bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1453: Specific detection of mutant Ras oncoproteins in cell and tissue lysates by multiplex immunoassay
Authors: Hwang Joseph B. , Saporita Anthony , Xiao Qiang
Published: September 2020

Ras proteins (HRAS, KRAS, and NRAS) are small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras-GTP activates downstream pathways, including MAPK, by binding to Raf kinase and phosphatidylinositol 3-kinase (PI3K) to promote cellular proliferation, survival, growth and differentiation. Oncogenic mutations in Ras are found in approximately 25% of human cancers. Furthermore, 90% of pancreatic cancers harbor KRAS mutations with 80% of KRAS mutations occurring at codon 12 with G12V and G12D being the most prevalent. The Ras-Raf-MEK pathway can also be activated downstream of Ras. BRAF is a well-established oncogene, with the BRAF V600E mutation being prevalent in melanoma. While targeting this BRAF oncoprotein has had therapeutic efficacy, treating cancers driven by Ras oncoproteins remains an urgent unmet clinical need. We developed a novel multiplex immunoassay capable of simultaneously detecting total Ras, RasG12V, RasG12D, phospho-MEK1 (S217/S221), phospho-BRAF (S446) and phospho-CRAF (S338) in a single well. This multiplex assay detects the H-Ras, N-Ras, and K-Ras isoforms for total Ras, RasG12D, and RasG12V. To confirm specific recognition of mutant Ras oncoproteins, this kit was able to detect the known KRAS G12V mutation found in the COR-L23 cell line and the NRAS G12D mutation found in THP-1 cell line, respectively. In addition, the response to BRAF inhibitors vemurafenib and dabrafenib was compared between MDA-MB-435S cells, which harbor the BRAF V600E mutation, and insensitive cell lines, using multiplex immunoassays. While COR-L23 and THP-1 cells were not responsive to vemurafenib and dabrafenib, MDA-MB-435S cells showed a dramatic reduction in pMEK1 and pERK, consistent with inhibition of mutant BRAF protein. We then analyzed commercially-sourced tissue lysates, using tumor and adjacent tissue from a patient with colon cancer harboring a KRAS G12D mutation. While multiplex protein analysis revealed a similar phosphorylation pattern for Raf and MEK proteins, the RasG12D oncoprotein was specifically observed in lysate from the tumor but not the adjacent tissue. Finally, exosomes were enriched from pancreatic cancer serum and tested using the Ras-Raf Oncoprotein panel. These exosomal preparations yielded detectable signal for Total Ras and phospho-BRAF. Our data demonstrate the utility of studying the effects of Ras oncoproteins by multiplex immunoassay in multiple sample types, including cell, tissue, and exosomal lysates.

Citation Format: Joseph B. Hwang, Anthony Saporita, Qiang Xiao. Specific detection of mutant Ras oncoproteins in cell and tissue lysates by multiplex immunoassay [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 1453.
Abstract 1466: Molecular insights of resistin associated proliferation by inducing hypercholesterolemia in breast cancer
Authors: Deb Ankita , Bhat Manoj
Published: September 2020

Introduction: Breast cancer is the most prevalent cancer in women impacting ~2.1 million women each year (WHO, 2019). It is the second leading cause of death after lung cancer in women. In spite of several therapeutic advances, further improvement in prognosis and survivability is still desired. According to numerous studies, Resistin, a proinflammatory cytokine, has a positive correlation with breast tumor progression. It plays multifaceted roles in diabetes, obesity, cardiovascular diseases as well as in several cancers. Despite several epidemiological studies showing an increase in serum resistin level in breast cancer patients, its relevance is not clearly understood. In the present study, we have focused on the role of resistin in breast cancer and emphasized on metabolic alterations occurring in this process.

Methods: In vitro studies include long term colony formation and LDL estimation assays, immunoblotting, immunofluorescence . For in vivo studies, female Balb/C mice were used as isograft breast cancer model.

Results: In vitro studies show that resistin facilitates progression of breast cancer through activation of JAK-STAT pathway. Downstream targets, c-Myc and cdc25A are also regulated via Resistin. Inhibition of JAK-STAT pathway abrogates resistin induced growth in breast cancer cells. Moreover, resistin also promotes cholesterol accumulation thereby inducing a hypercholesterolemic environment. In vivo studies indicate that with increase in tumor progression, resistin as well as LDL levels are also elevated in serum.

