bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6057: Discovery of a novel function for USP7 inhibitors: Reprogramming the tumor microenvironment
Authors: Jacq Xavier , Jurisic Anamarija , Daubriac Julien , Lobb Ian T. , Wappett Mark , Cranston Aaron , Sung Peggy , Gavory Gerald , O'Dowd Colin , Harisson Tim
Published: September 2020

During the transition from healthy tissue to cancer and metastasis, the tumor microenvironment (TME) initiates crucial changes that licenses the progression from primary tumors to aggressive and metastatic disease. Our understanding of the specific factors and molecular mechanisms underpinning these transitions in the TME is critical for influencing the potency and scope of existing cancer therapy strategies but is still evolving and subject to substantial and widespread investigations. Here we uncover a novel molecular mechanism actioned by fibroblasts in the TME to modulate the secretion of the vascular endothelial growth factor (VEFG) via the hypoxia-inducible transcription factor (HIF1α). Utilizing primary human cells co-culture systems in combination with highly potent and selective USP7 inhibitors, we demonstrate that the deubiquitinating enzyme USP7 modulates the stability of HIF1α in primary human fibroblasts and secreted VEGF levels in primary fibroblasts as well as cancer-associated fibroblasts. This USP7-mediated modulation of VEGF secretion in the TME does not occur in cancer cells, immune cells or endothelial cells, rather it is restricted to the fibroblastic compartment. Importantly, the modulation of VEGF secretion by USP7 in fibroblasts is not mediated via the Mdm2 axis: Mdm2 antagonists are unable to recapitulate the VEGF modulation in fibroblasts. Knock-down of USP7 in primary fibroblasts validates the phenotype obtained with potent and selective USP7 inhibitors. In line with their effects on the TME in primary cell cultures, potent, selective USP7 inhibitors demonstrated tumour growth inhibition in vivo in a syngeneic cancer xenograft model that does not respond to USP7 inhibitors in vitro. Collectively, our studies reveal a molecular mechanism whereby USP7 inhibition leads to TME reprogramming mediated by an increased destabilization of HIF1α leading to reduced levels of VEGF in the TME and improved anti-tumor activity. Our results support further investigations of combination of a USP7 inhibitor with immune checkpoint inhibitors, as well as other TME modulating agents.

Citation Format: Xavier Jacq, Anamarija Jurisic, Julien Daubriac, Ian T. Lobb, Mark Wappett, Aaron Cranston, Peggy Sung, Gerald Gavory, Colin O'Dowd, Tim Harisson. Discovery of a novel function for USP7 inhibitors: Reprogramming the tumor microenvironment [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 6057.
Abstract 6061: NR2F1 limits dissemination of early cancer mammary epithelial cells
Authors: Sosa Maria Soledad , Kale Nupura , Shrivastava Nitisha , Rodriguez-Tirado Carolina , Carlini Maria , Ji Jiayi , Bravo-Cordero Javier
Published: September 2020

Cancer cells have been found to disseminate during very early and sometimes asymptomatic stages of tumor progression. At secondary organs, early disseminated cancer cells (eDCCs) can enter a non-proliferative state for prolonged periods of time. Intriguingly, eDCCs also carry metastasis- initiating capacity. However, the mechanisms by which these early cancer cells complete all steps of metastasis including metastasis-initiating capacity are unknown. Here we report that the orphan nuclear receptor and lineage commitment regulator NR2F1 serves as an early barrier to early dissemination propelled by HER2 signaling. NR2F1 loss of function in HER2 early cancer cells induced cell motility, loss of E-cadherin junctions, disorganized laminin-V deposition, decreased β-catenin membrane localization and increased micro-invading cells with CK14+/CK18 phenotype. Importantly, upon NR2F1 depletion in HER2 early cancer acini, TWIST, ZEB1 and PRRX1 levels were upregulated. Our findings reveal a previously unrecognized function of NR2F1 as a suppressor of dissemination during early stages of breast tumor progression. Thus, therapies that restore and activate NR2F1 function might limit early dissemination and metastatic outgrowth.

Citation Format: Maria Soledad Sosa, Nupura Kale, Nitisha Shrivastava, Carolina Rodriguez-Tirado, Maria Carlini, Jiayi Ji, Javier Bravo-Cordero. NR2F1 limits dissemination of early cancer mammary epithelial 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 6061.
Abstract 6096: Bladder cancer microbiome and its association with chemoresponse
Authors: Ginwala Rashida , Nachman Erika , Peri Suraj , Franklin Jodie , Greathouse Leigh , Ingersoll Molly , Kutikov Alexander , White James R. , Abbosh Philip H.
Published: September 2020

Background: The microbiome is widely known to cause cancer, modify its behavior, and alter the way therapies interact with tumors. Despite the urinary microbiome being one of the most clinically significant microbiomes in human health, is one of the least well-described.

