bluuebunny/crossref_metadata_2025 huggingface dataset:
Cordyceps militaris Induces Immunogenic Cell Death and Enhances Antitumor Immunogenic Response in Breast Cancer
Authors: Quan Xingguo , Kwak Beom Seok , Lee Ji-Young , Park Jin Hee , Lee Anbok , Kim Tae Hyun , Park SaeGwang
Published: September 2020

Cordyceps militaris has been widely used as a traditional medicine in East Asia. Its effects against breast cancer have been reported previously. However, whether C. militaris‐induced breast cancer cell death is immunogenic remains unelucidated. This study aimed to determine whether ethanolic extracts of C. militaris (CM‐EE) could induce immunogenic cell death (ICD) in breast cancer immunotherapy to improve the efficacy of immune checkpoint inhibitors. Human and mouse breast cancer cells were treated with various concentrations of CM‐EE for 72 h, and cytotoxicity was measured using the sulforhodamine B assay. Flow cytometry was used to assess cell death with annexin V/7‐AAD staining and measure the surface exposure of damage‐associated molecular pattern (DAMP) molecules including calreticulin, HSP70, and HSP90. Western blot for cleaved poly (ADP‐ribose) polymerase (PARP) was used to confirm apoptotic cell death. The immunogenicity of CM‐EE‐induced dead cells was evaluated using the CFSE dilution assay. CM‐EE reduced the viability of human (MCF7, MDA‐MB‐231, HS578T, and SKBR3) and mouse (4T1‐neu‐HA, TUBO‐HA, and TUBO‐P2J‐HA) breast cancer cells. The IC50 was 25–50 µg/ml in human breast cancer cells and 10–50 µg/ml in mouse breast cancer cells at 72 h. CM‐EE‐treated breast cancer cells were positively stained by annexin V, cleaved PARP, and cleaved caspase 3/7 which were increased upon CM‐EE treatment. Surface exposure of DAMP molecules was increased in dose‐ and time‐dependent manners. The CFSE dilution assay revealed that dendritic cells fed with CM‐EE‐treated breast cancer cells successfully stimulated tumor‐specific T cell proliferation without inhibiting DC function and T cell proliferation. The expression of PD‐L1 mRNA and protein level was increased in dose‐dependent manners. In addition, CM‐EE also potentiated the cytotoxic activity of tumor‐specific T cells. CM‐EE can induce immunogenic and apoptotic cell death in breast cancer cells, and it is a good candidate for cancer immunotherapy and may improve the efficacy of immune checkpoint inhibitors.
Development of Prognostic Indicator Based on Autophagy‐Related lncRNA Analysis in Colon Adenocarcinoma
Authors: Zhou Weige , Zhang Shijing , Li Hui-biao , Cai Zheyou , Tang Shuting , Chen Li-xia , Lang Jian-ying , Chen Zheng , Chen Xin-lin
Published: September 2020

There were no systematic researches about autophagy‐related long noncoding RNA (lncRNA) signatures to predict the survival of patients with colon adenocarcinoma. It was necessary to set up corresponding autophagy‐related lncRNA signatures. The expression profiles of lncRNAs which contained 480 colon adenocarcinoma samples were obtained from The Cancer Genome Atlas (TCGA) database. The coexpression network of lncRNAs and autophagy‐related genes was utilized to select autophagy‐related lncRNAs. The lncRNAs were further screened using univariate Cox regression. In addition, Lasso regression and multivariate Cox regression were used to develop an autophagy‐related lncRNA signature. A risk score based on the signature was established, and Cox regression was used to test whether it was an independent prognostic factor. The functional enrichment of autophagy‐related lncRNAs was visualized using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Ten prognostic autophagy‐related lncRNAs (AC027307.2, AC068580.3, AL138756.1, CD27‐AS1, EIF3J‐DT, LINC01011, LINC01063, LINC02381, AC073896.3, and SNHG16) were identified to be significantly different, which made up an autophagy‐related lncRNA signature. The signature divided patients with colon adenocarcinoma into the low‐risk group and the high‐risk group. A risk score based on the signature was a significantly independent factor for the patients with colon adenocarcinoma (HR = 1.088, 95%CI = 1.057 − 1.120; P < 0.001). Additionally, the ten lncRNAs were significantly enriched in autophagy process, metabolism, and tumor classical pathways. In conclusion, the ten autophagy‐related lncRNAs and their signature might be molecular biomarkers and therapeutic targets for the patients with colon adenocarcinoma.
Abstract 3480: Potential impact of voluntary exercise on patterns of metastasis in a colorectal cancer model
Authors: Wijler Liza A. , Raats Daniëlle A. , van Dijk Miriam , May Anne M. , Kranenburg Onno
Published: September 2020

Background: Physical activity (PA) is associated with a lower risk of colorectal cancer (CRC) initiation and death from CRC. However, evidence for a causal link between PA and disease progression is lacking. This study assesses the effect of voluntary exercise on metastases formation in a novel pre-clinical mouse model for late-stage metastatic CRC.