Conclusions: In vitro as well as in vivo studies show that increase in serum resistin level is positively correlated with breast cancer progression and induction of hypercholesterolemia. Insights into resistin associated molecular events contributing to metabolic changes might facilitate progression of breast cancer. Targeting this pathway might contribute to a better therapeutic outcomes.

Citation Format: Ankita Deb, Manoj Bhat. Molecular insights of resistin associated proliferation by inducing hypercholesterolemia in 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 1466.
Abstract 1470: UGP2 is a critical regulator of protein glycosylation in pancreatic cancer
Authors: Wolfe Andrew L. , Galeas Jacqueline , Toska Eneda , Zhou Qing , Ku Angel , Koche Richard P. , Bandyopadhyay Sourav , Lebrilla Carlito B. , Scaltriti Maurizio , McCormick Frank , Kim Sung Eun
Published: September 2020

UDP-glucose pyrophosphorylase 2 (UGP2) is a lynchpin metabolic enzyme that rests at the convergence of multiple metabolic pathways regulating both glycogen synthesis and glycosylation modifications. Here we elucidated the essential role of UGP2-mediated glycosylation in the maintenance of KRAS-driven cancer using both in vitro and in vivo models. A key effector of KRAS oncogenic function is the transcription factor YAP. We identified the YAP/TEAD transcription factor complex as a major regulator of UGP2 mRNA expression and enzymatic activity using genomic perturbations, correlative protein immunohistochemistry in PDAC samples, and ChIP-seq and targeted ChIP-qPCR at the UGP2 promoter. Loss of YAP or UGP2 leads to a decrease in glycogen and defects in key glycosylation targets such as EGFR. In murine xenograft models using pancreatic cancer lines, knockdown of UGP2 prevented tumor growth and decreased expression of the critical glycosylation target EGFR. This essential role for UGP2 in cancer maintenance may lead to new avenues of therapy in otherwise intractable KRAS-driven cancers.

Citation Format: Andrew L. Wolfe, Jacqueline Galeas, Eneda Toska, Qing Zhou, Angel Ku, Richard P. Koche, Sourav Bandyopadhyay, Carlito B. Lebrilla, Maurizio Scaltriti, Frank McCormick, Sung Eun Kim. UGP2 is a critical regulator of protein glycosylation in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1470.
Abstract 1477: LRG1 blockade normalizes tumor vasculature and improves efficacy of chemotherapy
Authors: Pilotti Camilla , O'Connor Marie N , Kallenberg David , Dowsett Laura , George Jestin , Moss Stephen E. , Greenwood John
Published: September 2020

In cancer blood vessels are dysfunctional, poorly perfused and leaky. Malfunctioning vessels contribute to the pro-oncogenic environment and limit the efficacy of current systemically administered drugs. Normalizing the tumor vasculature to improve vessel permeability, reduce hypoxia and vascular leakage and enhance drug delivery, has become an experimental objective in cancer research. This study was carried out to investigate the effect of blocking the secreted glycoprotein leucine-rich alpha-2-glycoprotein 1 (LRG1) on tumor vascular function, and evaluate the impact it has on the efficacy of the common standard of care chemotherapeutic drug cisplatin. Under normal conditions LRG1 is mainly expressed in the liver but also in other tissues such as bone marrow and immune cells. LRG1 has been described in multiple reports to be a serum prognostic biomarker in several cancers, for example lung, prostate, colorectal and breast. LRG1 promotes dysfunctional vessel growth by disrupting TGFβ signaling. We demonstrate that in Lrg1-/- mice and following treatment with a LRG1 function-blocking antibody (15C4) tumor growth was inhibited. In addition, we show using RNAscope that following subcutaneous grafting of the B16F0 and LL2 tumor cell lines in mice, Lrg1 is induced in tumor endothelial cells. Despite having no effect on total vessel area, the density was decreased upon LRG1 blockade, with the persisting larger vessels exhibiting improved vessel structure as evidenced by increased pericyte and basement membrane endothelial cell coverage. Better mural cell association with tumor vascular endothelial cells and basement membrane coverage are also indicators of vessel stabilization and maturation. Using a systemically delivered fluorescent lectin tracer to mark perfused vessels, we observed a significant increase in tumor perfusion in mice treated with 15C4. Lastly, vessel normalization, through LRG1 antibody blockade, significantly enhanced the efficacy of cisplatin chemotherapy as shown by a slower tumor growth rate and increased tumor cell death compared to monotherapy. These data further corroborate the hypothesis that inhibition of LRG1 improves the delivery, and hence efficacy, of a cytotoxic drug. In conclusion, deletion or inhibition of LRG1 results in an improved vascular configuration and function, and the efficacy of chemotherapy. LRG1 blockade may therefore represent a novel strategy to enhance vessel health and improve the efficacy of cancer therapeutics.

