro and in vivo models, suggesting a potential benefit of molecular screening to improve clinical outcomes. Neither ATO nor cytotoxic chemotherapy was successful in inducing remissions in this particular subset of patients.

Conclusion: Well-defined treatment guidelines in high-risk APL and the need for alternative agents in patients resistant to ATO therapy are needed to improve clinical efficacy and outcomes.

1. Zhu, H.H.; Qin, Y.Z.; Huang, X.J. Resistance to arsenic therapy in acute promyelocytic leukemia. N Engl J Med 2014, 370, 1864-1866, doi:10.1056/NEJMc1316382.

Citation Format: Ravinder Singh Chale, Joaquin J. Jimenez. Acute promyelocytic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 639.
Abstract 6393: Targeting the increased endocytosis of Na/K ATPase α1 subunit as a new therapeutic approach to prostate cancer
Authors: Banerjee Moumita , Li Zhichuan , Gao Yingnyu , Huang Minqi , Cai Liquan , Gao Tianyan , Zijian Xie
Published: September 2020

Prostate cancer (PCa) is the second leading cause of cancer-related death in males and there is no effective therapeutics to treat aggressive metastatic PCa (~70% mortality rate). We previously reported that the α1 Na/K-ATPase (α1 NKA), the product of ATP1A1 gene, is a tumor suppressor protein whose expression was significantly reduced in primary tumor and further reduced in the bone metastatic lesions (≤10% of normal tissue). Here we show that loss of cell surface expression of ATP1A1 induces Epithelial Mesenchymal Transition (EMT), and promotes metastatic potential and tumor growth of PCa by decreasing E-cadherin expression and upregulating c-Myc expression via Src-dependent signaling. The progressive loss of α1 NKA in PCa is because of increased endocytosis by activating the α1 NKA/Src receptor complex. Using a unique α1 NKA screening platform, we have discovered MB5, an inverse agonist of receptor α1 NKA that can block ouabain, an agonist-induced signaling and endocytosis of α1 NKA. Functionally, MB5 treatment increased the expression of α1 NKA and E cadherin-which reversed EMT and decreased the invasion and growth of tumor xenografts. Thus, we have revealed a hitherto unrecognized mechanism that regulates the EMT and invasiveness, and identified α1 NKA inverse agonists such as MB5 as a potential drug candidate for the treatment of aggressive PCa.

Citation Format: Moumita Banerjee, Zhichuan Li, Yingnyu Gao, Minqi Huang, Liquan Cai, Tianyan Gao, Xie Zijian. Targeting the increased endocytosis of Na/K ATPase α1 subunit as a new therapeutic approach to prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6393.
Abstract 6394: Combined FLT3 and PARP inhibition induces synthetic lethality in non-small cell lung cancer cells
Authors: Ryu Hwani , Kim Hyo Jeong , Song Jie-Young , Hwang Sang-Gu , Ahn Jiyeon
Published: September 2020

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Fms-like receptor tyrosine kinase 3 (FLT3), a member of class III receptor tyrosine kinase, targeted inhibitors have not been well studied in lung cancer including NSCLC. Since recent studies have been reported that FLT3 inhibitors can suppress DNA repair activities, we investigated the antitumor activities of quizartinib, an FLT3 inhibitor, in NSCLC cells to expand the use of FLT3 inhibitors. Quizartinib inhibited the growth of NSCLC cells in a concentration-dependent manner, and induced DNA damage and reactive oxygen species (ROS) generation in A549 and H1299 cells. We found that quizartinib suppressed DNA damage repair genes through the downregulation of STAT5, resulting in the ‘BRCAness' or ‘DNA-PKness' phenotype in A549 and H1299 cells. As quizartinib-induced ‘BRCAness/DNA-PKness' and poly(ADP-ribose) polymerase inhibitors (PARP) inhibition can induce synthetic lethality, combined treatment with quizartinib and olaparib, a PARP inhibitor, showed synergistic inhibitory proliferation and apoptotic cell death in A549 and H1299 cells compared with either drug alone. Taken together, our findings suggest that quizartinib may be a synthetic lethal partner of PARP inhibitors to enhance therapeutic efficacy in NSCLC cells.

