bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1100: Genetic driver-mutations define composition and properties of tumor-associated myeloid cells in glioblastoma
Authors: Chen Zhihong , Hurting Cameron J. , Gabanic Ben , Murukuti Jyothi , Hambardzumyan Dolores
Published: August 2019

The GBM microenvironment is composed of numerous non-neoplastic cells, among which tumor-associated myeloid cells (TAMs) are the most abundant. TAMs are constituted of several different cell types, including brain microglia and bone-marrow-derived infiltrating cells, which can be further divided into inflammatory monocytes, monocyte-derived macrophages, and polymorphnuclear neutrophils. These myeloid cells are believed to promote tumor growth and immune evasion by expressing immune checkpoint proteins. Remarkable progress has been made in treating lung cancer and melanoma by using immune-checkpoint inhibitors; however, mixed results were obtained for its application in GBM. To examine how molecular-subtypes of GBM influence the composition and immunosuppression of tumor-associated myeloid cells, we used the RCAS_tv-a system, a somatic cell-specific gene transfer system, to generate de novo murine GBM (mGBM). By manipulating different known human oncogenic-driver mutations such as overexpressing PDGFB or EGFRVIII, or silencing NF1, we generated Proneural (PN), Classical (CL) and Mesenchymal (MES) GBMs respectively, which phenotypically resemble their human counterparts. We found that the majority (80%) of tumor-infiltrating myeloid cells in PN GBM are bone-marrow derived macrophages, whereas this number is significantly less in CL and MES mGBM. Interestingly, unique to MES, there is a significant presence (16%) of neutrophils in these tumors than in other subtypes (4%). Further characterization for subtype-specific expression of PD-L1, a druggable immune checkpoint molecule, by flow cytometry shows that MES has the highest expression of PD-L1), whereas the expression is lower in PN and CL subtypes. Treating GBM-bearing mice with anti-PDL1 antibodies did not prolong their survival. Concurrent treatment of irradiation and anti-PDL1 antibody did not confer significant survival advantage either compared to controls. These results reflect the unsuccessful trails of recent human clinical studies; however, it also emphasizes the importance of understanding the mechanism of resistance in GBM immunotherapy before effective immune treatment can be established. Our immunocompetent mouse models provide indispensable tools for these studies.

Note: This abstract was not presented at the meeting.

Citation Format: Zhihong Chen, Cameron J. Hurting, Ben Gabanic, Jyothi Murukuti, Dolores Hambardzumyan. Genetic driver-mutations define composition and properties of tumor-associated myeloid cells in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1100.
Abstract 1106: Co-administration of the iRGD tumor-penetrating peptide improves the tumor immunostimulatory effects of low-dose IL-2
Authors: Jarvelainen Harri , Botta Gregory P. , Mendoza Tatiana Hurtado De , Ruoslahti Erkki
Published: August 2019

Background: Recombinant IL-2 at high doses is an effective immunotherapy treatment for various types of solid tumors. However, its clinical utility has been limited by serious mechanism-based side-effects. The clinical-stage iRGD peptide specifically targets tumors and, via activation of the ‘CendR’ trans-tissue transport pathway, increases tumor penetration and accumulation of various types of co-administered drugs. By selectively increasing the drug delivery into tumors, but not to normal tissues, iRGD potentiates the pharmacological activity of various anti-cancer agents within tumor tissue, while reducing their toxicities. In this study we investigate whether co-administration of iRGD with low-dose IL-2 can improve the tumor immune microenvironment by increasing in the ratios of tumor-killing effector CD8 cells vs. the immunosuppressive T-regulatory cells.

Methods: Subcutaneous breast tumors (4T1) were generated in immunocompetent mice. Mice were treated with either vehicle control, iRGD, IL-2, or IL-2 + iRGD for 5 days. Tumors were preserved for immunohistochemistry (IHC) or enzymatically digested for fluorescence activated cell sorting (FACS) 16 hours after the last dosing. The FACS and IHC panels were designed to detect the percentage of total T cells, CD4 and CD8 T cells, and Treg cells.

Results: Low doses of IL-2 alone were found to increase Tregs within the tumor but had no effect in CD4 or CD8 effector T cells, compared to vehicle treatment. However, when Low dose IL-2 was co-administered with iRGD, we observed a significantly lower percentage of T regs and an increase in CD4 effector T cells. More importantly the ratio of CD8 / Treg cells was increased by at least 10-fold, compared to low dose IL-2 alone.

Conclusions: The combination treatment with iRGD and low doses of IL-2 is capable of favorably altering the tumor immune microenvironment such that immunosuppressive T reg cells were reduced, while there was an increase in the effector CD4 and CD8 T-cell populations. The tumor-selective IL-2 pharmacology benefit obtained with iRGD co-administration may provide new options for the use of the well-validated IL-2 in solid tumor patients, including a strategy to overcome the primary resistance to PD-1 blockade.