Results: To begin to annotate the urinary microbiome present in bladder cancers, we analyzed raw sequencing data from the Cancer Genome Atlas (TCGA) from 116 tumors (duplicates from 22 tumors), 22 adjacent normal bladder tissues, and 99 blood samples to identify reads originating from known microbiota. Twenty-seven viral and bacterial species were found to be enriched in the tumor samples. Bacterial species such as B. thetaiotamicron, A. mucinaphila and Bifidobacterium spp. with known positive association with immune checkpoint blockade therapy against CTLA-4 and PD-L1 were frequently found in these tumor samples. We also found sex-specific enrichment of genera such as Lactobacillus and Prevotella in female bladder cancer patients which also are prevalent in the normal female genitourinary tract. A. prevotii and F. magna were also enriched in females. We also found a correlation between Prevotella and TCGA subtypes III and IV (basal expression subtypes), suggesting a link between enrichment of certain microbial species and bladder cancer subtypes. Finally, single sample gene set enrichment analysis using the Hallmark and KEGG gene sets revealed the association of Mycoplasma with the Hallmark DNA repair gene set and with the KEGG pyrimidine metabolism pathway that can be further studied to ascertain its effects on DNA damaging chemotherapeutic agents. To corroborate these findings, we performed 16S metagenomics from urine pellets of 40 bladder cancer patients undergoing radical cystectomy on a prospective clinical trial (NCT02968732). We found one predominant genus consistent with prior urinary metagenomics studies in samples from most of the patients. Thirty eight of the 58 genera identified in the TCGA analyses were also present in these urine samples. Further correlation with response to clinical metadata is ongoing.

Conclusions: Description of the bladder cancer microbiome is the first step in approaches to manipulate it for therapeutic gain.

Citation Format: Rashida Ginwala, Erika Nachman, Suraj Peri, Jodie Franklin, Leigh Greathouse, Molly Ingersoll, Alexander Kutikov, James R. White, Philip H. Abbosh. Bladder cancer microbiome and its association with chemoresponse [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 6096.
Abstract 6116: Role of secretory factors from obese-derived omentum in supporting ovarian cancer progression
Authors: Waters Jennifer , Robinson Mikella , Gilbert Samuel F. , Alexander Logan J. , House Carrie D.
Published: September 2020

Ovarian cancer is the most lethal gynecological malignancy in the United States, with a known predilection for metastasizing to the omentum, a sheet of fatty peritoneal tissue that encloses the abdomen. This finding coincides with some evidence that ovarian cancer is more aggressive in the obese setting with worse overall survival. While it has been shown that ovarian cancer cells invade towards adipocytes in vitro, the mechanism by which they support formation of secondary tumors is not well understood. We hypothesize that soluble factors enriched in the obese setting are activating signaling pathways in tumor cells to drive migration and proliferation. To evaluate this hypothesis we co-cultured primary human omental adipocytes derived from obese female donors with ovarian cancer cell lines and profiled secretory factors (cytokines and adipokines) responsible for increasing tumor initiation in vivo and clonogenicity, proliferation, and drug resistance in vitro. Secretion of cytokines IL-6, IL-8, and CCL2 by adipocytes is enhanced when co-cultured with ovarian cancer cells. A subcutaneous tumor model showed that low dilutions of ovarian cancer cells require adipocytes for engraftment and tumor formation. A transplantation assay showed that ovarian cancer cells require the presence of adipocytes for sustained tumor-initiation capacity. Studies are underway to investigate adipocyte-secreted factors in this process. Our findings highlight the role of adipocytes in the ovarian tumor microenvironment and uncover signaling factors that may enhance ovarian cancer progression. Understanding these processes will enable the design of more effective therapies for treating ovarian cancer.

Citation Format: Jennifer Waters, Mikella Robinson, Samuel F. Gilbert, Logan J. Alexander, Carrie D. House. Role of secretory factors from obese-derived omentum in supporting ovarian cancer progression [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 6116.
Abstract 6139: Sex-dependent development of anal squamous cell carcinoma in Kras-mutant mice
Authors: Walcheck Morgan , Noel Jessica , Nukaya Manabu , Matkowskyj Kristina , Ronnekleiv-Kelly Sean
Published: September 2020

A historical over-reliance on male animals in research studies has clouded sex differences in pathologic diseases. This led to a mandate from the NIH requiring the use of both male and female animals in preclinical studies. Our study began with the aim to evaluate both sexes of KC mice (LSL-KrasG12D/+; Pdx1-Cre), a standard pre-clinical model for pancreatic ductal adenocarcinoma (PDAC). This was because previous characterization of this model to advanced age (9-12 months) only included males, and evaluating both sexes was an essential foundation for subsequent studies of PDAC progression. Twenty-eight KC mice have been evaluated, comprised of 15 males and 13 females. KC mice developed the expected pancreatic cancer pre-cursor lesions called PanINs (100% PanIN1, 7% PanIN 2 and 7% PanIN 3) and PDAC (14%) at an established time point (age 9 months). Surprisingly, female KC mice developed anal lesions, a finding that has not previously been described. Histopathologic analysis demonstrated anal squamous cell carcinoma (SCC), with all 13 KC females developing anal SCC compared to 0/15 in KC males and 0/83 control mice (Table 1). Genotyping for activated Kras mutation confirmed the presence of KrasG12D, indicating Pdx1 expression (Pdx1-Cre) in the anal tumor tissue. To understand the distinct sex differences and connection to sex-dependent steroids (estrogen and testosterone), female mice were ovariectomized (n = 5) and males castrated (n = 6). Remarkably, while 5/5 females developed anal SCC (i.e. unchanged phenotype), 6/6 males developed anal SCC, a stark contrast to the absence of tumor development in non-castrated KC males. This suggests a novel mechanism in which Kras driven anal SCC tumor development is suppressed through testosterone / dihydrotestosterone (DHT) exposure. Follow-up studies will focus on the mechanism by which testosterone or DHT suppresses Kras-driven anal SCC formation. These findings may ultimately have therapeutic implications for the subset of patients with Kras-mutant anal SCC.