Citation Format: Camilla Pilotti, Marie N O'Connor, David Kallenberg, Laura Dowsett, Jestin George, Stephen E. Moss, John Greenwood. LRG1 blockade normalizes tumor vasculature and improves efficacy of chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1477.
Abstract 1480: CCL4 promotes oral squamous cell carcinoma angiogenesis by targeting the angiopoietin-2 signal pathway
Authors: Lien Ming-Yu
Published: September 2020

The inflammatory chemokine (C-C motif) ligand 4 (CCL4) plays an important role in the pathogenesis and progression of cancer, with higher serum CCL4 levels in patients with oral squamous cell carcinoma (OSCC) marking a more advanced stage of disease. OSCC accounts for approximately 95% of oral cancer in Taiwan and this disease has a poor prognosis, with aggressive local invasion and metastasis predisposing to recurrence and additional morbidity. Angiogenesis, a physiological process that enables new blood vessels to form from pre-existing vessels, is involved in the growth, maintenance, and metastasis of most tumors. Angiopoietin-2 (ANGPT2) induces angiogenesis and is a potent endothelial cell-specific mitogen. Until now, it has been unclear as to whether levels of CCL4 expression are a marker of angiogenic activity in patients with OSCC and are correlated with higher levels of ANGPT2 expression. Our investigations have found that treating OSCC cells with CCL4 increases ANGPT2 expression and that CCL4 treatment promotes angiogenesis in a

murine model of OSCC. Thus, CCL4 may be a novel molecular therapeutic target for inhibiting angiogenesis and metastasis in OSCC.

Citation Format: Ming-Yu Lien. CCL4 promotes oral squamous cell carcinoma angiogenesis by targeting the angiopoietin-2 signal pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1480.
Abstract 1489: Exosomes are key players in nicotine-induced angiogenesis
Authors: Singh Shubhangi , Saranyutanon Sirin , Khan Mohammad Aslam , Srivastava Sanjeev Kumar , Singh Seema , Singh Ajay P.
Published: September 2020

Nicotine, an addictive component of cigarette smoke and tobacco, have been shown to promote cancer pathogenesis. Angiogenesis is an important hallmark of cancer that sustains the supply of nutrients and oxygen to the fast proliferating cancer cells through the formation of new blood vessels. The goal of present study was to examine the direct and indirect effect of nicotine on endothelial cell growth, their migration and invasion, and capillary forming ability. We treated the human umbilical vein endothelial cells with two doses of nicotine within physiological range of smokers (200nM and 400nM) and examined the effect on their growth. In parallel, we treated the prostate cancer cells with similar doses of nicotine or vehicle for 8 h and subsequently replaced the media. After next 48 h, conditioned media (CM) was collected from vehicle- (Veh-CM) and nicotine- (Nic-CM) treated prostate cancer cells and used for the treatment of endothelial cells. The growth of endothelial cells increased by ~19 % and ~22 % upon direct treatment of nicotine at 200nM and 400nM doses, respectively. However, a far greater increase was recorded (~128% and ~ 146%, respectively), when endothelial cells were treated with the conditioned media derived from nicotine-treated cancer cells at 200nM and 400nM doses. In next of experiments, we fractionated Veh-CM and Nic-CM (200nM dose) into soluble and exosomal fractions and examined their effect on endothelial cells. We observed that exosomal fraction was more potent (~89%) in inducing endothelial cell growth as compared to the soluble fraction (~46%). In addition, we also observed significant greater increase in motility, invasion and capillary-forming ability of endothelial cells that were pre-treated with Nic-Exo as compared to those treated with Veh-Exo. Studies are currently ongoing to examine the comparative efficacy of nicotine-treated soluble and exosomal factions in potentiating motility, invasion and capillary formation and define the underlying molecular mechanism(s). Our findings thus support a novel pathogenic role of nicotine in cancer where exosomes act as key players in promoting angiogenesis.