Citation Format: Harri Jarvelainen, Gregory P. Botta, Tatiana Hurtado De Mendoza, Erkki Ruoslahti. Co-administration of the iRGD tumor-penetrating peptide improves the tumor immunostimulatory effects of low-dose IL-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1106.
Abstract 113: c-Kit-dependent tissue resident macrophage progenitors drive cancer progression
Authors: Pathria Paulina , Takahashi Hideyuki , Kaneda Megan , Pu Minya , Messer Karen , Shepard Ryan M. , Louis Tiani L. , Shih Ann , Bertagnolli Mark , Wrasidlo Wolfgang , Cheresh David A. , Varner Judith A.
Published: August 2019

Macrophages play a key role in promoting tumor growth and resistance to therapy. Here we show that Tissue Resident Macrophages (TRM) as well as Bone Marrow-Derived Macrophages (BMDM) play critical but unique roles in promoting tumor growth. TRM were recently shown to originate in the yolk sac or fetal liver during embryogenesis; these cells self-maintain in post-natal tissues independent of hematopoietic stem cells. We found that BMDM are CD11b+Gr1+F4/80loCX3CR1loCCR2+and are recruited to tumors in a CCR2-dependent manner. In contrast, CD11b+Gr1-F4/80hiCX3CR1hiCCR2- TRMs accumulate in tumors independently of the trafficking receptor CCR2. Gene expression and functional studies indicate that tumor-derived TRM are highly proliferative, immune suppressive and distinct from BMDM. We show that TRM develop from c-Kit/c-KitL - dependent TRM progenitors that are abundant in tumors but not in normal tissues; purified progenitors form macrophages and potently stimulate tumor growth when adoptively transferred into mice. Tumor cells induce the expansion of TRM progenitors by secreting Stem Cell Factor (SCF/c-KitL). Notably, in vitro and in vivo proliferation of TRM progenitors and tumor growth are significantly inhibited by SCF and c-Kit inhibitors, including a novel, allosteric dual inhibitor of cKit and CDK8/19 that dramatically suppresses tumor growth by targeting both TRM and tumor cells. As cKit inhibitors synergize with other immune therapy regimens to suppress tumor growth, our studies identify cKit as a valuable target for immune therapy of solid tumors.

Citation Format: Paulina Pathria, Hideyuki Takahashi, Megan Kaneda, Minya Pu, Karen Messer, Ryan M. Shepard, Tiani L. Louis, Ann Shih, Mark Bertagnolli, Wolfgang Wrasidlo, David A. Cheresh, Judith A. Varner. c-Kit-dependent tissue resident macrophage progenitors drive cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 113.
Abstract 1146: [18F]FPyGal: A novel ß-galactosidase specific PET tracer for in vivo imaging of tumor senescence
Authors: Krueger Marcel A. , Cotton Jonathan M. , Zhou Benyuan , Wolter Katharina , Schwenck Johannes , Kuehn Anna , Fuchs Kerstin , Maurer Andreas , Fougere Christian La , Zender Lars , Pichler Bernd J.
Published: August 2019

Senescence influences treatment outcome in cancer. Therefore it is of great interest to develop a diagnostic tool to detect senescent cells in cancer patients. Here we describe the evaluation of the PET tracer FPyGal for non-invasive imaging of ß-galactosidase as a surrogate marker for senescence. In vitro, we induced senescence in HCT116 cells by doxorubicin, in a liver progenitor cell line by p53-reactivation and in two liver carcinoma cell lines by a ribosomal checkpoint inhibitor (RCI). The cells were then incubated with FPyGal and tracer uptake was determined in a gamma-counter. For in vivo testing, the described cell lines were injected s.c. in mice. The tumor bearing mice were then treated appropriately to induce senescence. PET/MR scans were performed after i.v. injection of FPyGal for assessment of tracer uptake in the tumors. Ex vivo autoradiographies and X-Gal stainings were prepared to correlate intratumoral distribution of ⇓-galactosidase activity and tracer uptake. After toxicology studies, a pilot first-in-man study was performed in a cancer patient treated with Alisertib. Compared to the respective control cells, in vitro tracer uptake was significantly increased in all four cellular models tested. The strongest increase was observed in one of the liver carcinoma cell lines with a >3-fold increase in senescent cells. FPyGal tracer uptake increased in control HCT116 tumors from 1.1 to 1.7%ID/cc in senescent cells in in vivo PET/MRI studies and in the HRas model from 0.9 to 1.5 %ID/cc. In one of the liver tumor models we could observe a 2-fold increase in treated mice. Ex vivo ⇓-galactosidase staining and IHC of tumor tissue confirmed induction of senescence. Autoradiography and X-Gal staining showed an intratumoral correlation of ⇓-gal activity and FPyGal uptake. The compound passed the toxicology tests. A first patient study revealed high and heterogeneous tracer uptake in a liver metastasis. FPyGal shows increased in vitro and in vivo uptake in senescent cells and tumors. No toxicity of the compound could be detected and a first-in-man study showed promising results. Clinical trials including histological cross-validation are currently being prepared.

Citation Format: Marcel A. Krueger, Jonathan M. Cotton, Benyuan Zhou, Katharina Wolter, Johannes Schwenck, Anna Kuehn, Kerstin Fuchs, Andreas Maurer, Christian La Fougere, Lars Zender, Bernd J. Pichler. [18F]FPyGal: A novel ß-galactosidase specific PET tracer for in vivo imaging of tumor senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1146.
Abstract 1147: A phage display identified peptide selectively binds to kidney injury molecule-1(KIM-1) and detects KIM-1-overexpressing tumors in vivo
Authors: Yoon Jae-Won , Lee Soo-woong , Haque Md. Enamul , Park Rang- Woon , Baek Moon-Chang , Kim Dong-Kyu , Kim Sang Kyoon , Lee Byungheon
Published: August 2019