Table 1:Characterization and Incidence of Anal SCC TumorsGroupMouseAnal SCC IncidenceControlC57Bl6 Male (n=12)0/12 (0%)ControlC57Bl6 female (n=11)0/11 (0%)ControlKrasG12D/+ Female (n=9)0/9 (0%)ControlKrasG12D/+ Male (n=18)0/18 (0%)ControlPDX1-Cre male (n=10)0/10 (0%)ControlPDX1-Cre Female (n=23)0/23 (0%)KC MiceKC Male (n=15)0/15 (0%)KC MiceKC Female (n=13)13/13 (100%)Study KC MiceCastrated KC Male (n=6)6/6 (100%)Study KC MiceOvariectomized KC Female (n=5)5/5 (100%)

Citation Format: Morgan Walcheck, Jessica Noel, Manabu Nukaya, Kristina Matkowskyj, Sean Ronnekleiv-Kelly. Sex-dependent development of anal squamous cell carcinoma in Kras-mutant mice [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 6139.
Abstract 6171: High throughput screening identified new radiosensitizers which inhibit DNA damage repair pathway
Authors: Tamari Keisuke , Li Zhihao , Matsutani Hideki , Seo Yuji , Takahashi Yutaka , Minami Kazumasa , Isohashi Fumiaki , Ogawa Kazuhiko
Published: September 2020

Radiotherapy (RT) plays a crucial role in the treatment of cervical cancer. However, the efficacy of existing radiosensitizers in clinical applications is limited. The aim of this study was to established an efficient method of identifying new radiosensitizers and to investigated the specific mechanisms when used in combination with RT. An effective platform for radiosensitizers discovery was created based on high-throughput screening (HTS) technology. We established an automated and powerful HTS approach to identify potent radiosensitizers from the LOPAC1280 chemical library. We used the Geldanamycin for validation of our HTS method, which had been known as a radiosensitizer for cervical cancer cells. Z'-score of our HTS platform was 0.6. From 1280 compounds, we identified four hit compounds. Among them, we found Dihydroouabain (DHO) and Ro90-7501 which have not been previously known as radiosensitizers. Clonogenic survival assay showed that DHO and Ro90-7501 significantly enhanced radiosensitivity in cervical cancer cells. Western blot analysis showed that DHO inhibited the phosphorylation of Chk1, and Ro90-7501 inhibited the phosphorylation of ATM. These results indicated that DHO and Ro90-7501 inhibited DNA damage response pathway. In conclusion, we established the HTS method for radiosensitizer screening, and found DHO and Ro90-7501 as new radiosensitizers targeting DNA damage repair pathway.

Citation Format: Keisuke Tamari, Zhihao Li, Hideki Matsutani, Yuji Seo, Yutaka Takahashi, Kazumasa Minami, Fumiaki Isohashi, Kazuhiko Ogawa. High throughput screening identified new radiosensitizers which inhibit DNA damage repair pathway [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 6171.
Abstract 6177: Triple negative breast cancer patients treated with radiation therapy: Improvement in survival and local control with adjuvant chemotherapy compared to neoadjuvant chemotherapy
Authors: Nittala Mary R. , Mundra Eswar K. , Packianathan Satya , Mehta Divyang , Smith Maria L. , Woods William C. , McKinney Shawn , Craft Barbara S. , Vijayakumar Srinivasan
Published: September 2020

Purpose: The objective of this study is to evaluate local control and survival rates of node negative triple negative breast cancer patients (TNBCs) treated with radiotherapy (RT) in our institution.

Methods: This is a retrospective analysis of 67 TNBCs (2007 - 2017) at an academic state institution who underwent lumpectomy and /or mastectomy (surgery) followed by adjuvant irradiation to median total dose of 50 Gy (range 40.5-50.40 Gy). Chemotherapy was administered in a neoadjuvant (31) or adjuvant setting (36). For all 67 TNBCs, local control (LC), overall survival (OS) and disease free survival (DFS) were estimated using the Kaplan-Meier method. The significance of survival variables were analyzed using the Cox univariate and multivariate proportional hazards model. A p-value of less than 0.05 was considered statistically significant. The SPSS 24.0 software was used for data analysis.

Results: The baseline characteristics of all 67 TNBCs were measured with median follow up of 58 months (range 10-142 months). Patients were stratified into two groups (neoadjuvant chemotherapy-RT (31) vs. adjuvant chemotherapy-RT (36)). The median rates for LC, DFS and OS were 27 % vs. 68 % (p= 0.001), 27.2% vs. 63.2 % (p= 0.034), and 63.4% vs. 92.5% (0.001) respectively. On Cox multivariate analysis, patients who received adjuvant chemotherapy were associated with statistically improved significant LC (p= 0.002) and OS (p=0.005). The variables included were: ethnicity (p=0.251), age (p=0.984), BMI (p=0.032), lump boost (p=0.300), TNM (p=0.162) and tumor grade (p=0.336).