Citation Format: Shubhangi Singh, Sirin Saranyutanon, Mohammad Aslam Khan, Sanjeev Kumar Srivastava, Seema Singh, Ajay P. Singh. Exosomes are key players in nicotine-induced angiogenesis [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 1489.
Abstract 1523: Novel role of TBK1 in breast cancer EMT
Authors: Maan Meenu , Dutta Mainak , Jaiswal Neha , Saavedra Harold , Chellappan Srikumar
Published: September 2020

TANK Binding Kinase 1 (TBK1) regulates interferon signaling and NFκB function by acting as a non-canonical IκB kinase. TBK1 also participates in RalB-mediated inflammatory responses and is essential for the survival of non-small cell lung cancers (NSCLC) driven by oncogenic KRAS. Recent evidence suggests a role for TBK1 as a driver of cancer progression and it is overexpressed in breast, colon, lung and pancreatic cancers. Our published results demonstrate that TBK1 is essential for proper microtubule dynamics and regulation of mitosis. Here we show that knockdown of TBK1 results in a decreased expression of mesenchymal genes involved in EMT and a re-distribution of β-catenin in breast cancer cells. Similarly, expression of matrix metalloproteinases 2 and 9 was altered. This led us to hypothesize that TBK1 acts as an EMT driver in cancer. Inhibition of TBK1 resulted in decreased adherence dependent as well as independent colony formation and stem cell self-renewal, while inducing senescence like phenotypes. We propose that TBK1 mediated EMT is mediated through its interaction with the transcription factor E2F1; the interaction was detected in breast cancer cell lines as well as breast cancer Tissue Micro Arrays. To get a holistic view of the changes that take place in the cell after TBK1 knockdown, we performed a global metabolomics and RNAseq analysis of breast cancer cell lines. An integrated analysis of this data suggested significant alterations in glucose, galactose and nucleotide metabolism upon TBK1 knockdown. Two of the genes that were markedly altered were aldoketoreductase1 B10 (AKR1B10) and thymidine phosphorylase (TP); attention was focused on these genes, as both have been reported to be cancer drivers and their over-expression results in poor prognosis. We observed a downregulation of AKR1B10 and TP in response to the TBK1 knockdown. Experiments are under way to investigate whether the pro-cancer role of TBK1 is dependent on AKR1B10 and TP, especially in breast cancer cells. These results suggest that TBK1 mediated EMT might be a function of the altered expression of mesenchymal proteins and the metabolic profile of the cell brought about by TBK1.

Citation Format: Meenu Maan, Mainak Dutta, Neha Jaiswal, Harold Saavedra, Srikumar Chellappan. Novel role of TBK1 in breast cancer EMT [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 1523.
Abstract 1569: TGFβ signaling suppresses alternative end joining associated with genomic mutations and PARP Inhibition sensitivity
Authors: Liu Qi , Palomero Luis , Moore Jade , Guix Ines , Pujana Miquel Angel , Barcellos-Hoff Mary Helen
Published: September 2020

Differential use of DNA repair pathways in cancer leads to distinct clinical outcomes following genotoxic therapies. We found that deficient TGFβ signaling due to human papilloma virus (HPV) in head and neck squamous cell carcinoma (HNSCC) results in impaired DNA repair proficiency, increased use of alternative end joining (alt-EJ) and that a 50-gene TGFβ signature is negatively correlated with prognosis (Clin Ca Res 2018, 24(23): 6001). To assess the molecular consequence of TGFβ signaling in the DNA damage response (DDR), we performed a Nanostring assay on SAS HNSCC and U251 glioblastoma cell lines using the 180 gene DNA damage and repair panel. The results showed that treatment with TGFβ type 1 receptor kinase inhibitor LY2157299 or TGFβ profoundly altered DDR gene expression profiles. TGFβ inhibition for 24 hr significantly (p<0.05) increased critical alt-EJ genes, POLQ (DNA polymerase theta) and PARP1 (poly(ADP-ribose) polymerase 1), which was confirmed by qRT-PCR. To interrogate the relationship between TGFβ and alt-EJ in The Cancer Genome Atlas (TCGA), we used the literature to curate a 36 gene alt-EJ signature. Single specimen gene set enrichment analyses (ssGSEA) showed that the alt-EJ signature was significantly anti-correlated with the TGFβ signature in 15 of 17 cancer types (PCC = -0.35; p =10-16). This anti-correlation was also observed in 875 cancer cell lines in Catalogue of Somatic Mutations in Cancer (COSMIC) database, suggesting a conserved, cancer cell intrinsic mechanism by which TGFβ regulates alt-EJ. Alt-EJ is an error prone DNA repair mechanism that generates gross genomic alterations. Consistent with this, low ssGSEA score for TGFβ and high ssGSEA score for alt-EJ are correlated with increased deletions or insertions in genome sequence of patient tumors in TCGA as well as cancer cell lines in COSMIC (p<0.05). PARP1 requirement prompted analyses of drug sensitivity data in the Sanger database, which showed that cell lines with high alt-EJ and low TGFβ ssGSEA score are significantly more sensitive to PARP1 inhibitors, olaparib and ABT-888, (p < 0.0001). These data lead to the novel hypothesis that TGFβ signaling actively suppresses alt-EJ. If true, the TGFβ/alt-EJ signature may be useful as a biomarker for precision cancer medicine with genotoxic agents, TGFβ or PARP inhibitors.