Citation Format: Hwani Ryu, Hyo Jeong Kim, Jie-Young Song, Sang-Gu Hwang, Jiyeon Ahn. Combined FLT3 and PARP inhibition induces synthetic lethality in non-small cell lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6394.
Abstract 6395: VHL deficiency sensitizes CC-RCC to inhibition of small GTPases by statins
Authors: Razorenova Olga V. , Thompson Jordan M. , Alvarez Alejandro , Singha Monika K. , MW Matthew W. Pavesic , Nguyen Quy H. , Nelson Luke J. , Fruman David A.
Published: September 2020

Clear cell renal cell carcinoma (CC-RCC) type of kidney cancer has limited therapeutic options available for advanced stages. The objective of this study was to investigate statins as potential therapeutics for CC-RCC. Importantly, treatment with statins was found to be synthetically lethal with the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in 90% of CC-RCC driving the disease. Treatment with statins causes a profound cytostatic effect at nanomolar concentrations and becomes cytotoxic at low micromolar concentrations in VHL-deficient CC-RCC. The synthetic lethal effect can be fully rescued by both mevalonate and geranylgeranylpyrophosphate, but not by squalene, indicating that the effect is due to disruption of small GTPase isoprenylation and not the inhibition of cholesterol synthesis. Rho and Rho kinase (ROCK) inhibition by siRNAs, as well as C3-toxin (for Rho) and Y27637 (for ROCK) mimic the statin-mediated synthetic lethality effect, suggesting the involvement of Rho/ROCK pathway in the mechanism. Overactivation of hypoxia-inducible factor signaling resulting from VHL loss is required for statin-mediated toxicity. Since both VHL and ROCK were previously reported to control mitosis fidelity, we show that mitotic catastrophe is one of the mechanisms of action of compounds targeting the Rho/ROCK pathway in VHL-deficient CC-RCC. Finally, statin treatment is able to inhibit both tumor initiation and progression of subcutaneous 786-OT1-based CC-RCC tumors in mice. Thus, statins represent potential therapeutics for the treatment of VHL-deficient CC-RCC.

Citation Format: Olga V. Razorenova, Jordan M. Thompson, Alejandro Alvarez, Monika K. Singha, Matthew W. Pavesic MW, Quy H. Nguyen, Luke J. Nelson, David A. Fruman. VHL deficiency sensitizes CC-RCC to inhibition of small GTPases by statins [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6395.
Abstract 6396: Sarcoma treatment using HSP90 inhibitors in combinatorial synergy with doxorubicin
Authors: Kirkham Matt , Luelling Sarah , Fatema Kaniz , Barrott Jared
Published: September 2020

Synovial sarcoma, a rare muscle cancer, has few targeted therapies, and is most commonly treated using doxorubicin (DOX). Even as a primary treatment agent, successful treatment and remission rates are low, and the significant cardiotoxicity of DOX limits dosage options. Synovial sarcoma, and other sarcomas, have a high expression of HSP90, a protein that acts as a chaperone molecule to stabilize and help fold other proteins within the cellular matrix. Under high stress situations, HSP90 is upregulated and acts to stabilize other more thermally labile proteins, including many oncogenes. Using FDA approved drug panels, it was determined that HSP90 is a suitable target for treatment. We look to use a novel HSP90 inhibitor (HS10) compound, to target the high expression of HSP90 within synovial sarcoma and osteosarcoma in combination with the chemotherapy standard DOX to determine if a synergistic effect is observed. This synergistic effect would allow for an overall decreased dose to induce effective cancer cell targeting, reducing overall systemic toxicity within patients. Our results indicate such synergism does occur between DOX and HS10, at a dosage of 5-25 nM DOX and 1000 nM HS10, reducing cell survival to 37-68%, dependent on cell line. DOX dosage alone reduced cell survival to 74% in the most effective case, with no reduction in the least effective case, at a dose of 100 nM. This dose is four times stronger than the highest DOX synergistic dose. It can be concluded, then, that synergistic combination does exist between DOX and HS10, and dosage of DOX can be reduced with more effective targeting for synovial and osteosarcoma cell lines. Protein expression was evaluated using immunoblotting to isolate protein fractions within the cytoplasm and cell membrane, showing high expression within the cytoplasm. Isolated blood plasma from mice with and without tumors was analyzed through ELISA, showing elevated HSP90 presence in mice with tumors (236.4±190pg) vs mice without tumors (6.5±3.5pg). This confirmation of elevated HSP90 expression within sarcoma patients suggests targeting using HS10 to induce synergy to be a viable approach for advanced sarcoma treatment.