Methods: Murine colorectal tumor organoids were transplanted into the livers of immunocompetent C57Bl/6 mice using microsurgery. Animals were fed ad libitum. Voluntary exercise in tumor-bearing (TB) mice was modeled by offering running wheels for 0h/day (n=12), 3h/day (n=12) or 24h/day (n=12). Running wheel revolutions were cumulatively measured every 30min. PA was monitored by infrared cameras. Tumor progression was monitored using in vivo Bioluminescent Imaging. Food intake was measured by weighing. Body composition was monitored by echoMRI. All animals were sacrificed 12 weeks after tumor initiation.

Results: We have developed a novel model for exploring the effects of exercise on metastatic tumor progression in CRC. The model combines micro-surgical transplantation of tumor organoids, yielding spontaneously metastasizing CRC, with voluntary exercise. Voluntary exercise in both exercise groups led to an on average running distances of 2,3±0,3 km/night (total 221±29 km) in TB-3h animals and 6,6±3,0km/night (total 600±290km) in TB-24h animals. TB-3h and TB-24h animals developed more lean mass and lower fat mass compared to sedentary animals. Intermittent exercise had no effect on primary tumor growth and reduced metastasis. Vigorous exercise increased primary tumor growth (229606±186012) compared to TB-3h (152821±125902) and sedentary animals (165171±250993). Moreover, vigorous exercise non-significantly increased lung metastasis formation (2382±3539) compared to intermittent exercise (753±852) and to sedentary (1333±2902), while reducing metastatic spread to the liver and the peritoneal cavity was found.

Conclusions: These results suggest that exercise has the potential to influence the patterns and extent of metastasis in CRC, and that the degree of exercise is likely to be an important variable. Confirmation of these results in additional models, for instance using patient-derived organoids, is now warranted.

Citation Format: Liza A. Wijler, Daniëlle A. Raats, Miriam van Dijk, Anne M. May, Onno Kranenburg. Potential impact of voluntary exercise on patterns of metastasis in a colorectal cancer model [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 3480.
Abstract 4582: Tumor matrix-targeted polymeric glyco-adjuvant for in situ cancer vaccination
Authors: Gray Laura T. , Wilson D. Scott , Ishihara Jun , Sasaki Koichi , Raczy Michal , Swartz Melody , Hubbell Jeffrey
Published: September 2020

Cancer vaccination is challenging due to a lack of strong, clinically approved adjuvants and difficulty in identifying cancer-specific antigens. Here, in order to address these challenges, we report the creation of a targeted therapeutic cancer vaccine with two components: (1) a mannosylated, toll-like receptor 7 (TLR7)-agonizing polymer, p(Man-TLR7), and (2) a fusion protein consisting of serum albumin (SA) fused to a collagen binding domain (CBD), namely the A3 domain of von Willebrand factor. p(Man-TLR7) has been shown to elicit strong CD8+ T cell responses, which are vital for the success of cancer vaccines. As such, we hypothesized that chemically linking p(Man-TLR7) to a tumor-targeting protein would localize our polymeric glyco-adjuvant to the tumor, promoting the immunogenic processing of endogenous tumor antigens and bypassing the need to pre-identify tumor-specific antigens. In the approach presented here, CBD-SA acts as our tumor-targeting protein by actively binding to collagen in the tumor microenvironment via the CBD, in addition to utilizing the passive targeting of SA. Upon successful and reproducible generation of CBD-SA-p(Man-TLR7) conjugates, we observed that the conjugates retained the ability to bind both collagen I and collagen III. We then assessed the in vivo anti-tumor efficacy of our conjugates in B16F10 tumor-bearing mice, testing both murine CBD-SA-p(Man-TLR7) and human CBD-SA-p(Man-TLR7) conjugates to evaluate the translatability of our approach. We observed an 84% decrease in average B16F10 tumor size and improved overall survival compared to untreated controls when mice were treated with intravenously-delivered murine CBD-SA-p(Man-TLR7) in combination with anti-PD-1 and anti-CTLA-4 antibodies. In the case of intravenously-delivered human CBD-SA-p(Man-TLR7), a 60% decrease in average tumor size compared to untreated controls was observed when mice were treated with our vaccine as a monotherapy. Further improved anti-tumor efficacy (an 85% decrease in average tumor size compared to untreated controls) and improved overall survival were observed when we combined our human CBD-SA-p(Man-TLR7) vaccination with anti-PD-1 and anti-CTLA-4 antibodies. In conclusion, in situ vaccination with CBD-SA-p(Man-TLR7) is a translatable approach that synergizes with checkpoint antibody therapy to provide therapeutic benefit in treatment of a poorly immunogenic melanoma model.