Conclusion: Among TNBC patients undergoing RT, adjuvant chemotherapy appears to be better than neoadjuvant chemotherapy in determining the clinical outcomes.

Citation Format: Mary R. Nittala, Eswar K. Mundra, Satya Packianathan, Divyang Mehta, Maria L. Smith, William C. Woods, Shawn McKinney, Barbara S. Craft, Srinivasan Vijayakumar. Triple negative breast cancer patients treated with radiation therapy: Improvement in survival and local control with adjuvant chemotherapy compared to neoadjuvant chemotherapy [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 6177.
Abstract 6206: The xl313 cryptic collagen epitope regulates ovarian tumor growth by a yap dependent mechanism
Authors: Han Xianghua , Caron Jennifer M. , Brooks Peter C.
Published: September 2020

Structural changes in the extra cellular matrix (ECM) can lead to the exposure of cryptic regulatory sites that play important roles in angiogenesis, inflammation, tumor growth, and tumor metastasis. Previously, we discovered that a unique cryptic regulatory site (XL313 cryptic epitope) containing the amino acid sequence RGDKGE, is generated during tumor development, and we found that this collagen epitope promotes angiogenesis and inflammation in vivo. Here, we provide the first evidence that ovarian carcinoma cells can bind the XL313 collagen epitope through integrin alpha-V beta-3 and that selectively blocking cellular interactions with the XL313 epitope significantly inhibits ovarian tumor growth and the formation of ascites fluid in vivo. To understand the cellular mechanism(s) by which antagonists of the XL313 epitope inhibit these pathological processes, we examined down-stream signaling events that were altered following blocking ovarian tumor cell interaction with the XL313 collagen epitope. Importantly, selectively targeting the XL313 epitope significantly reduced nuclear accumulation of the transcriptional co-activator YAP. In addition, blocking cellular interaction with the XL313 epitope resulted in enhanced YAP phosphorylation at serine-127. Interestingly, specific targeting of the XL313 epitope also enhanced LATS1 phosphorylation, which is a key effector molecule in the hippo- signaling pathway. Taken together, our data provide new cellular and molecular insight for understanding the role of the XL313 cryptic collagen epitope in controlling ovarian tumor growth, and suggest that selectively targeting the XL313 epitope-integrin-signaling cascade may represent a novel strategy to inhibit ovarian cancer progression.

Citation Format: Xianghua Han, Jennifer M. Caron, Peter C. Brooks. The xl313 cryptic collagen epitope regulates ovarian tumor growth by a yap dependent mechanism [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 6206.
Abstract 6216: Therapeutic potential of IAP inhibitor APG-1387 in combination with PARP- or MEK-targeted therapy, or chemotherapy in pancreatic cancer
Authors: Li Qiang , Fang Douglas D. , Gu Shoulai , Yang Dajun , Zhai Yifan
Published: September 2020

APG-1387 is a small molecule antagonist of the inhibitor of apoptosis proteins (IAPs) which blocks the activity of IAPs family proteins (XIAP, cIAP-1, cIAP-2, and ML-IAP) and consequently induces apoptosis. It is currently in phase I clinical development in patients with solid tumors, including pancreatic cancer. Targeting IAPs has been reported to effectively inhibit tumor growth in the preclinical models of pancreatic cancer. Currently, PARP and MEK inhibitors are also in clinical development for pancreatic cancer with BRCA1/2 or KRAS mutations. To further explore the therapeutic potential of APG-1387 in combination with PARP or MEK inhibitors, a genomic biomarker guided avatar mouse trial (n=2) using pancreatic cancer patient-derived xenografts (PDX) was conducted to evaluate antitumor activity of APG-1387 in combination with (i) the PARP inhibitor, olaparib, in PDXs carrying BRCA1/2 mutations, and (ii) the MEK inhibitor, trametinib, in PDXs carrying KRAS mutations.

The results revealed that APG-1387 improved the antitumor activity of olaparib in 4 out of 6 (67%) BRCA1/2 mutant PDXs with a 44% reduction of T/C (%) value (T, treatment group, C, vehicle control), including one partial tumor regression (PR). In combination with trametinib, the antitumor activity was improved in 4 out of 9 (44%) KRAS mutant PDXs, showing a 34% reduction in the T/C value. Furthermore, synergy of the combinations of APG-1387 with PARP or MEK inhibitor was confirmed in a follow-up efficacy study (n=5) using a PDX model carrying both BRCA2 and KRAS mutations. The molecular mechanisms underlying the synergy are currently being investigated.

In summary, our results suggest that APG-1387 is a potential agent for pancreatic cancer treatment, ascribing to its potential synergistic antitumor effect by combining with either PARP inhibitors in BRCA1/2 mutant or MEK inhibitors in KRAS mutant pancreatic cancers. Overall, our data provide the scientific rationale for the future clinical development of these combinations in pancreatic cancer patients with distinct genomic alterations.