Citation Format: Matt Kirkham, Sarah Luelling, Kaniz Fatema, Jared Barrott. Sarcoma treatment using HSP90 inhibitors in combinatorial synergy with doxorubicin [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6396.
Abstract 6407: A live cell method to assess E3 ligase and target protein occupancy for PROTACs
Authors: Vasta James , Corona Cesear , Wilkinson Jennifer , Ingold Morgan R. , Zimprich Chad , Schwinn Marie , Machleidt Thomas , Hartnett Jim , Cong Mei , Fan Frank , Somberg Richard L. , Robers Matthew
Published: September 2020

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that hijack ubiquitin E3 ligases and induce degradation of intracellular proteins through a tightly regulated proteosomal mechanism. Although several successful PROTACs have been developed against key intracellular target classes including bromodomains, kinases, and nuclear hormone receptors, these bivalent molecules often suffer from poor cell permeability due to high molecular weight. To enable a high-throughput, quantitative readout for PROTAC permeability and E3 ligase occupancy in living cells, we have developed a panel of target engagement (TE) assays for key E3 ligase including CRBN, VHL, XIAP, cIAP, and MDM2. These intracellular assays are the first biophysical method to enable the quantitative determination of compound occupancy, potency, and residence time for specific target proteins inside living cells. The assays provide a direct measure of compound binding or occupancy to a target under physiological conditions using bioluminescent resonance energy transfer (BRET).

Citation Format: James Vasta, Cesear Corona, Jennifer Wilkinson, Morgan R. Ingold, Chad Zimprich, Marie Schwinn, Thomas Machleidt, Jim Hartnett, Mei Cong, Frank Fan, Richard L. Somberg, Matthew Robers. A live cell method to assess E3 ligase and target protein occupancy for PROTACs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6407.
Abstract 6408: E3scan™ ligand binding assay platform for targeted protein degradation and PROTAC discovery
Authors: Katsenelson Ksenya Cohen , Gonzalez Luis , Pallares Gabriel , King Alastair J. , Treiber Daniel K.
Published: September 2020

E3 ligases have emerged as pivotal targets for a next generation protein degradation-based drug discovery paradigm. This new paradigm includes both ligand binding-directed “reprogramming” of E3 substrate specificity approaches and a more directed approach, using small molecule proteolysis-targeting chimeras (PROTACs), to selectively degrade disease-driving proteins. As there are hundreds of diverse putative E3 ligases with differentiated tissue expression, this new paradigm may well define a next dimension of precision medicine defined by an axis of tissue-specific activity. While there have been some early successes, the E3 drug discovery field has a significant unmet need for a standardized biochemical ligand binding assay platform. A platform is required that: 1) Can measure ligand binding across the E3 family using a standardized method enabling “apples to apples” comparisons; 2), Is highly scalable and rapid; 3) Has an exquisite dynamic range for the measurement of accurate KD values as low as digit picomolar (pM). Eurofins DiscoverX herein presents its novel E3scan™ technology that addresses each of these unmet needs. E3scan, based upon well-established KINOMEscan® technology, has been successfully applied to diverse E3 ligases, including CRBN, VHL, MDM2, MDMX, cIAP1, cIAP2, and XIAP, with many other E3 assays in progress. We shall present assay validation data for these targets, including data for ligands with KD values in the low to mid pM range. In conclusion, we present Eurofins DiscoverX's novel E3scan platform that shall enable accelerated screening and SAR analysis in the E3 drug discovery field, with rapid turnaround times for discovery library screens (20 business day TAT) and weekly SAR (5 business day TAT) and the largest assay panel available on a single technology platform.

Citation Format: Ksenya Cohen Katsenelson, Luis Gonzalez, Gabriel Pallares, Alastair J. King, Daniel K. Treiber. E3scan™ ligand binding assay platform for targeted protein degradation and PROTAC discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6408.
Abstract 6426: Novel mechanism of PIM2 inhibition leads to significant effect on solid tumors
Authors: Katsuta Eriko , Takabe Kazuaki , Adjei Alex , Opyrchal Mateusz
Published: September 2020

Background: PIM2 is overexpressed in hematopoietic and solid cancers, and is involved in multiple signaling pathways. In hematologic malignancies, PIM2 has been shown to be a pro-survival factor and demonstrated to be a possible therapeutic target. However, the role of PIM2 as a therapeutic target in solid cancers has not been fully elucidated.

Methods: PIM2 expression was compared between normal and cancer tissues in The Cancer Genome Atlas (TCGA) datasets. Pan-PIM inhibitor treatment, as well as PIM2 overexpression/knockdown technique was utilized to investigate the PIM2 role in solid cancers. Multiple cancer models were utilized for in vitro and in vivo experiments to investigate if PIM2 as a therapeutic target and examine mechanism of anti-tumor activity by JP11646.