Kidney injury molecule-1 (KIM-1), also known as hepatitis A virus cellular receptor-1 and T cell Ig mucin-1, is a transmembrane protein, which is not overexpressed in normal kidneys, but is significantly upregulated in damaged kidney tubular epithelial cells in toxic and ischemic acute kidney injury. Of interest, KIM-1 is overexpressed in most cases of renal cell carcinoma (RCC) and in other types of tumors including ovarian clear cell carcinoma and lung cancer. The extracellular domain of KIM-1 can be identified in the urine of patients with RCC and can thus be used as a biomarker for the detection of RCC. This study was carried out to identify a peptide that selectively binds to KIM-1 by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1-overexpressing tumors in vivo. Biopanning of a phage-displayed peptide library was performed on KIM-1-coated plates. Compared to other phage clones selected after biopanning, a phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 as examined by fluorometry and KIM-1-overexpressing 769-P renal tumor cells 1 as examined by ELISA. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin as examined by fluorometry and to KIM-1-overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1-low expressing HEK293 normal cells when examined by flow cytometry. Co-localization and competition assays using an anti-KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. Whole-body fluorescence imaging demonstrated selective homing of the CNWMINKEC peptide to KIM-1-overexpressing A498 renal tumor compared to KIM-1-low expressing HepG2 liver tumor in mice. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 h in the presence of serum. These results suggest that the CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1-overexpressing tumors.

Keywords In vivo imaging, kidney injury molecule-1, peptide, phage display, renal cell carcinoma

Note: This abstract was not presented at the meeting.

Citation Format: Jae-Won Yoon, Soo-woong Lee, Md. Enamul Haque, Rang- Woon Park, Moon-Chang Baek, Dong-Kyu Kim, Sang Kyoon Kim, Byungheon Lee. A phage display identified peptide selectively binds to kidney injury molecule-1(KIM-1) and detects KIM-1-overexpressing tumors in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1147.
Abstract 1154: DCLK1 monoclonal antibody-based CAR-T cells as novel treatment strategy against multiple myeloma
Authors: Sureban Sripathi M. , Berahovich Robert , Zhou Hua , Xu Shirley , Wu Lijun , Golubovskaya Vita , Qu Dongfeng , May Randal , Bannerman-Menson Edwin , Houchen Courtney W.
Published: August 2019

Multiple Myeloma (MM) is characterized by the clonal expansion of malignant plasma cells in the bone marrow, and is the second most common hematological cancer. Cellular therapies including chimeric antigen T receptor cell (CAR-T) have emerged as a promising therapeutic strategy to combat this devastating disease. MM are derived from a rare population of cancer stem cells (CSC) that are responsible for initiation, progression, resistance to conventional therapy and recurrence. To date there have been no CAR-T therapies directed at CSC-specific antigens. In this report, we describe the effects of a novel CAR-T targeting the CSC marker DCLK1 on MM cells. Using mouse models, it has been demonstrated that DCLK1 marks the cell of origin of colon and pancreatic cancers. DCLK1 regulates epithelial to mesenchymal transition (EMT), and its upregulation is associated with poor overall survival in many cancer subtypes. Inhibition of DCLK1 activity following RNA silencing resulted in tumor xenograft growth arrest. These features lead us to explore the therapeutic potential of targeting DCLK1+ cells in MM. In order to create a novel DCLK1 CAR, we generated a new humanized DCLK1 monoclonal antibody, clone CBT-15 #8. This antibody recognizes a small extra cellular C-terminal epitope on human isoform 2 and 4 of DCLK1. Novel DCLK1 CAR-T cells containing the clone were generated and detected with anti-human FAB antibody by flow cytometry. Furthermore, we observed cell surface expression of DCLK1 on >93 percent of MM Cells (RPMI8226) by flow cytometry. The CAR-T cells had significantly higher cytotoxicity in MM (RPMI8226 and MM1S) cells than in leukemia K562 cells. The CAR-T cells secreted significantly higher levels of IFN-γ in vitro than T cells (p=0.0036). Further studies are underway to determine its efficacy in inhibiting tumor xenografts in vivo. This report is the first to link the TSC marker DCLK1 to MM and may represent a novel therapeutic strategy for MM and other hematologic malignancies.

Citation Format: Sripathi M. Sureban, Robert Berahovich, Hua Zhou, Shirley Xu, Lijun Wu, Vita Golubovskaya, Dongfeng Qu, Randal May, Edwin Bannerman-Menson, Courtney W. Houchen. DCLK1 monoclonal antibody-based CAR-T cells as novel treatment strategy against multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1154.
Abstract 1156: Skeletal muscle progenitor cell-derived exosomes have therapeutic potential in inhibition of prostate cancer cell proliferation
Authors: Sinha Krishna M. , Yarmand Rozita , Kahraman Nermin , Duzagac Fahriye , Lopez Gabriel , Ozpolat Bulent , Huard Johnny
Published: August 2019