Citation Format: Qiang Li, Douglas D. Fang, Shoulai Gu, Dajun Yang, Yifan Zhai. Therapeutic potential of IAP inhibitor APG-1387 in combination with PARP- or MEK-targeted therapy, or chemotherapy in pancreatic 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 6216.
Abstract 622: Rapid non-uniform adaptation to conformation-specific KRAS G12Cinhibition
Authors: Xue Jenny Y. , Zhao Yulei , Aronowitz Jordan , Mai Trang T. , Vides Alberto , Qeriqi Besnik , Kim Dongsung , Li Chuanchuan , de Stanchina Elisa , Mazutis Linas , Risso Davide , Lito Piro
Published: September 2020

KRAS GTPases are activated in one-third of cancers and KRAS G12C is the most common activating alteration in lung adenocarcinoma. KRAS G12C-specific inhibitors (G12Ci) are in Phase-I clinical trials and early data show only partial responses in lung cancer patients. How cancer cells bypass inhibition, to prevent maximal responses to therapy, is not understood. Because KRAS G12C cycles between an active and inactive conformation, and the covalent G12Ci only bind to the latter, we tested whether isogenic cell populations respond non-uniformly by studying the effect of treatment at a single-cell resolution. Using single-cell RNA sequencing and a fluorescent quiescence biosensor, we show that shortly after treatment, most cancer cells are sequestered in a quiescent state with low KRAS activity, while a small population reactivates KRAS to resume proliferation. This rapid divergent response is due to synthesis of new, drug-free KRAS protein, resulting from increased KRAS transcription in response to suppressed MAPK signaling. Combining cell fate-specific gene expressions and results from a CRISPR-Cas9 screen, we identified that adaptive signals such as epidermal growth-factor receptor and aurora kinase A signaling modulate the heterogeneous response to treatment with G12Ci. These upstream signals help to maintain new KRAS G12C protein in its active, drug-insensitive state, which restores KRAS signaling and transcriptional output in a subset of cells to allow escape from G12Ci-induced quiescence. Cells without these adaptive changes (or cells where they are pharmacologically inhibited) remain sensitive to G12Ci treatment, because new KRAS G12C is either not available, or it exists in its inactive, drug-sensitive state. Combined inhibition of these adaptive signals along with KRAS G12C produced more potent antitumor effects in xenograft models. The direct targeting of KRAS oncoproteins has been a longstanding objective in precision oncology. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome to maximize the therapeutic potential of conformation-specific KRAS G12C inhibitors in the clinic.

Citation Format: Jenny Y. Xue, Yulei Zhao, Jordan Aronowitz, Trang T. Mai, Alberto Vides, Besnik Qeriqi, Dongsung Kim, Chuanchuan Li, Elisa de Stanchina, Linas Mazutis, Davide Risso, Piro Lito. Rapid non-uniform adaptation to conformation-specific KRAS G12Cinhibition [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 622.
Abstract 6225: Addition of TRAIL receptor agonists after treatment with ONC201 or ONC212 converts pancreatic cancer cells from anti-proliferative to apoptotic in vitro
Authors: Jhaveri Aakash V. , Zhou Lanlan , Ferarrini Isacco , Lee Young , El-Deiry Wafik S.
Published: September 2020

The 7% five-year survival rate for pancreatic cancer patients has remained unchanged for decades; the need for novel therapeutic interventions is imminent. ONC201 is the founding member of the impridone class, a class of orally bioavailable small molecules which show efficacy in multiple tumor types. ONC201 and ONC212, a fluorinated imipridone, have been shown to be potent inducers of the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. Both compounds are currently in clinical trials. In the context of pancreatic cancer, ONC201 and ONC212 induce either apoptotic or anti-proliferative effects. Additionally, both compounds activate the integrated stress response leading to DR5 upregulation in various cell types. We hypothesized that exogenous addition of TRAIL receptor agonists after pre-treatment with ONC201 or ONC212 could convert cells from anti-proliferative to apoptotic. Here, we examined both rhTRAIL and TLY012, a novel TRAIL receptor agonist with a substantially improved half-life. Pancreatic cancer cell lines were screened for synergy when treated with ONC201 or ONC212 for 72 hours followed by either rhTRAIL or TLY012 for 4 hours. Four cell lines (AsPC-1, BxPC3, Capan-1, and PANC-1) showed potent synergy with both rhTRAIL and TLY012. Growth inhibition was assessed through both Cell-Titer Glo and colony assays. Apoptotic synergy was determined using Cell-Titer Glo, western blots for PARP cleavage, and sub-G1 flow cytometric assays. Further analysis showed that ONC201 activates the integrated stress response and primes pancreatic cancer cells for TRAIL-induced apoptosis through regulation of key proteins. Upregulation of the integrated stress response and downregulation of XIAP, c-FLIP, and Bcl-XL was observed after treatment with ONC201 in BxPC3 and PANC-1 cell lines. The combination of ONC201 and TRAIL receptor agonists was non-toxic to human foreskin fibroblasts. Future experiments aim to examine G1 arrest after treatment with ONC201 and ONC212 via western blot and flow cytometry, to confirm surface expression of TRAIL and DR5 via flow cytometry, and to determine efficacy in pancreatic cancer xenografts. Taken together, these data indicate a novel therapeutic strategy for treatment of patients with ONC201 and ONC212 resistant pancreatic tumors.