Citation Format: Qi Liu, Luis Palomero, Jade Moore, Ines Guix, Miquel Angel Pujana, Mary Helen Barcellos-Hoff. TGFβ signaling suppresses alternative end joining associated with genomic mutations and PARP Inhibition sensitivity [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 1569.
Abstract 1582: A pre-existing inflammatory immune microenvironment predicts the clinical and immunological response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 peptide vaccination
Authors: Abdulrahman Ziena , de Miranda Noel F. , van Poelgeest Mariette I. , van der Burg Sjoerd H.
Published: September 2020

Background: Vulvar High-grade Squamous Intraepithelial Lesion (vHSIL) is predominantly induced by high-risk Human Papilloma Virus type 16 (HPV16). In two independent trials, therapeutic vaccination against the oncoproteins of HPV16 with synthetic long peptides (SLP) resulted in vHSIL regression in about half of the patients after 12 months. Several studies have shown that the immune microenvironment influences therapy outcome. Therefore, a thorough investigation of the vHSIL immune microenvironment before and after SLP vaccination was performed, and its impact on clinical response was studied.

Methods: Two novel multiplex immunofluorescence panels were designed and fully optimized for formalin-fixed paraffin-embedded tissue, one for T cells (CD3, CD8, Foxp3, Tim3, Tbet, PD-1, DAPI) and one for myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI). Pre- and post-vaccination biopsies of 29 vHSIL patients, as well as 27 healthy vulva excisions, were stained, scanned with the Vectra multispectral imaging system, and automatically phenotyped and counted with inForm advanced image analysis software.

Results: A pre-existing pro-inflammatory tumor microenvironment, marked by high numbers of CD4+ and CD8+ T cells expressing Tbet and/or PD-1 as well as CD14+ inflammatory macrophages, is a strong predictor for good clinical response to therapeutic HPV16 SLP vaccination. A clear stepwise increase in pre-vaccination infiltrating Tbet+, CD4+, CD8+ T cells and CD14+ macrophages, and decrease in Foxp3+ regulatory T cells was observed as response increased from non to partial to complete response. Moreover, the pre-vaccination immune microenvironment of complete responders resembled healthy vulva. Vaccination further increased infiltrating CD4+ and Tbet+ T cells and CD14+ macrophages, and decreased Foxp3+ regulatory T cells and CD68+CD163+ M2 macrophages in the complete and partial responders, but not in the non responders.

Conclusion: Clinical responsiveness to therapeutic HPV16 SLP vaccination requires a pre-existing inflamed type 1 immune contexture in vHSIL. Hence, only patients with an inflamed microenvironment should be selected for monotherapy by therapeutic peptide vaccination, since this strategy is incapable of overcoming an immunologically cold tumor microenvironment.

Citation Format: Ziena Abdulrahman, Noel F. de Miranda, Mariette I. van Poelgeest, Sjoerd H. van der Burg. A pre-existing inflammatory immune microenvironment predicts the clinical and immunological response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 peptide 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 1582.
Abstract 1608: Adaptive resistance to PD-1 blockade upregulates Tim-3 through STAT3 pathway in regulatory T cells in melanoma
Authors: Huang Lili , Xu Yu , Fang Juemin , Liu Weixing , Liu Zhuqing , Xu Qing
Published: September 2020