Citation Format: Laura T. Gray, D. Scott Wilson, Jun Ishihara, Koichi Sasaki, Michal Raczy, Melody Swartz, Jeffrey Hubbell. Tumor matrix-targeted polymeric glyco-adjuvant for in situ cancer vaccination [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 4582.
Abstract 5236: Complete response to selpercatinib (LOXO-292), a highly selective RET inhibitor, in a patient with RET fusion-positive breast cancer
Authors: Takeda Masayuki , Watanabe Satomi , Rothenberg S. Michael , Kherani Jennifer , French Pearl P. , Olek Elizabeth
Published: September 2020

Background: Activating RET gene fusions have been reported in diverse solid tumors but <0.1% of breast cancers. Selpercatinib (LOXO-292), an investigational, highly selective and potent small molecule RET kinase inhibitor, has demonstrated significant anti-tumor activity in diverse RET fusion-positive solid tumor patients in an ongoing clinical trial (LIBRETTO-001, NCT03157128). Here we describe the first patient with RET fusion-positive breast cancer treated with selpercatinib.

Case summary: A 46-year-old Japanese woman presented with FDG-avid right axillary, right neck and mediastinal lymphadenopathy on PET-CT imaging. Ultrasound identified a hypoechoic nodule in the right breast; biopsy revealed malignant cells, ER(+), PgR(-), and HER2(-). She was diagnosed with stage IV adenocarcinoma of the right breast. Genomic testing results of a lymph node specimen identified a CCDC6-RET fusion with no other reported alterations, including none in BRCA1 or BRCA2. Consistent with local standard of care, she received treatment with tamoxifen plus goserelin from March through June 2019, but these were discontinued for progression in the right breast and new lesions in the left lower lung. Re-biopsy revealed that the molecular biology changed into triple-negative at the time of progression. On 10-Jul-19 she started treatment with selpercatinib at the recommended Phase 2 dose of 160 mg twice daily in the ongoing Phase 1/2 trial of selpercatinib. She experienced rapid clinical improvement, with resolution of right breast and neck pain and erythema. Spiral CT imaging on 31-Jul-2019 and 4-Sep-2019 demonstrated a partial response by RECIST 1.1 (tumor reductions -30% and -30%), with reduction in multiple right breast masses, axillary, neck and mediastinal lymphadenopathy and left lung metastases; repeat imaging on 30-Oct-19 demonstrated a complete response. At the time of this writing, she remains in response and on treatment for >150 days, with all adverse events grades 1-2, and most recovered and none requiring dose interruption or modification.

Conclusion: Selpercatinib demonstrated anti-tumor activity in a patient with RET fusion-positive breast cancer. To our knowledge, this is the first report of a breast cancer patient with a sustained response to selective, RET-targeted therapy and adds to the diversity of RET fusion-positive tumor types that may benefit from selective RET inhibition.

Citation Format: Masayuki Takeda, Satomi Watanabe, S. Michael Rothenberg, Jennifer Kherani, Pearl P. French, Elizabeth Olek. Complete response to selpercatinib (LOXO-292), a highly selective RET inhibitor, in a patient with RET fusion-positive breast 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 5236.
Abstract 5471: CNSigs: An R package for the identification of copy number signatures
Authors: Tallman David , Stockard Sinclair , Weber Zachary T. , Asad Sarah , Collier Katharine , Adams Elizabeth , Bey Jerome , Stover Daniel G.
Published: September 2020

Copy number aberrations (CNAs) are gains and losses of large genomic segments present across most cancer types and are a hallmark of cancer genomic alterations. However, the processes underlying CNAs and characteristic patterns of CNAs are poorly understood. Using single nucleotide variant (SNV) data, bioinformatic advances have identified underlying mutational signatures resulting from distinct mutational processes. Mutational signatures have led to a variety of discoveries, several of which are being investigated in clinical management of cancer. The development of algorithms able to uncover similar signatures for CNAs, rather than SNVs, is still in its infancy. Here we present an analysis package for the R programming language called CNSigs that allows for the robust and reproducible derivation of copy number signatures. Based on a list of extracted copy number features previously verified in ovarian cancer, we utilize mixed model approaches and non-negative matrix factorization to derive CNA signatures across cancer types. The development of a package to derive signatures from copy number data allows further investigation of underlying processes that may be responsible for these CNA fingerprints. To verify the reproducibility of the signatures, we derived signatures from two independent breast cancer datasets that use distinct copy number segmentation approaches. From these independent datasets we were able to recover the same signatures with high accuracy. We identified five signatures that are distinct from known breast cancer receptor-based or expression-based subtypes, yet reveal unique associations with underlying mutations, mutational processes, and transcriptional programs. To validate robustness, we applied the pipeline to 11 different cancer types from the TCGA dataset and showed that we were able to derive signatures from all of these cancer types of varying sample sizes. We identified certain signatures that cut across tumor types, while others are distinct to individual cancers. This work lays the groundwork for further analysis into the underlying molecular processes leading to these copy number signatures seen across cancer types. The CNSigs package also allows researchers to easily analyze their own samples to look for signatures in their copy number profiles and to compare these to signatures previously derived for their cancer type.