Citation Format: Aakash V. Jhaveri, Lanlan Zhou, Isacco Ferarrini, Young Lee, Wafik S. El-Deiry. Addition of TRAIL receptor agonists after treatment with ONC201 or ONC212 converts pancreatic cancer cells from anti-proliferative to apoptotic in vitro [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 6225.
Abstract 6226: Sapanisertib interferes with the NRF2-mediated stress response to synergize with Obatoclax and extend AT/RT survival
Authors: Parkhurst Ashlyn , Wang Sabrina , Kaur Harpreet , Arnold Antje , Eberhart Charles , Raabe Eric , Rubens Jeffrey
Published: September 2020

Atypical teratoid rhabdoid tumors (AT/RT) are the most common malignant brain tumors of infancy, highly resistant to all therapies, and responsible for one of the greatest numbers of life years lost due to cancer. We have previously demonstrated that Sapanisertib combines synergistically with cisplatin to extend survival in AT/RT. To understand the mechanisms underlying this synergy and to identify further novel combination therapies to improve AT/RT survival, we performed RNASeq on AT/RT cell models 4 hours after Sapanisertib treatment. Pathway analysis revealed that Sapanisertib disrupts the NRF2-mediated stress response and apoptotic signaling pathway leading to a pro-death phenotype. Activating mutations in the NRF2 coding gene, NFE2L2, have been identified as a possible biomarker predicting Sapanisertib efficacy in lung cancer clinical trials. Short hairpin knockdown of NRF2 led to decreased expression of the anti-apoptotic proteins MCL-1, BCL-2, and BCL-xL suggesting that interfering with NRF2 may drive this pro-death phenotype in AT/RT. Obatoclax is a synthetic small molecule inhibitor of the BCL-2 family of proteins that was well tolerated in phase II clinical trials and readily crosses the blood brain barrier. Obatoclax treatment of NRF2 knockdown AT/RT cell models slows cell growth, induces high rates of apoptosis, and leads to increased oxidative stress compared to short hairpin scramble controls (cell viability assay, t-test p<0.05; ANNEXIN V cell sorting assay t-test p<0.05, MUSE oxidative stress assay t-test p<0.05). We treated AT/RT cell models with Sapanisertib and showed that treatment decreases expression of NRF2 and MCL-1 (western blot). Sapanisertib combines synergistically with Obatoclax to increase oxidative stress, slow cell growth, and increase apoptosis (MUSE oxidative stress assay ANOVA p<0.05; cell viability assay, ANOVA p<0.05; ANNEXIN V cell sorting assay ANOVA p<0.05, Compusyn model of synergy, CI<1.0). We next developed orthotopic mouse models of AT/RT using our luciferase-labeled CHLA06 and BT37 AT/RT cell models. We administered Sapinisertib and Obatoclax using mouse equivalent dosing to human clinical trials (Sapinisertib 6mg/kg oral weekly, Obatoclax 10mg/kg oral twice a week). Combination treatment was well tolerated and more than doubled median survival. Our data supports a new clinical trial combining Sapinisertib with Obatoclax to extend survival in AT/RT.

Citation Format: Ashlyn Parkhurst, Sabrina Wang, Harpreet Kaur, Antje Arnold, Charles Eberhart, Eric Raabe, Jeffrey Rubens. Sapanisertib interferes with the NRF2-mediated stress response to synergize with Obatoclax and extend AT/RT survival [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 6226.
Abstract 6237: Bone-targeted nanoparticle containing protein therapeutics as an effective delivery system for bone metastasis
Authors: Niu Yimin , Gao Cuicui , Yang Hongbin , Huo Qiang , Wang Andrew , Xu Ming , Liu Yang
Published: September 2020

Background: Protein drugs are one of the most promising new areas in drug research and development, however, great challenges are still faced in the drug delivery in vivo. Previously, we constructed bone targeted micelles for bone metastases. In order to address the issues of protein delivery in vivo and enhanced cancer bone metastasis treatment, we engineered and evaluated a bone-targeting nanoparticles by using metal organic framework nanoparticles (MOFs), bone-targeting agents (Alendronate) and hyaluronic acid (HA) as materials and a protein toxin as therapeutic.

Methods: Alendronate (ALN) were conjugated to HA through PEG as linker and ALN-PEG-HA were obtained. Cytochrome C, as modle proteins, were caged in the MOFs (ZIF-8) by selfassembly of Zn2+ and organic ligands (2-methylimidazole) to form protein-encapsulated nanoparticles (ZIF8-CC). And ALN-PEG-HA were further decorated on the surface of ZIF8-CC nanoparticles via the coordination effect between its carboxyls and metal ions in ZIF-8 to result in active-targeting ALN-HA@ZIF8-CC nanopartilces. The materials and nanoparticles were synthesized and characterized by NMR, FT-IR, TEM, DLS, XRD, TGA and N2 adsorption−desorption isotherms analysis. The in vitro release profiles, stability and cytocompatibility were also studied.