Tregs play an opposing role in anti-tumour immunity by suppressing effective immunotherapies in melanoma. Thus, understanding the precise mechanisms of Tregs in immune therapy is important. TILs separated from freshly excised melanoma tumours and PBMCs obtained from healthy donors were used to assess the expression levels of Tim-3 and IL-10/TGF-β of Tregs by multi-colour flow cytometry and immunohistochemistry in the presence or absence of an anti-PD-1 antibody or STAT3 inhibitor in vitro and in vivo. The mRNA level of STAT3 gene was closely associated with the expression of PD-1 and Tim-3 in melanoma patients. Tim-3 expression was significantly upregulated on Tregs after PD-1 blockade in mice treated with either induced mouse and human melanoma TILs or healthy donor PBMCs. The levels of IL-10 and TGF-β secretion in the TIL group increased compared with those in the control group, supporting a potential escape mechanism from anti-PD-1 therapy in the tumour microenvironment. Furthermore, in the presence of the STAT3 pathway inhibitor Stattic, Tim-3 and PD-1 expression on Treg was regulated on TILs from respondent mice melanoma, suggesting that STAT3 could mediate Tim-3 and PD-1 expression on Treg in tumor microenvironment. Therefore, our findings reveal the mechanism of PD-1 and Tim-3 expression in a STAT3-dependent manner on Tregs in the TME, providing further support for STAT3 as a potential transient small molecular inhibitor targets for patients suffering from melanoma.

Citation Format: Lili Huang, Yu Xu, Juemin Fang, Weixing Liu, Zhuqing Liu, Qing Xu. Adaptive resistance to PD-1 blockade upregulates Tim-3 through STAT3 pathway in regulatory T cells in melanoma [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 1608.
Abstract 1611: MYB and the MYB-NFIB fusion induce adenoid cystic carcinoma
Authors: Hu Yuan , Zhu Xiaoyuan , Luo Han , El-Naggar Adel , Caulin Carlos
Published: September 2020

Adenoid cystic carcinoma (ACC) is the second most common malignancy of the salivary glands, also found at lower frequency rates in other secretory glands such as the mammary and lacrimal glands. Salivary ACCs are slow-growing but aggressive neoplasms, with propensity to perineural invasion and high recurrence rates. MYB is the most frequently altered gene in ACCs, as approximately 40% of the ACCs contain MYB-NFIB gene fusions, and the full-length MYB is overexpressed in over 50% of the fusion-negative ACCs. However, it is still unclear whether MYB activities promote ACC development. To assess the oncogenic potential of MYB in vivo, we generated transgenic mice that overexpress MYB using the TetO system. In these mice, focal MYB overexpression was directed to salivary and mammary glands using the activator line MMTV-tTA. We found that 25% of these mice developed tumors with an average latency of 23 months. Remarkably, all the tumors that developed in these mice were ACCs, and the three main phenotypic variants of human ACCs (cribriform, tubular and solid) were represented in this cohort. Using the same transgenic system, we found that the MYB-NFIB fusion also induced ACCs, with similar incidence and lower latency. Immunohistochemical analysis of these mouse ACCs for several biomarkers of human ACC, including p63 and c-kit, provided additional evidence of the similarity of the mouse ACCs that developed in these models, and human ACCs. RNAseq global expression profiling uncovered the activation of several cancer-associated pathways in MYB-induced mouse ACCs. Most notably, activation NOTCH signaling was highly enriched in these tumors. Additional analysis for the presence of the NOTCH intracellular domain demonstrated that all of the MYB- and MYB-NFIB-induced ACCs contain NOTCH activation. These findings, and the recent identification of activating mutations in NOTCH1 and NOTCH2 in human ACCs, suggest that NOTCH activities may contribute to ACC development. To test this hypothesis, ongoing studies in the lab are directed to assess the role of NOTCH signaling in ACC development, by generating mouse models in which constitutive activation of NOTCH is induced in mice alone or in combination with MYB alterations. Overall, these studies demonstrate that MYB activities are the main oncogenic driver of ACC. Genomic rearrangements that result in MYB-NFIB fusions may enhance the oncogenic potential of MYB.