Results: PIM2 was overexpressed in several types of cancer tissues, including breast, esophageal, head-neck, kidney, liver and endometrial cancers in TCGA. PIM2 overexpression promoted tumor growth in in vivo breast cancer mouse model. PIM2 inhibition by siRNA as well as pan-PIM inhibitor JP11646 activated apoptosis as seen by increased cleaved PARP. JP11646 treatment inhibited cancer cell proliferation in a dose-dependent manner in vitro in various types of solid cancers, including head-neck, ovarian, breast, prostate, liver, pancreatic, colorectal and non-small cell lung cancers. Interestingly, treatment with pan-PIM inhibitor JP11646 resulted in selective downregulation of PIM2 protein expression, but not PIM1 or PIM3 in breast cancer cell lines, BT549 and MDA-MB-231. The treatment also resulted in downregulation of phosphorylated downstream targets, 4EBP1 and TSC2 in breast cancer cells. However, additional proteasome inhibitor treatment blocked PIM2 downregulation and apoptosis activation was not seen. The result suggests that proteasome complex is required for PIM2 degradation which is necessary to induce apoptosis. JP11646 treatment inhibited tumor growth in breast, liver, pancreas, and non-small cell lung cancer in vivo xenograft models and was compared to multiple standard of care treatments showing advantage in lung tumors.

Conclusion: We found that PIM2 promoted cancer progression in solid tumors. We show novel mechanism of JP11646 targeting PIM2 for degradation and induction of apoptosis. We show that PIM2 may serve as a novel therapeutic target for multiple types of solid cancers with enhanced therapeutic effect through protein degradation rather than kinase inhibition alone.

Citation Format: Eriko Katsuta, Kazuaki Takabe, Alex Adjei, Mateusz Opyrchal. Novel mechanism of PIM2 inhibition leads to significant effect on solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6426.
Abstract 643: Cdk4/6 inhibitor Abemaciclib overcomes resistance to BET inhibitor in leukemic cells with MLL-AF5q31 fusion gene
Authors: Amari Keigo , Toda Yuki , Hosogi Shigekuni , Imamura Toshihiko , Ashihara Eishi
Published: September 2020

Mixed-lineage leukemia (MLL)-related leukemia which has MLL rearrangement is an intractable disease. Recently, bromodomain and extra-terminal (BET) inhibitors are promising agents, and clinical trials are undergone. However, drug resistance against BET inhibitors has been reported, therefore, it is important to elucidate its mechanism. In this study, we established OTX015-resistant leukemic cells with MLL-AF5q31 fusion gene (OTX015-R cells) and identified molecules that contribute to drug resistance. In addition, we evaluated whether identified molecules are effective as therapeutic target for BET inhibitor-resistant MLL-related leukemia.

To generate OTX015-R cell line, we cultured MLL-AF5q31 cells in the complete medium containing OTX015 with stepwise increasing concentrations. OTX015-R cells exhibited cross-resistance to various BET inhibitors. In addition, protein expressions of bromodomain-containing protein 4 (Brd4) and Brd4-regulated molecules c-Myc, Cdk6 and Bcl-2 were remarkably increased in OTX015-R cells compared with the parental cells. Furthermore, treatment of Cdk4/6 inhibitor Abemaciclib showed a significant antitumor effect on OTX015-R cells and protein expressions of Brd4 and Brd4-regulated molecules were decreased after Abemaciclib treatment. Abemaciclib exhibits antitumor effects on the parental MLL-AF5q31 cells by induction of cell cycle arrest. On the other hand, interestingly, apoptosis in the OTX015-R cells was induced without cell cycle arrest. These results suggest that Abemaciclib induces apoptosis in OTX015-R cells by another mechanism different from cell cycle regulation. We are going to clarify this mechanism.

In conclusion, BET inhibitor-resistant MLL-AF5q31 cells upregulated expression levels of BRD4 and its downstream molecules. We suggest that Cdk6 is a potent therapeutic target for BET inhibitor-resistant MLL-related leukemias.

Citation Format: Keigo Amari, Yuki Toda, Shigekuni Hosogi, Toshihiko Imamura, Eishi Ashihara. Cdk4/6 inhibitor Abemaciclib overcomes resistance to BET inhibitor in leukemic cells with MLL-AF5q31 fusion gene [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 643.