Adult stem cells have been used as regenerative medicine for treating human diseases through gene therapy approach. These stem cells secrete cytokines, growth factors and extracellular vesicles including exosomes, which may induce antitumor activities. We have demonstrated that muscle progenitor cells (MPCs) from adult (murine and human) skeletal muscle have a high regenerative potential to repair bone, skeletal muscle, and cartilage. However, to date, the potential therapeutic use of MPCs in cancers has not yet been investigated. The objectives of this study were to identify and examine the roles of secreted factors including exosomes from MPCs on the proliferation/survival, motility/migration and invasive potential of prostate cancer (PCa) cells. Here we showed that co-culture of PCa cell lines, PC3 and C42B, with MPCs in trans-well inserts strongly suppressed PCa cell proliferation and survival by activating CDK inhibitors (p16 and p27) and apoptotic BBC3 and NOXA genes. Mass spectrometry analysis of factors secreted by MPCs identified several proteins with known anti-proliferative functions, including IGFBP6/7, pigment epithelium-derived factor (PEDF), HPCAL1, NO, and SPARC. Conditioned media (CM) from MPCs significantly increased docetaxel-induced cytotoxicity of PCa cells by suppressing the expression of key genes including AR, MLL, FOXA1, TWF1, and TGIF. Next, we isolated exosomes from cultured mouse and human MPCs through step-wise ultracentrifugation methods and tested their effect in the proliferation of cancer cells. We observed that treatment of PCa cells with MPC-derived exosomes inhibited cell proliferation as assayed with MTT and down-regulated the expression of AR and its target NKX3.1 genes. We also observed that the migration ability of ovarian cancer cells (OVAC5) and pancreatic cancer (Panc1), was markedly reduced following MPC-derived exosome treatments compared to untreated cells. Taken together, our data suggest that MPCs can suppress the proliferation and survival of cancer cells through secreted factors including exosomes. Thus, adult stem cell-based therapy along with existing therapies may be a promising strategy in treating various solid cancers as well as regenerating cancer-induced bone and muscle tissue lesions.

Citation Format: Krishna M. Sinha, Rozita Yarmand, Nermin Kahraman, Fahriye Duzagac, Gabriel Lopez, Bulent Ozpolat, Johnny Huard. Skeletal muscle progenitor cell-derived exosomes have therapeutic potential in inhibition of prostate cancer cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1156.
Abstract 1166: TDO2 overexpression is associated with cancer stem cells and poor prognosis in esophageal squamous cell carcinoma
Authors: Pham Thang Quoc , Oue Naohide , Sekino Yohei , Yamamoto Yuji , Sakamoto Naoya , Honma Ririno , Sentani Kazuhiro , Yasui Wataru
Published: August 2019

Objective: Esophageal cancer is one of the deadliest cancers in the world, and the main subtype is esophageal squamous cell carcinoma (ESCC), which comprises 90% of cases. We previously analyzed the gene expression profile of spheroid colonies and parental cells derived from gastric cancer (GC) cell lines by microarray analysis. Among the genes upregulated in spheroid colonies, tryptophan 2,3-dioxygenase (TDO2) is dramatically upregulated in MKN-1 cells (derived from adenosquamous cell carcinoma) compared with other GC cell lines. Expression of TDO2, an enzyme involved in tryptophan catabolism, has been linked with tumor survival and poor prognosis of brain and breast cancer. However, no studies have investigated the potential role of TDO2 in esophageal cancer. Here we explored the expression and biological significance of TDO2 in ESCC.

Methods: To explore the expression of TDO2 in cancer and normal samples, we used an online analytical tool, the Broad Institute TCGA Genome Data Analysis Center, http://firebrowse.org/. For quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we used 10 ESCC samples (tumor tissues and the corresponding non-neoplastic tissue). TDO2 protein expression was evaluated in 90 ESCC tissue samples by immunohistochemistry. TDO2 function in ESCC cell lines and spheroid colony formation was evaluated by RNA interference (RNAi). Cell growth was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Modified Boyden chamber assays were performed to examine cell invasiveness.

Results: TDO2 expression was upregulated in most cancers except for liver cancer and pancreas cancer. The fold change expression of TDO2 between normal and cancer tissue was the highest in esophageal cancer. TDO2 overexpression was associated with tumor stage, recurrence status and the CD44 cancer stem cell marker in ESCC. TDO2 overexpression was correlated with poor outcome of ESCC patients. Inhibition of TDO2 expression by RNAi in TE-10 and TE-11 cell lines reduced both the number and the size of spheroid colonies as well as cell proliferation and invasive activity. Knockdown of TDO2 expression also induced inactivation of the EGFR signaling pathway.

Conclusion: Our results imply that TDO2 could play an important role in the progression of ESCC. Furthermore, TDO2 may be a potential therapeutic target in ESCC.

Citation Format: Thang Quoc Pham, Naohide Oue, Yohei Sekino, Yuji Yamamoto, Naoya Sakamoto, Ririno Honma, Kazuhiro Sentani, Wataru Yasui. TDO2 overexpression is associated with cancer stem cells and poor prognosis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1166.
Abstract 1170: Designing a biomarker-based bedside tool-kit for the clinical evaluation of bladder cancer
Authors: Dabke Isha R. , Pearce Richard , Kallifatidis Georgios , Lokeshwar Balakrishna
Published: August 2019

Cancer of the urinary bladder (UBC) ranks second in incidence and mortality among the genitourinary cancers causing over 16,000 deaths annually. Currently, the precise diagnosis of bladder cancer involves cystoscopy and cytology, both expensive and painful procedures. Morphologically, malignancies of the bladder can be divided into two subtypes: luminal and basal, which express distinct cytokeratin and stem cell markers and have differing sensitivities to therapy. Although diagnosis based on molecular signatures has the potential to be effective, a verification of their expression in strictly compartmentalized epithelial subtypes is not presently available. The implications of this project are the formation of a bedside tool-kit based on prognostic stem cell biomarkers that can be used following biopsy to efficiently evaluate cancer subtype and provide a preliminary diagnosis.