Citation Format: David Tallman, Sinclair Stockard, Zachary T. Weber, Sarah Asad, Katharine Collier, Elizabeth Adams, Jerome Bey, Daniel G. Stover. CNSigs: An R package for the identification of copy number signatures [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 5471.
Abstract 5542: ASTX660, a non-peptidomimetic antagonist of cIAP1/2 and XIAP, enhances immune mediated tumor killing and induction of immunogenic cell death
Authors: Ferrari Nicola , Ward George , Munck Joanne , Jueliger Simone , Davis Matthew , Gewinner Christina , Ferraldeschi Roberta , Lyons John , Sims Martin John
Published: September 2020

Introduction: ASTX660 is a potent, non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), which is currently being tested in a first in human phase I-II study in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor receptor superfamily mediated cell death and enhance anti-tumor immune responses. Herein, we describe the profile of ASTX660 in a range of preclinical tumor models and evaluate ASTX660's ability to enhance immune mediated killing of tumor cells, both in vitro and in vivo.

Methods: ASTX660 was tested in a panel of human and mouse tumor cell lines, assessing proliferation, apoptosis and immunogenic cell death (ICD). ASTX660 was tested alone or with recombinant Death Receptor ligands. Additionally, we used a novel co-culture system of tumor cell lines with anti-CD3 activated human peripheral blood mononuclear cells (PBMC) to assess ASTX660 effects on immune mediated cell killing. Target engagement along with induction of apoptosis and ICD markers were analysed by Western blotting and flow cytometry. Murine tumor models in immunocompetent and immunocompromised mice were utilised to evaluate the efficacy of ASTX660 in the presence or absence of an effective immune response.

Results: ASTX660 antagonised IAPs in cell lines, as indicated by a decrease in cIAP1 protein levels and disruption of the XIAP:SMAC protein complex. In murine T cell lymphoma cell lines ASTX660 treatment was associated with an increase in apoptosis and ICD. In immunocompetent mice, administration of ASTX660 delivered a complete regression of BW5147 tumor growth which was not seen in mice deficient in T and B cells. To confirm human relevance, ASTX660 also enhanced anti-CD3 stimulated PBMC-dependent killing of tumour cell lines.

Conclusion: The combination of the direct effects of ASTX660 on tumor cells and ASTX660 dependent effects on immune responses adds to the description of ASTX660's mode of action and our ongoing understanding of the clinical efficacy in T cell Lymphoma(1). Reference: A Hollebecque et al. 2019 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

Citation Format: Nicola Ferrari, George Ward, Joanne Munck, Simone Jueliger, Matthew Davis, Christina Gewinner, Roberta Ferraldeschi, John Lyons, Martin John Sims. ASTX660, a non-peptidomimetic antagonist of cIAP1/2 and XIAP, enhances immune mediated tumor killing and induction of immunogenic cell death [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 5542.
Abstract 5652: Dual CD73 enzyme blocker to reverse immune suppression
Authors: Yuan Jijun
Published: September 2020

Abstract:

CD73 is a cell surface enzyme that converts adenosine monophosphate to adenosine, a well-known immunosuppressive mediator that inhibits the cytotoxic function of T cells and natural killer cells. Tumors utilize this mechanism to escape attack by the immune system. Many attempts were made to antagonize the enzyme activity of CD73, including antibody drugs and small molecules. However, most current CD73 enzyme blockers were screened against tumor membrane CD73 and focused only on the membrane format of CD73. Our recent findings show that not only the membrane enzyme on tumor cells, but also the soluble format of CD73 shed off by tumor cells play important roles in generating immunosuppressive adenosine. To reverse the immune suppression exerted by CD73, both the membrane and the soluble format need to be blocked. We screened hundreds of anti-CD73 antibodies against soluble CD73 presented in tumor patient (CRC/LC/HCC/Melanoma) serum, and two candidates GB7002-01 and GB7002-04 were selected with great inhibition potency against tumor membrane CD73 and soluble CD73 in patient serum. GB7002-01/04 exhibited superior T cell proliferation promoting abilities in immune suppressive environment. To be precise, GB7002-01/04 totally reversed the T cell proliferation suppressive effects of pathological concentration of AMP. Moreover, this release of immune suppression was translated to better tumor killing by PBMC in the presence of GB7002-01/04. Humanized GB7002-01/04 has been put into in vivo efficacy study, preliminary toxicity and PK study. As we are observing anti-tumor efficacy, safety, and stability of these two candidates, they are promising agents for extended clinical exploration in several cancer indications.

Citation Format: Jijun Yuan. Dual CD73 enzyme blocker to reverse immune suppression [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 5652.
Abstract 6209: Monocytes phagocytose neutrophil extracellular traps (NETs) and differentiate into pro-inflammatory macrophages
Authors: Saito Akira , Ozawa Hideyuki , Takahashi Kazuya , Kimura Yuki , Tojo Mineyuki , Kumagai Yuko , Kanamaru Rihito , Tsukui Hidenori , Sata Naohiro , Kitayama Joji
Published: September 2020

Background: Macrophages are multipurpose phagocytes with various functions. Neutrophil extracellular traps (NETs) are web-like structures ensnare microbes which were composed of dsDNA, histones, and antimicrobial peptides. NETs are generally degraded by DNAse and macrophages, however, it is unknown how the degradation of NETs affects the local immune response thereafter. Here, we investigated the interaction between NETs and peripheral blood monocytes (PBMo).