Citation Format: Yuan Hu, Xiaoyuan Zhu, Han Luo, Adel El-Naggar, Carlos Caulin. MYB and the MYB-NFIB fusion induce adenoid cystic carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1611.
Abstract 1624: Lrig1 is an Egfr-dependent tumor suppressor in mouse duodenal and colonic neoplasia
Authors: Coffey Robert J. , Niitsu Hiro , Lu Yuanyuan , Huh Won Jae , Franklin Jeffrey L.
Published: September 2020

Leucine-rich repeats and immunoglobulin-like domain 1 (Lrig1) is a pan-ErbB negative regulator. Lrig1 null mice develop spontaneous duodenal adenomas by 3-4 months of age but they exhibit no overt colonic pathology (AE Powell Cell 149:146-158, 2012). To examine a possible tumor suppressor role for Lrig1 in colonic neoplasia, we utilized Lrig1CreERT2 and Lrig1Apple mice in which a tamoxifen-inducible Cre recombinase or an Apple fluorescent reporter were inserted into the translational start site of endogenous Lrig1, thus creating Lrig1 null mice. Colonic tumors were generated by administration of AOM/DSS to Lrig1CreERT2/CreERT2 mice and by administration of tamoxifen (TAM) to Lrig1CreERT2/Apple;Apcflox/+ mice to induce loss of one Apc allele. Compared to littermate control mice, there was a significant increase in colonic tumor number in both Lrig1 null cohorts (p=0.016 and p=0.007, respectively).

We previously reported Egfr was the most upregulated ErbB family member in Lrig1 null duodenal tumors (Y Wang Am J Path 185:1123-1134, 2015). To directly implicate Egfr in both Lrig1 null duodenal and colonic tumors, we administered TAM at 6 weeks of age to Lrig1CreERT2/CreERT2;Egfrflox/flox mice. At 6 months of age, all oil-injected mice developed tumors, whereas tumors were not detected in 8/21 (38.1%) of TAM-injected mice; the tumor volume in TAM-injected mice was reduced to 7.78 mm3 (73.5%) compared to (10.58 mm3) in the oil-injected group as measured by MRI scanning (p<0.05). In the AOM/DSS colonic tumor model, administration of TAM at 6 weeks of age to Lrig1CreERT2/CreERT2;Egfrflox/flox mice resulted in a significant decrease in tumor number (p=0.021) but not size compared to oil-injected mice. These results indicate that Lrig1 is an Egfr-dependent tumor suppressor in both mouse duodenal and colonic neoplasia.

Citation Format: Robert J. Coffey, Hiro Niitsu, Yuanyuan Lu, Won Jae Huh, Jeffrey L. Franklin. Lrig1 is an Egfr-dependent tumor suppressor in mouse duodenal and colonic neoplasia [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 1624.
Abstract 1626: A novel mouse model of hepatocellular carcinoma induced by c-myc overexpression
Authors: Wang Jinjin , Feng Dan , Zhu Haiyan , Ci Lei , Wan Zhipeng , Sun Ruilin
Published: September 2020

Hepatocellular carcinoma is a fatal liver cancer, and there are limited treatment options for this cancer. c-Myc is a key oncogene in liver cancer, but the specific mechanism of carcinogenesis in liver is unknown. Animal models are widely used to explore the molecular pathogenesis of HCC and test new drug candidates. In this poster, we reported a c-Myc conditional over-expression mouse model (LSL-Myc), which inserts the CAG-Loxp-stop-Loxp-c-Myc-polyA expression cassette into the H11 site. The expression of the Myc gene in this mouse model is regulated by Cre recombinase, which is not normally expressed, but only expressed in Cre-expressing tissue cells. LSL-Myc / Alb-cre double-positive mice were produced by crossing LSL-Myc hybrid mice with Alb-cre mice. In this mouse model, c-Myc was specifically expressed in liver cells. LSL-Myc / Alb-cre double-positive mice show a typical HCC phenotype and show accelerated tumor initiation and rapid HCC progression. Mice can detect significant liver tumorigenesis at 2 months of age, and 100% mice can occur hepatocellular carcinoma. The liver cancer tissues from LSL-Myc / Alb-cre double-positive mice can be transplanted to C57BL/6 mice to establish a mouse-derived PDX model. In summary, this model has the advantages of rapid oncogenesis and high incidence of liver cancer. It is an ideal model for studying the mechanism of c-Myc-driven liver cancer and c-Myc targeted therapy.