Towards that goal, we analyzed mRNA expression profiles in five established bladder cancer cell lines (RT-4, 5637, T24, HT-1376, 253J), ranging from a grade I (RT-4) to a grade IV cancer cell line (253J). Based on existing literature, these cell lines were classified into either the luminal or basal subtype of bladder cancer. Through RT-PCR and Western Blot studies, genomic expression levels of eleven biomarkers (luminal: UPK, GATA-3, RAB-25, E-Cadherin; and basal: CK-6, p63, CD44S, CD44V, CyclinB1, EGFR, CD49) were analyzed in these cell lines.

Cell viability following exposure to two chemotherapy drugs: Gemcitabine and Cisplatin, commonly used to treat bladder cancer, were assayed by colorimetric and clonogenic colony-forming assays. Literature states that though basal cancers are more aggressive, they are paradoxically more sensitive to chemotherapy compared to luminal UBC.

Less aggressive/luminal UBC cell lines showed higher mRNA levels for luminal biomarkers, and expressed GATA-3 100% of the time. The more invasive/basal cell lines showed only a slight upregulation of basal biomarkers, but expressed CD44S 100% of the time.

Furthermore, MTT Assays showed a significant toxic effect of Gemcitabine on basal cell lines earlier (t=24 hr vs 48/72 hr) than luminal cell lines. However, Cisplatin did not show a significant difference in luminal vs. basal UBC suggesting that the two subtypes of UBC have similar sensitivity to Cisplatin.

The study presented here suggests that a PCR-based, biomarker toolkit can aid in an efficient classification of bladder cancers at the bedside, and treatment can be personalized based on the UBC subtype.

Citation Format: Isha R. Dabke, Richard Pearce, Georgios Kallifatidis, Balakrishna Lokeshwar. Designing a biomarker-based bedside tool-kit for the clinical evaluation of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1170.
Abstract 1199: Identification of candidate regulators of genomic instability in human lung adenocarcinoma through a new cross-species in silico analysis
Authors: Rao Chinthalapally V. , Farooqui Mudassir , Zhang Yuting , Kumar Gaurav , Panneerselvam Janani , Yarla Nagendra , Asch Adam S. , Yamada Hiroshi Y.
Published: August 2019

(a) Chromosome instability (CIN), a type of genomic instability that is mitotic error-driven, has a deep impact on carcinogenesis and cancer recurrence. High CIN is a marker for poor prognosis in various cancers, including lung adenocarcinoma. In the tumor genome, CIN can be indicated with Copy Number Alterations (CNA) as its surrogate indicator. Although a diverse array of genes can cause CIN at the cellular level, genes responsible for tumor CNA remain elusive. In this study, we set out to identify genes whose expression shows correlation with CNA in human lung adenocarcinoma.

(b) We employed a novel cross-species in silico analysis. Our previous lung RNAseq results from lung cancer-prone CIN mice indicated 348 misregulated genes (92 up-regulated, 256 down-regulated; 2-fold cutoff, P<0.05). We hypothesized that some of the genes misregulated in CIN mice are involved in genomic instability in tumors. With in silico analysis with a human cancer genome database, we identified five human genes as candidate regulators of CNA in human lung adenocarcinoma: MMP13, NUF2, CCL22, ZNF366, and GPR114. High expression of MMP13, CCL22, ZNF366, and GPR114 was correlated with low CNA, suggesting their role in suppressing CIN. High expression of NUF2 was correlated with high CNA, suggesting its role in increasing CIN.

(c) MMP13 (Matrix Metalloprotease 13) is a protease involved in the breakdown of extracellular matrix. NUF2 (yeast NUF2 Kinetochore Protein homolog) is involved in the mitotic process, the misregulation of which directly leads to CIN. CCL22 (C-C Motif Chemokine Ligand 22), ZNF366 (Zinc Finger Protein 366), and GPR114 (G protein-coupled receptor 114, aka ADGRG5) are generally markers for subsets of immune cells (likely dendritic cells). This result led us to further hypothesize that infiltration of CCL22-, ZNF336-, and/or GPR114-positive immune cells inhibits pre-tumor cells and tumor cells with high CNA.

(d) The removal mechanism of cells with genomic instability is poorly understood. This is the first time that candidate markers for the immune cells directly or indirectly involved in removal of CIN cells have been identified. This study may aid in fine-tuning cancer-preventive immunotherapy that targets lung cells with genomic instability.