Results: The synthesis of ALN-PEG-HA was confirmed by 1H-NMR and FT-IR. TEM, XRD and DLS showed that ALN-HA@ZIF8-CC nanopartilces had been prepared, and the experiment of washing nanoparticles with SDS confirmed that protein was encapsulated in ZIF-8 instead of adsorbed on its surface. TGA and N2 absorption-degradation isotherms analysis also confirmed the formation of the crystal structure and porous structure, respectively. HA coated nanoparticles have good stability while ZIF8-CC were easy to aggregate. The results of in vitro release showed that the nanoparticles have acid sensitive release properties and excellent cell compatibility. In addition, In vitro bone fragment targeting experiment confirmed that nanoparticles have strong bone affinity.

Conclusions: Our data demonstrated that bone-targeted protein drug-encapsulated MOFs were constructed.

Citation Format: Yimin Niu, Cuicui Gao, Hongbin Yang, Qiang Huo, Andrew Wang, Ming Xu, Yang Liu. Bone-targeted nanoparticle containing protein therapeutics as an effective delivery system for bone metastasis [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 6237.
Abstract 6247: Drug delivery with real time customizable micro-scaled continuous liquid interface printed 3D devices
Authors: Hagan C. Tilden , Bloomquist Cameron J. , DeSimone Joseph M. , Wang Andrew Z.
Published: September 2020

Background: Surgery is a key treatment for cancer. However, local recurrence after surgery is common and can lead to significant morbidity and mortality. Current strategies to prevent recurrence include intraoperative radiation or systemic chemotherapy. We hypothesize that local chemotherapy delivery can prevent recurrence while reducing toxicity. To accomplish this, we propose to engineer 3D printed devices that can deliver chemotherapy in a localized and prolonged fashion.

Methods: Paclitaxel and a cisplatin prodrug were dissolved into a liquid resin mixture of PEG550-DMA and HEMA. Custom 3D patches were printed on a Carbon 3D continuous liquid interface printer with microneedles protruding from one side of a flat surface substrate. A non-drug loaded flat backing was also printed from the same resin, and adhered to back side of the microneedle patch. Tumor growth recurrence inhibition post surgical resection was assessed in NU/NU mice in both 344SQ/Luc and A431 tumor models. Tumors were inoculated subcutaneously (106 344SQ/Luc, 5 × 105 A431) and allowed to grow until they reached 5-7mm in length. Tumors were then surgically resected and mice were either given an IV drug treatment, or had a 3D printed patch implanted in the surgical pocket. Tumor recurrence was evaluated with both luciferase bioluminescence imaging and physical measurement of tumor size. Toxicity parameters were analyzed with enzyme and CBC tests.

Results: All drug loaded patches showed significant tumor growth inhibition compared to their corresponding IV drug treatment groups. While single drug loaded patches were significant in their inhibition of tumor growth compared to IV delivery of the same drug, dual drug loaded patches showed the most significant tumor inhibition compared to either single IV drug treatment or a dual IV drug treatment. There were no significant differences in measured toxicity parameters between groups.

Conclusions: Our data demonstrated that local drug delivery through a customizable 3D continuous liquid interface printed drug loaded patch can improve tumor growth recurrence inhibition post surgical resection.

Citation Format: C. Tilden Hagan, Cameron J. Bloomquist, Joseph M. DeSimone, Andrew Z. Wang. Drug delivery with real time customizable micro-scaled continuous liquid interface printed 3D devices [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 6247.
Abstract 6265: Combining arsenic and topoisomerase inhibitors synergistically inhibit gastric cancer
Authors: Kang Ting , Ge Maolin , Lu Shumin , Wang Qing , Zhu Shouhai , Zhu Chuanying , Zheng Leizhen , Liu Han , Chen Siyu
Published: September 2020

Objectives: Arsenic compounds have cytotoxic activities in gastric cancer cells, however, the underlying mechanism is unclear and the clinical potential need to be explored. This study was aimed to investigate the mechanism of arsenic on gastric cancer and find a novel regimen for the treatment of gastric cancer.

Methods: Gastric cancer cell lines were treated with arsenics and/or chemotherapeutic drugs. MTT assay, FACS, Western blotting, q-RT-PCR and CHIP were used to detect proliferation, modulation of proteins and genes.

Results: We previously showed that NFATc3 was a target of Arsenic sulfide and its inhibition blocks gastric cancer growth. Here we show that the NFATc3 expression is critically important for the growth and survival of gastric cancer cells because knockdown of NFATc3 resulted in DNA damage and cell cycle arrest. Arsenic sulfide can target NFATc3 in gastric cancer cells and cause cell double-strand DNA breaks (DSB). However, overexpression of NFATc3 can block the DNA damage. Abolishing NFATc3 expression, whether by shRNA-induced silencing or Arsenic sulfide treatment, directly upregulated recombination activating gene 1 (RAG1, an endonuclease which is essential for immunoglobulin V(D)J recombination), leading to fatal DSB. NFATc3 targets the promoter of RAG1 for transcriptional inhibition. Moreover, we find that Arsenic sulfide and topoisomerase inhibitors (Irinotecan and Epirubicin, Doxorubicin, Etoposide and SN38) synergistically inhibit gastric cancer. Arsenic sulfide, as well as Arsenic Trioxide, enhanced the cytotoxic effect of Irinotecan and they synergistically induced NFATc3 degradation, resulted in disability of DNA repair system, as reflected by the decrease in the phosphorylated ATM/CHK2/CHK1 protein level and the dramatic increase in the phosphorylated H2AX protein level. More inspiring, a patient with advanced gastric cancer with liver and lymph-node metastasis who failed in first-line treatment achieved PR with 2 months treatment of Irinotecan combined with Realgar-Indigo Naturalis Formula, the main component of which is Arsenic sulfide. The progression-free survival was 4 months with no additional adverse event.