Method: 5 × 105 Polymorphonuclear leukocytes(PMNs), PMNs irradiated with UV for 15 minutes (UV-PMN) and PMNs stimulated by PMA for 15min and extensively washed (PMA-PMN) were placed on 6 well plate and cultured for 2 hours and NETs formation was visualized by SYTOX green. Peripheral blood was obtained from healthy volunteers and CD14+ PBMo were purified by positive selection using the magnetic cell separation system. The PBMo were co-cultured with those PMNs at 37°C in 5% CO2 conditions for 3 days. The differentiation of macrophage was evaluated by the expression of M1/M2 markers by flowcytometry. Cytokine production was evaluated using the Cytokine Array system. The macrophages were co-cultured with autologous T cells stimulated with anti-CD3 mAb and the effect on T cell proliferation was evaluated with CFSE dilution assay.

Results: PMA-PMN, but not PMN or UV-PMN, produced massive SYTOX(+) NETs within 2 hours. Co-culture of the NETs with PBMo stained with PKH26 showed NETs are phagocytosed by monocytes within 24 hours. PBMo co-cultured with PMA-PMN showed significantly enhanced expression of CD86, and reduced expression of CD163 as compared to those co-cultured with PMNs or UV-PMN. However, the expression of CD86 was suppressed if PMA-PMN were pretreated with 100 u/ml DNase I. The supernatant of the co-culture with PMA-PMN showed markedly increased levels of IL-1β and TNF-α. PBMo co-cultured with PMA-PMN strongly promote the proliferation of autologous T cells. However, the enhanced T cell growth were significantly suppressed by the pretreatment of NETs with DNase I.

Conclusion: Monocytes appear to differentiate to proinflammatory macrophages after the phagocytosis of NETs. In tumor-bearing host, NETs are known to be increased in circulating blood, which may promote tumor associated inflammatory process.

Citation Format: Akira Saito, Hideyuki Ozawa, Kazuya Takahashi, Yuki Kimura, Mineyuki Tojo, Yuko Kumagai, Rihito Kanamaru, Hidenori Tsukui, Naohiro Sata, Joji Kitayama. Monocytes phagocytose neutrophil extracellular traps (NETs) and differentiate into pro-inflammatory macrophages [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 6209.
Abstract 6213: Extracellular vesicles MDM2-DNA derived from dedifferentiated liposarcoma induces MMP2 production from preadipocytes
Authors: Casadei Lucia , Calore Federica , Deshmukh Ameya , Zewdu Abeba , Fadda Paolo , Braggio Danielle , Wabitsch Martin , Croce Carlo M. , Lev Dina , Pollock Raphael
Published: September 2020

Dedifferentiated liposarcoma (DDLPS) is molecularly characterized by wt p53 and MDM2 gene amplification causing MDM2 protein over-production, the key oncogenic process in DDLPS. Commonly located in fat-bearing retroperitoneal areas, almost 60% of DDLPS patients undergo multifocal recurrence, typically amenable to palliative treatment only, and occasionally develop distant metastasis. These factors lead to an abysmal 10% 10 year overall survival rate. Tumor cell-derived extracellular vesicles (EVs) can facilitate loco-regional malignancy dissemination by depositing molecular factors that participate in the development of pre-metastatic niches for tumor cell implantation and growth. High number of MDM2 DNA molecules was identified within EVs from DDLPS patient serum (ROC vs normal; 0.95) as well as from DDLPS cell lines. This MDM2 DNA could be transferred to preadipocytes (P-a), a major and ubiquitous cellular component of the DDLPS tumor microenvironment (TME), with subsequent P-a production of matrix metalloproteinase 2 (MMP2), a critical component in the metastatic cascade. Since multifocal loco-regional DDLPS spreading is the main cause of the remarkably high lethality of this disease, a better understanding of the underlying oncogenic processes and their regulatory mechanisms is essential to improve the outcome of this devastating disease.

Citation Format: Lucia Casadei, Federica Calore, Ameya Deshmukh, Abeba Zewdu, Paolo Fadda, Danielle Braggio, Martin Wabitsch, Carlo M. Croce, Dina Lev, Raphael Pollock. Extracellular vesicles MDM2-DNA derived from dedifferentiated liposarcoma induces MMP2 production from preadipocytes [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 6213.
Abstract 625: Selective inhibition of NF1 loss-of-function driven oncogenic signaling in melanomas
Authors: Abecunas Cara , Fallahi-Sichani Mohammad , Leopold Judith , Whitehead Christopher , Ziemke Elizabeth
Published: September 2020