Citation Format: Chinthalapally V. Rao, Mudassir Farooqui, Yuting Zhang, Gaurav Kumar, Janani Panneerselvam, Nagendra Yarla, Adam S. Asch, Hiroshi Y. Yamada. Identification of candidate regulators of genomic instability in human lung adenocarcinoma through a new cross-species in silico analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1199.
Abstract 1248: Not BCL2 but BFL1 is overexpressed and have strong correlation with NF-kB pathway in diffuse large B-cell lymphoma
Authors: Lee Chansub , Song Hyojin , Song Seulki , Kim Daeyoon , Park Jihyun , Lee Sungyoung , Yun Hongseok , Kim Sheehyun , Yoon Sung-Soo , Koh Youngil
Published: August 2019

Venetoclax, anti-apoptotic BCL2-selective inhibitor, has demonstrated efficacy in B-cell multiple lymphoid malignancies. However, several studies have shown that the other anti-apoptotic BCL2 family genes can increase the risk of venetoclax resistance. Accordingly, compounds targeting other BCL2 family are actively being developed. So it is necessary to find which BCL2 family genes is overexpressed in certain tumors. We profiled BCL2 family genes expression with RNA-seq across different types of lymphoid malignancies (follicular lymphoma, burkitt lymphoma, diffuse large b-cell lymphoma, multiple myeloma, and acute leukocyte leukemia). Log2-transformed TMM-normalized RPKM was used for expression levels. As is known, BCL2 and MCL1 was highly expressed in follicular lymphoma and multiple myeloma, respectively. Interestingly, we discovered BFL1, one of anti-apoptotic BCL2 family genes, is overexpressed in both activated B-cell (ABC) and germinal center B-cell (GCB) type diffuse large B cell lymphoma (DLBCL). It has been well known that BFL1 is a pro-survival NF-kB target gene and indeed NF-kB pathway is highly activated in ABC-DLBCL. Also recent studies have shown that activation of this pathway can occur in GCB-DLBCL. Using gene set enrichment analysis, we confirmed that high expression of BFL1 is associated with negative regulation of intrinsic apoptosis and activation of NF-kB in B-cell. Also we discovered that NF-kB pathway activation in correlation with BFL-1 is especially dominant in DLBCL compared to other lymphoid malignancies. Among NF-kB signal related molecules (TNF receptors or Toll-like receptors), CD40 expression significantly correlated with BFL1 expression. In contrast, BCL2, another important apoptosis related gene, showed a negative association with the NF-kB pathway in DLBCL. We could validate our observation using microarray data. In conclusion, BFL1, which may controls intrinsic apoptosis and have strong correlation with NF-kB, is overexpressed in DLCBL. We suggest not venetoclax but BFL1 inhibitor may exert clinical benefit in DLBCL.

Citation Format: Chansub Lee, Hyojin Song, Seulki Song, Daeyoon Kim, Jihyun Park, Sungyoung Lee, Hongseok Yun, Sheehyun Kim, Sung-Soo Yoon, Youngil Koh. Not BCL2 but BFL1 is overexpressed and have strong correlation with NF-kB pathway in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1248.
Abstract 1249: The role of PRMT5 in MYC-driven medulloblastoma
Authors: Chaturvedi Nagendra K. , Kesherwani Varun , Kling Matthew J. , Ray Sutapa , McGuire Timothy R. , Joshi Shantaram S. , Sharp J. Graham , Coulter Don W.
Published: August 2019

Medulloblastoma (MB) is the most common pediatric brain tumor, accounting for about 20% of all brain tumors in children. Children with Group 3 MB (aberrant MYC) show the worst prognosis, with <50% survival. Recently, the role and association of protein arginine methyl transferase (PRMT) 5 have been closely associated with aberrant MYC function in various cancers including other brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in MB have not been explored. Here we provide preliminary data indicating PRMT5 as a novel regulator of MYC and implicating PRMT5 as a potential therapeutic target in MYC-driven MB. Using cell lines, our results showed PRMT5 overexpression in MYC-driven MB compared to non-MYC MB. The results from co-immunoprecipitation experiment in MYC-driven MB cells demonstrated that MYC physically associates with PRMT5 by direct protein-protein interaction. In addition, cycloheximide chase experiment showed that PRMT5 regulates MYC stability. Knockdown of PRMT5 using siRNA in MYC-driven MB cells significantly decreased cell growth and MYC expression. We also tested the therapeutic potential of targeting PRMT5 against MB cell lines using a small molecule inhibitor EPZ015666. We observed a dose-dependent efficacy of EPZ015666 in suppressing cell growth in MYC-driven MB cell lines. We also observed a superior efficacy of this inhibitor against MYC-driven MB cells compared to non-MYC MB cells. Together, our results implicate the regulation of MYC oncoprotein by PRMT5, and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven MB and other MYC-driven cancers.

Citation Format: Nagendra K. Chaturvedi, Varun Kesherwani, Matthew J. Kling, Sutapa Ray, Timothy R. McGuire, Shantaram S. Joshi, J. Graham Sharp, Don W. Coulter. The role of PRMT5 in MYC-driven medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1249.
Abstract 1272: Whole genome Crispr screening to identify potential SERD molecule resistance mechanisms
Authors: Marcireau Christophe , Berthelot Karine , Williart Alice , Fournet Hamida , Debono Delphine , Thill Gilbert , Erasimus Helene , Chassin Dorine , Lanneau Christophe , Yaligara Veeanagouda , Orsini Cecile , Didier Michel , Mikol Vincent , Bouaboula Monsif , Debussche Laurent
Published: August 2019

Metastatic breast cancer results in substantial morbidity and mortality for women afflicted with this disease. Approximately 80% of breast cancers express the estrogen receptor. The cancer cell survival and proliferation is driven by the activation of the estrogen receptor. SAR439859 is an effective endocrine therapy for breast cancer selectively and effectively degrading the estrogen receptor. A phase 1/2 study of SAR439859 alone and/or in combination with palbociclib in postmenopausal women with estrogen receptor positive advanced breast cancer was initiated last year by Sanofi.