Conclusions: Our research links arsenic compounds to the regulation of DNA damage repair and RAG1 expression as a mechanism for the cytotoxic effect. The combination of arsenic with topoisomerase inhibitors may represent a novel regimen for the treatment of gastric cancer and warrants further studies.

Citation Format: Ting Kang, Maolin Ge, Shumin Lu, Qing Wang, Shouhai Zhu, Chuanying Zhu, Leizhen Zheng, Han Liu, Siyu Chen. Combining arsenic and topoisomerase inhibitors synergistically inhibit gastric 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 6265.
Abstract 6293: Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K-Akt pathway proteins
Authors: Kazakova Irina G. , Tran Crystal , Silver Anita , Dermody Jessica
Published: September 2020

The PI3K-Akt signaling pathway modulates cell growth, survival and apoptosis, and this pathway is frequently altered in human cancers, contributing resistance to radiation and chemotherapy treatment. Akt is a target for specific inhibition and recently, a number of small molecules have been developed to improve the pharmacologic properties of known inhibitors like wortmannin and LY294002. However, pan-Akt inhibitors can result in unanticipated side effects due to the lack of specificity for Akt isoforms 1, 2, and 3. Therefore, detection and quantitation of Akt isoforms and their downstream targets for both expression levels and phosphorylation states is crucial for therapeutic drug development. Here we demonstrate application of the Multiple Detection Approach to characterize the PI3K-Akt signaling pathway. This approach uses sequential analysis of proteins separated and immobilized in a capillary, by performing either dual immunoassays or immunoassay with total protein on the Simple Western platform using chemiluminescence detection. Assays with control and LY294002 inhibitor-treated samples were developed. Proteins were first separated based on molecular weight via capillary electrophoresis, followed by immobilization via UV-crosslinking. Next, PI3K-Akt pathway targets were sequentially probed in the same capillary with total and phospho-specific antibodies, to determine the phosphorylated fraction relative to the total fraction. Primary antibodies from the first immunoprobe were removed after the detection step with >95% efficiency, as confirmed by re-probing with the same secondary antibody. Target protein loss was negligible due to covalent immobilization to the capillary wall, which was confirmed with re-probing, thus validating the quantitative data generated using this sequential approach. In addition, total protein normalization was performed in tandem with the immunoassay in the same capillary. This approach enables normalization of phosphorylation levels and/or target abundance in cell line or tissue samples, correcting for change in protein content due to treatment, loading, and/or other systematic errors. These results present the utility of the Multiple Detection Approach to quickly characterize and quantify proteins involved in signaling pathways targeted during development of cancer therapies.

Citation Format: Irina G. Kazakova, Crystal Tran, Anita Silver, Jessica Dermody. Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K-Akt pathway proteins [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 6293.
Abstract 6294: Absolute, quantitative and high throughput measurement of tissue biomarkers in FFPE specimens using QDB method
Authors: Zhang Jiandi
Published: September 2020

Absolute quantitation of tissue biomarkers in FFPE samples to aid drug discovery efforts. Tissue protein biomarkers play irreplaceable role in clinical diagnosis, prognosis and prediction. It is also heavily used in drug discovery to evaluate the effectiveness of a drug candidate, or to monitor its potential side effects at both preclinical stage and during clinical trials. In recent years, several tissue protein biomarkers are used as companion diagnostics to identify right patients for the right treatment.

However, while the consistency and objectivity are the prerequisites for any assays to be used in scientific studies, the current prevailing method for tissue biomarker assessment, immunohistochemistry (IHC), is known to be associated with inconsistency and subjectivity. For example, there were studies demonstrating as much as 20% inaccuracy in Her2 assessment for breast cancer patients using IHC method. Conceivably, this practice leads to significantly increased cost and efforts in drug discovery.

In this study, we demonstrated the usage of Quantitative Dot Blot (QDB) method to achieve absolute and quantitative measurements of tissue biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples in high throughput format. We were able to develop multiple QDB-based immunoassays using clinically validated antibodies for IHC in several types of cancers including breast cancer, lung cancer and prostate cancer. In the case of Her2, when the quantitatively measured results were converted into dichotomous variables using Receiver Operative Characteristics, we were able to achieve concordance with IHC at 94.8% (n=748), and 94.2% with Fluorescence in situ hybridization (FISH) (n=141). QDB-based assays also require minimum amount of total protein lysates, ranging from 0.1 to 0.5 μg per sample. Total protein lysates extracted from 2X5μm is sufficient to measure at least 5 to 10 protein biomarkers in triplicate.

The introduction of QDB method in drug discovery promises significantly increased objectivity and consistency of the results. The high throughput aspect of QDB method also suits well the daily activity in drug development. We believe the adoption of this method should significantly speed up the drug discovery process while reduce the related costs and efforts.

Citation Format: Jiandi Zhang. Absolute, quantitative and high throughput measurement of tissue biomarkers in FFPE specimens using QDB method [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 6294.