Oncogenic activation of the MAPK signaling pathway occurs across >90% of melanomas, motivating the clinical evaluation of MAPK-targeted therapies in these tumors. However, only BRAFV600E/K-mutated melanomas show consistent clinical responses to MAPK-targeted therapies. Inactivation of the tumor suppressor gene neurofibromin 1 (NF1) is among the mechanisms that drive hyperactivated RAS signaling in >10% of melanomas. These tumors, however, have variable dependency on RAS-mediated pathways, such as MAPK and PI3K/mTOR, and thus exhibit inconsistent sensitivity to targeted inhibitors of these pathways. It is of paramount importance, therefore, to identify new therapeutic strategies to block tumor cell growth or improve MAPK- or PI3K-targeted therapy response in NF1 loss-of-function (NF1LoF) melanomas. To identify actionable vulnerabilities in NF1LoF melanoma cells, we apply a systematic approach, combining multiplexed high-throughput immunofluorescence microscopy, single-cell measurements and computational analysis, to study the responses of differentially mutated human melanoma cell lines to a variety of RAS-related pathway inhibitors. Our analysis led to the identification of a multi-kinase inhibitor, MTX-216, which is efficacious in induction of tumor cell death and inhibition of NF1LoF melanoma growth both in vitro and in vivo, but does not exhibit cytotoxicity in BRAFV600E melanomas and wild-type melanocytes. Single-cell analysis of downstream RAS-mediated signaling markers (e.g. phospho-S6) and proliferation markers (e.g. Ki-67) demonstrated cell-to-cell heterogeneity in NF1LoF melanoma cells treated with commercially available kinase inhibitors, resulting in incomplete drug responses. MTX-216 reduces this single-cell variability, providing evidence that co-suppression of signaling and proliferation pathways is correlated with drug-induced cytotoxicity. Our systematic analysis, together with ongoing studies to uncover the mechanism of action of MTX-216 in NF1LoF melanomas, has the potential to identify new and effective treatment strategies for NF1-mutant melanomas.

Citation Format: Cara Abecunas, Mohammad Fallahi-Sichani, Judith Leopold, Christopher Whitehead, Elizabeth Ziemke. Selective inhibition of NF1 loss-of-function driven oncogenic signaling in melanomas [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 625.
Abstract 6281: Differential response of irradiated breast carcinoma cells to DNA-PK inhibition: Spheroid response is governed by cell adhesion effects rather than changes in radiosensitivity
Authors: Yu Jing , Lu Ryan , Nedrow Jessie , Sgouros George
Published: September 2020

Agents that increase tumor radiosensitivity are of interest in improving outcomes in radiotherapy (XRT). DNA-PKcs inhibitors lead to radiosensitization and also to alterations in cell adhesion proteins. Using a HER2 positive mammary carcinoma cell line, NT2.5, we investigated the impact of Nu7441, a potent and selective DNA-PKcs inhibitor together with external beam irradiation in both 2D monolayer and 3D spheroid cell culture systems. Colony formation assays were performed for cells irradiated as monolayers and also for cells disaggregated from spheroids. Cells grown in monolayer culture and as spheroids were incubated with Nu7441 (5 µM) for 24 h after different doses of external beam irradiation. In monolayer culture cells, α/β increased from 3.0 ± 0.2 Gy (XRT alone) to 6.9 ± 0.2 Gy when XRT was followed by Nu7441 incubation. Corresponding α/β values for cells obtained by disaggregating treated spheroids were 3.6 ± 0.7 Gy (XRT alone) and 3.5 ± 0.2 Gy (XRT+Nu7441). In contrast to the minimal change in α/β, spheroids survival was highly sensitive to Nu7441 incubation. 21 days after 0, 2, 4, 6, 8, or 10 Gy XRT alone 100, 83, 75, 63, 56, and 31%, respectively, of the irradiated spheroids remained intact; when Nu7441 treatment was involved, 56% remained intact at 2 Gy and 13% at 4 Gy, no spheroids survived to 3 weeks at 6 Gy or more. We also found a progressive increase in fragmentation for spheroids exposed only to Nu7441 (0 Gy): 3/24, 9/24 and 17/24 fragmented after 14, 21 and 28 days of incubation, respectively. The discrepancy between the minimal change in α/β from cells derived from spheroids and the spheroid growth response was not related to poor penetration of Nu7441 since the colony formation assay results for different sizes of spheroids (180 to 400 µm) treated with 4 Gy with or without Nu7441were not significantly different. The results suggest that spheroid response to DNA-PKcs inhibitors is driven by their known inhibition of cell-cell adhesion pathways and potentially less so by radiosensitization.

Citation Format: Jing Yu, Ryan Lu, Jessie Nedrow, George Sgouros. Differential response of irradiated breast carcinoma cells to DNA-PK inhibition: Spheroid response is governed by cell adhesion effects rather than changes in radiosensitivity [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 6281.
Abstract 6309: Development of protein conformational array ELISA for p53 and SHP2 allosteric regulator screening and analysis
Authors: Wang Xing
Published: September 2020

P53 and SHP2 plays important role during cancer development. For p53, more than 50% of cancers were shown to have gene mutation in this tumor suppressor, however how these gene mutations impact the p53 protein conformation and function have not be studies in much detail. A Protein Conformational Array ELISA was developed with 17 polyclonal antibodies covering the whole p53 molecule, by measuring the linear epitope distribution on the surface of the p53 protein, the conformational (Higher Order Structure or HOS) can be described quantitively. Initial studies have shown that in lung cancer cells, the level of p53 is significantly increased and more interestingly, the p53 HOS has changed at its N-terminal as compared with the wild type. For SHP2, a protein phosphatase, a 27-antibody panel was developed to characterize the HOS changes. Because of the generic nature of the phosphate binding site, it has been very challenging to develop phosphatase inhibitors, however studies have shown that allosteric regulators can be identified with excellent anti-tumor activities. Currently, some of the allosteric regulators identified are being tested in the clinic. The SHP2 Conformational Array ELISA will provide a robust platform for the screening and testing of potential allosteric regulators with its molecular resolution and high-throughput capability. This presentation will describe the development of the two conformational arrays and their applications in the study of p53 and SHP2 from different cancer cell lines and the characterization of the allosteric regulators identified.