CRISPR/Cas9 is a recent and revolutionary technology for efficient and directed alterations of the genome. CRISPR allows users to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs (SgRNA). These double strand breaks can be repaired by non-homologous end joining DNA repair mechanism. This DNA repair mechanism is an error prone DNA repair process which can lead to gene invalidation.

We aimed at investigating the potential SAR439859 resistance mechanisms. A better understanding of resistance mechanisms is needed to overcome this potential problem and to propose complementary therapeutic solutions. We conducted a whole genome CRISPR screening in a breast tumor cell line with a thermofisher commercial SgRNA library (ref: M04305) to identified genes involved in potential SAR439859 resistance phenomena. CRISPR screening steps will be presented from the cell line selections to results analysis.

Citation Format: Christophe Marcireau, Karine Berthelot, Alice Williart, Hamida Fournet, Delphine Debono, Gilbert Thill, Helene Erasimus, Dorine Chassin, Christophe Lanneau, Veeanagouda Yaligara, Cecile Orsini, Michel Didier, Vincent Mikol, Monsif Bouaboula, Laurent Debussche. Whole genome Crispr screening to identify potential SERD molecule resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1272.
Abstract 1282: Understanding the kinomic contributions to tyrosine kinase inhibitor resistance in triple negative breast cancer
Authors: Lefebvre Cory , Litchfield David , Allan Alison
Published: August 2019

There is an increasing need to develop targeted therapies for triple negative breast cancer (TNBC), as conventional chemotherapy, anti-hormone or anti-HER2 therapies are ineffective at combatting systemic disease. TNBC tumors often have increased expression of receptor tyrosine kinases such as EGFR (epidermal growth factor receptor) and the hepatocyte growth factor (HGF) receptor, c-Met; presenting as potential targets for treatment. However, targeted anti-Met and anti-EGFR therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that dynamic changes in the kinome contribute to the acquired resistance of TNBC tumors to c-Met and EGFR inhibitors. Using MDA-MB-468 and MDA-MB-231 TNBC cell lines, we investigated the effects of the c-Met inhibitors cabozantinib and tivantinib and the EGFR inhibitor erlotinib on cell proliferation and activation of target receptors and downstream signalling pathways. We observed that cabozantinib and erlotinib significantly inhibited the proliferation (p≤0.05) of one and/or both TNBC cell lines in a cytostatic fashion. Similarly, tivantinib demonstrates growth inhibitory effects but in a mostly cytotoxic manner; consistent with recent reports of its mechanism in inhibiting microtubule polymerization. Exposure to erlotinib (24 hrs) resulted in decreased expression of phosphorylated and total EGFR in both TNBC cell lines and a decrease in activation (phosphorylated:total expression) of ERK1/2 in MDA-MB-468 cells (p≤0.05). Tivantinib and cabozantinib did not appear to have any effects on activation of c-Met or ERK1/2 in MBA-MB-231 or MDA-MB-468 cells following 24-hour exposure. Ongoing studies are aimed at implementing a novel kinomics strategy using SILAC and multiplexed inhibitor beads for kinase enrichment to investigate the dynamic changes in the kinome of susceptible and resistant cell lines in response to erlotinib and cabozantinib. With better understanding of the underlying mechanisms, we can potentially discover targets that could attenuate induced resistance to tyrosine kinase inhibitors.

Citation Format: Cory Lefebvre, David Litchfield, Alison Allan. Understanding the kinomic contributions to tyrosine kinase inhibitor resistance in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1282.
Abstract 1284: Resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanoma
Authors: Patel Hima , Mishra Rosalin , Joan Garrett
Published: August 2019

Melanoma five year survival rates are only 15% for patients with advanced disease. About 50% of melanomas contain the BRAFT1799A transversion which encodes for the constitutively active BRAFV600E oncoprotein. This opened up avenues for development of BRAF inhibitors which resulted in a response rate of about 50% and median progression-free survival of about 5 months. Co-targeting the downstream MEK protein with a MEK inhibitor along with BRAF inhibitor resulted in response rates of about 70% and are now approved as standard of care for patients harboring the BRAF-mutant melanoma. In spite of these successes, their long-term efficacy has been limited due to acquired resistance to these inhibitors. In order to study mechanisms underlying resistance, we generated dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) resistant WM115 (WM115 TDR) and WM983 (WM983 TDR) cell lines by adding increasing concentration of the drugs to WM115 and WM983 cells with BRAFV600E mutation (parental cells). We observed that the resistant cells undergo a change in their morphology and continue to remain viable over increasing doses of dabrafenib and trametinib as compared to parental cells demonstrated by MTT cell proliferation assay and Matrigel 3D assay. TDR cells demonstrated maintenance of p-ERK levels at 4 and 24 hours which under the same conditions p-ERK was inhibited in the parental cells. Current studies underway include RNA sequencing in order to decipher differentially expressed genes in the parental and TDR which could mediate resistance to BRAF and MEK inhibitors. Additionally we will examine phosphorylated receptor tyorsine kinases (RTKs) using an array to identify divergences among activation of RTKs in parental versus TDR cells. Deciphering causes of resistance to BRAF and MEK inhibitors will help in the development of novel mechanism-based interventions aiding long-term patient prospects in BRAF-mutant melanoma.

Citation Format: Hima Patel, Rosalin Mishra, Garrett Joan. Resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1284.
Abstract 1326: WSD0922: A BBB penetrable EGFR/EGFRVIII inhibitor for the treatment of GBM and metastatic CNS tumor
Authors: Zhong Wei , Zhang Jinqiang , Mu Ahihua , Sun Claire
Published: August 2019

Aberrant activity caused by EGFRm+ (EGFRvIII/Del19/L858R) is implicated in the brain tumors such as GBM and DIPG, and metastatic CNS tumors of NSCLC, and cancer of HNSCC. To date, approved EGFRi has limited efficacy against brain tumors due to insufficient BBB penetration and/or poor activity against EGFRvIII. Moreover, GBM is a whole-brain disease and needs a BBB penetrable therapy for killing tumor cells beyond intact BBB. We report that WSD0922, a novel selective EGFRi, displays potent activity, excellent CNS penetration, good safety profile and preclinical anti-tumor efficacy. The IC50 of WSD0922 against EGFRm+ <10nM and the ex-vivo GI50 in GBM PDX with EGFRvIII is ~20nM. WSD0922 is highly selective over other kinases. WSD0922 treatment resulted in dose dependent inhibition on EGFRvIII phosphorylation and AKT phosphorylation in GBM PDX model and the in vivo PD modulation correlated well with PK and efficacy. WSD0922 treatment significantly prolonged the survival of mice with intracranial tumor of EGFRm+ NSCLC. WSD0922 treatment resulted in tumor regression on cetuximab resistant PDX model of HNSCC. In screening platform of squamous lung cancer PDX models, WSD0922 demonstrated remarkable tumor regression in 3 PDX models (platinum sensitive/insensitive/EGFRm+). WSD0922 is well tolerated in GLP tox at dose ~10x clinical equivalent dose. In addition, WSD0922 is predicted to be CNS penetrable with decent PK in human. The phase 1 clinical trial in US will be initiated in early 2019 with a design of 3+3 basket trial. Taken together, our data provide a good rationale for WSD0922 to be developed toward clinic for the treatment of patients with GBM, HNSCC (EGFR and/or EGFRvIII) and CNS metastasis of NSCLC (EGFRm+).

Citation Format: Wei Zhong, Jinqiang Zhang, Ahihua Mu, Claire Sun. WSD0922: A BBB penetrable EGFR/EGFRVIII inhibitor for the treatment of GBM and metastatic CNS tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1326.
Abstract 1368: Simultaneous cell-free RNA PD-L1 expression and MSI from the same single-tube of blood in solid tumors
Authors: Weiss Glen J. , Walker Paul , Aktas Dilek , Ford Andrew , Brown Charmaine , Yeh Chen-Hsiung , Javadi Nader
Published: August 2019

Background: Liquid biopsy is a minimally invasive technique available in the clinic using next-generation sequencing (NGS) of plasma circulating cell-free DNA (cfDNA) and cfRNA. Here we report liquid biopsy results from solid tumors using the Circulogene Theranostics Personalized Gene Profile (CGP, 50-gene panel), as well as, microsatellite instability (MSI) testing. CGP uses proprietary in situ enrichment requiring minimal input volume of 20 uL plasma (cfDNA), 400 uL (cfRNA), and 100 uL buffy coat per case from the same single-tube of blood.

Methods: cfRNA PD-L1 expression, MSI, and co-occurring cfDNA mutations were retrospectively compiled from CGP ordered at multiple centers. Ct value cutoff for 1 copy PD-L1 (positive) was 42. MSI was performed using paired buffy coat gDNA (normal) and plasma cfDNA (tumor) on 5 microsatellite mononucleotide markers, BAT-25, BAT-26, NR-21, NR-24 and MONO-27 (Promega; current an industry standard that has been used in KEYNOTE pembrolizumab trials) and analyzed by capillary electrophoresis genetic analyzer. Based on batched processing, Circulogene’s turnaround time (TAT) is 5 business days on average. Data was summarized and the correlation between PD-L1 and MSI was measured.

Results: 397 patients (median age 67 years, range 27-96; 210 men: 187 women) underwent CGP testing for both PD-L1 and MSI (11/2017-10/2018). The majority of cancer types were lung (n=187), colorectal (n=43), breast (n=40), pancreatic (n=32), and prostate (n=30). 59/397 (14.9%) were PD-L1 positive, in the range of expected PD-L1 positivity across solid tumors. 110/397 (27.7%) were MSI-High (MSI-H). There was no correlation between PD-L1 and MSI results (r^2 <0.01). There was a median of 3 mutations/patient (range 0-19), with a 0% sample failure rate. Overall, 23 cases had no detectable mutations (5.8%), but 11 of these 23 (47.8%) had either PD-L1 positivity or MSI-H.

Conclusions: Simultaneous cfRNA PD-L1 and MSI testing from the same single-tube blood is feasible in a liquid biopsy and their positivity has frequency in the expected range for solid tumors (Xang et al. Onco Targets Ther 2016, Zhang et al. AMP Conference 2018, Abstract ST009). Despite the low plasma sample input, there were no sample failures, suggesting CGP in situ enrichment is robust and has a rapid TAT. Assay results with linkage to clinical outcomes is warranted.

Citation Format: Glen J. Weiss, Paul Walker, Dilek Aktas, Andrew Ford, Charmaine Brown, Chen-Hsiung Yeh, Nader Javadi. Simultaneous cell-free RNA PD-L1 expression and MSI from the same single-tube of blood in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1368.