Citation Format: Xing Wang. Development of protein conformational array ELISA for p53 and SHP2 allosteric regulator screening and analysis [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 6309.
Abstract 6311: An enabling technology to detect low abundance targets in cells and tissues
Authors: Patel Deven , Liu Jixiang , Dong Pengfei , Kan Shu , Wu Haiyan , Zhao Qin , Diwu Zhenjun
Published: September 2020

The rapid advances in sequencing, genomics and bioinformatics have generated a huge amount of genomic data available at our disposal. However, some proteins, the products of those genes, are expressed in cells or tissues at extremely low levels, making the identification and characterization of proteins the bottle neck for biomedical research field. Approximately, 65% of the human proteome is expressed at less than a few thousand copies. Thus, the availability of protein detection probes and tools becomes a critical element to measure lower abundance protein targets in the cells and tissues to identify clinical significance of the particular target proteins. There are several common signal amplification methods available for detecting low abundance protein targets. However, in some cases, their sensitivities are still not satisfactory. Biotin/avidin amplification system has been predominantly used to enhance detection sensitivity but it is still sometimes not sufficient, leading to extremely challenging to accurately measure the low expressed protein targets. There is urgently unmet need to develop a new signal amplification system with enhanced detection sensitivity. We develop the Power Styramide Signal Amplification (PSA) system that can detect extremely low-abundance targets in cells and tissues. The PSA technique is much more sensitive than the traditionally used biotin/avidin and TSA amplification methods. PSA uses a concept that utilizes the catalytic activity of horseradish peroxide (HRP) for covalent deposition of Styramide in situ. The radicals generated from the Styramide based substrates have much higher reactivity towards the target, thus producing a more robust signal and achieve much higher sensitivity towards lower expressed targets. When this technique was compared to directly conjugated antibody, we saw a significant increase in the signal and improved detection limit by 10-100 folds. Another advantage using Styramide-based substrates is that the need of either primary or secondary antibodies gets significantly reduced. You can get robust signal just by using very little antibodies without sacrificing the quality of the fluorescent images. Overall, this new technology enables us to detect low expressed targets in cells or tissues using lower antibody concentration with high detection limit.

Citation Format: Deven Patel, Jixiang Liu, Pengfei Dong, Shu Kan, Haiyan Wu, Qin Zhao, Zhenjun Diwu. An enabling technology to detect low abundance targets in cells and tissues [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 6311.
Abstract 6346: Optimizing precision combination therapies based on single-cell profiling of brain tumor biopsies
Authors: Babikir Husam , Wang Lin , Shamardani Karin , Catalan Francisca , Kohanbash Gary , Aghi Manish K. , Phillips Joanna J. , Diaz Aaron A.
Published: September 2020

Glioblastomas (GBM) display dramatic levels of intra-tumoral heterogeneity. This heterogeneity contributes significantly to resistance to standard and emerging treatments. As a result, GBM remains the deadliest primary tumor of the adult brain. Moreover, most preclinical studies of GBM use models of primary GBM, most studies of clinical specimens use tissue derived from primary GBMs, despite the fact that most clinical trials enroll patients at recurrence. There is a paucity of information about recurrent GBM. To address these critical barriers, we devised a strategy to optimize precision drug combinations based on an understanding of a patient's intratumor heterogeneity, ascertained via single-cell profiling. We conducted single-nucleus RNA sequencing (snRNA-seq) and/or single-nucleus assay for transposon-accessible chromatin (snATAC-seq) sequencing for 16 recurrent GBMs. For 6 of these cases we also derived cell lines from patient biopsies. We identified lineage hierarchies of proliferating cells and their more differentiated progeny. Based on this, we performed an in vitro drug-combination screen using FDA-approved chemotherapies, targeting pathways learned from these single-cell signatures. We found that targeting distinct cellular states in the inferred lineage hierarchy of a patient's tumor provides a more complete treatment than targeting one state alone. Additionally, we will present the results of our ongoing in vivo validation activities and profiling efforts. This work presents a novel paradigm for identifying precision combinations of chemotherapies targeted to phenotypes learned from single-cell profiling of a patient's tumor biopsy.

Citation Format: Husam Babikir, Lin Wang, Karin Shamardani, Francisca Catalan, Gary Kohanbash, Manish K. Aghi, Joanna J. Phillips, Aaron A. Diaz. Optimizing precision combination therapies based on single-cell profiling of brain tumor biopsies [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 6346.
Abstract 6356: Discovery of seleno allyl hybrid compounds, the Se incorporated mimics of naturally occurring garlic based allylic compounds, as potential anticancer agents
Authors: Talavera Irene , Astrain Nora , Barajas Miguel , Encío Ignacio , Sharma Arun K. , Sanmartín Carmen , Plano Daniel
Published: September 2020

Many synthetic derivatives of natural products have been found to exhibit biological activity towards cancer. One prominent example of such natural products are the garlic-based compounds. Given the well stablished preventive effects shown by garlic and some of its allylic components and a well-established chemopreventive and therapeutic effects shown by selenium (Se) compounds, we pursued to design Se-allyl hybrid compounds in order to increase efficacy as compared to naturally occurring allylic compounds. A total of 18 new selenoallyl (SeAl) compounds were designed following two different synthetic approaches. SeAl functionality was chosen to mimic the garlic bioactive allylic components. All the synthesized compounds were screened against colorectal (HT29 and HCT116) and mammary (MCF-7) cancer cell lines (48h treatment). Four most active compounds identified were submitted to the NCI Developmental Therapeutic Program (DTP). Two out of the four compounds were selected for the dose-response assay against a panel of 60 cancer cell lines. Both the compounds exhibited impressive cytotoxicity - lethal dose 50 (LD50) values below 9.2 µM in most of the melanoma and ovarian cancer cell lines tested (8 out of 9 for melanoma and 5 out of 7 for ovarian cancer cells for both compounds). Additionally, the Se-allyl derivative bearing an aspirin substituent presented a significant tumor size inhibition in a castration-resistant prostate cancer xenograft model at a dose of 4 mg/kg given intraperitoneally. Thus, this compound has the potential to be a promising candidate for prostate cancer treatment and potentially other cancers. To conclude, we firmly believe, based on the evidences obtained and presented herein, that structural modifications involving the hybridization approach with Se and naturally occurring allylic compounds could stand as a novel toolkit for anticancer drug development.

Citation Format: Irene Talavera, Nora Astrain, Miguel Barajas, Ignacio Encío, Arun K. Sharma, Carmen Sanmartín, Daniel Plano. Discovery of seleno allyl hybrid compounds, the Se incorporated mimics of naturally occurring garlic based allylic compounds, as potential anticancer agents [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 6356.
Abstract 6494: Personalize My Treatment (PMT): A pan-Canadian initiative integrating precision oncology across Canada - PMT-001 pilot project
Authors: Marques Maud , Gambaro Karen , de Tertre Mathilde Couetoux , McNamara Suzan , Hoffer Cyrla ,   The Exactis Network, Fajzel Richard , Batist Gerald
Published: September 2020

More than 90% of cancer therapeutics in development are targeted therapies. Although the number of patients who can benefit from targeted therapies is increasing, many of the biomarkers are identified in only a small fraction of patients. Identifying and enrolling these rare cancer patient sub-populations into clinical trials presents an enormous challenge to both pharmaceutical companies and clinicians. Furthermore, in Canada, the lack of harmonized standard of care molecular testing for cancer patients results in a discordance in the number of genes tested across Hospital sites. Identifying patients with biomarkers of interest across Canada is essential to attract precision oncology clinical trials to Canada and to offer cancer patients better therapeutic options and improved outcomes. Founded in 2014, Exactis Innovation is a non-profit Academic Research Organization which aims to integrate precision oncology across Canada. Exactis has consolidated a pan-Canadian network of 13 cancer care centre sites and 4 federated laboratories. Network sites host an REB-approved molecular cancer patient registry (PMT), through which patients can be profiled, identified as carrying a biomarker, followed throughout their disease trajectory and be re-contacted if their molecular profile matches a clinical trial or an approved therapy. Here we report the results of Exactis' first profiling initiative (PMT-001), performed across 8 Exactis Network sites within 3 Canadian provinces in a four months' time period. In summary, 447 individual tumors specimens were collected from breast, colorectal, lung and ovary cancer patients and 382 were profiled using the Oncomine comprehensive assay v3 (OCAv3) panel DNA (n=365) and OCAv3 DNA/RNA (n=151). At least one aberration was identified in 346 participants. As expected, the top mutated genes across the 4 cohorts were TP53 (50%), PIK3CA(15%), KRAS (12.5%) and BRCA1/2 (12%/11%). At the fusion level, we identified EML4-ALK (5.2%), KIF5B-RET(1.7%) and MET exon 14 skipping (3.4%) in NSCLC participants and we identified RSPO3 (3%) and RSPO2 (2%) fusion in colorectal cancer participants. The profiling pilot of the PMT initiative shows how strong foundations within the Exactis pan-Canadian network is resulting in fast, high quality and meaningful molecular data for Canadian cancer patients.

Citation Format: Maud Marques, Karen Gambaro, Mathilde Couetoux de Tertre, Suzan McNamara, Cyrla Hoffer, The Exactis Network, Richard Fajzel, Gerald Batist. Personalize My Treatment (PMT): A pan-Canadian initiative integrating precision oncology across Canada - PMT-001 pilot project [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 6494.