Citation Format: Jinjin Wang, Dan Feng, Haiyan Zhu, Lei Ci, Zhipeng Wan, Ruilin Sun. A novel mouse model of hepatocellular carcinoma induced by c-myc overexpression [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 1626.
Abstract 1628: Activation of the Nrf2E79Q mutation in mouse lung accelerate development of lung squamous dysplasia
Authors: Hamad Samera H. , Montgomery Stephanie , Bowman Brittany , Murphy Ryan , Major Ben , Weissman Bernard
Published: September 2020

NFE2-related factor 2 (Nrf2) is a transcription factor that regulates more than 500 genes in human cells to protect them from oxidative damage. Somatic mutations in NRF2 resulting in constitutively Nrf2 signaling have been implicated as driver mutations in cancer, especially lung cancer. However, the mechanisms by which NRF2 activating mutations drive tumor development remains unresolved. Therefore, we used a novel genetically engineered mouse model (GEMM), LSL-Nrf2E79Q/+, to understand the role of this activating mutation (one of the most common mutations found in human tumors) in the development of lung tumors. We compared tumor development in two groups of mice: p53fl/fl; p16fl/fl; LSL-Nrf2+/+ (WT, n=38) and p53fl/fl; p16fl/fl; Nrf2E79Q/+ (Het, n=37). We used intranasal instillation of Adenoviral Cre at 6-8 weeks of age to activate the Nrf2E79Q allele and inactivate p53 and p16. Among other tumors, lung tumors were the most common tumors that were found in the GEMM, with major subtypes of lung squamous dysplasia (LSD: pre-malignant lesions of lung squamous cell carcinoma (LSCC)) and lung adenocarcinoma (LADC). Additionally, Het mice showed significantly higher LSD (30%) compared to WT mice (11%) implicating Nrf2E79Q/+ in LSD initiation. However, no significant difference in LADC was observed in Het mice compared to WT mice. Immunohistochemistry (IHC) including Nrf2, TTF1, CK7 and p63 was used to differentiate lung tumors. LADC of WT and Het mice showed negative Nrf2 staining despite the presence of the recombined Nrf2E79Q/+ allele in the Het tumors. However, LSDs developed by Het mice showed positive Nrf2 staining, while those in WT mice were negative, suggesting the mutant Nrf2 increased the frequency of LSDs. LADC of both groups was positive for TTF1 (A primary-non small lung cancer marker, mostly LADC) and CK7 (Cytokeratin7: a marker of epithelial cells). However, LSD lesions were also positive for TTF1. No p63 (a marker of LSCC) stain was shown for LADC of both groups; while p63 was negative to mildly positive in the case of LSD. Results of this study appear consistent with TCGA results where LADC predominantly contained Keap1 loss, while Nrf2 mutations frequently appeared in LSCC. This is the first study to show that a common activating mutation of Nrf2 promotes initiation of LSD that could further develop into LSCC.

Citation Format: Samera H. Hamad, Stephanie Montgomery, Brittany Bowman, Ryan Murphy, Ben Major, Bernard Weissman. Activation of the Nrf2E79Q mutation in mouse lung accelerate development of lung squamous dysplasia [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 1628.
Abstract 1659: Development of an orthotopic murine model of a patient-derived pancreatic adenocarcinoma to evaluate the tissue specificity of two novel fluorescent compounds
Authors: Duchamp Olivier , Berthet Cyril , Provent Peggy , Chatel Elodie Marie Dit , Longin Jordan , Urbain Stanislas , Scherninski François
Published: September 2020

Fluorescent dyes have been used in medical diagnostics for over 50 years. The emergence of new fluorescent imaging technology in support of surgery and the need for increased sensitivity and precision have driven the search for novel fluorescent molecules. Orthotopic patient-derived pancreatic tumor models provide a closely related human tumor within organ-specific tumor microenvironment, which was well suited to the evaluation of the tissue specificity of two novel fluorescent compounds.

To this end, we have developed an orthotopic tumor model of IM-PAN-15 (PDX model developed within the IMODI consortium), a patient derived pancreatic adenocarcinoma, in mice. Tumor cells were amplified subcutaneously in SCID mice and the resulting fragments subsequently surgically implanted into the pancreas of irradiated BALB/c nude mice. Tumor development was monitored in vivo by MRI at three time points. Fluorescent compounds were injected intravenously 36 days after tumor fragment implantation, when mean tumor volumes were approximately 70 mm³.

Ex-vivo fluorescent imaging showed that 2h after injection, the two novel fluorescent compounds were present in the pancreas and tumor at roughly equivalent amounts. However, 48h after injection, a clear preferential distribution for the tumor was observed, with both compounds producing an approximately four-fold higher fluorescent signal in the tumor than in the surround pancreatic tissue. This effect persisted six days after injection, although the signal decreased over time. In comparison, indocyanine green, a dye widely used in medical diagnostics, showed no specific accumulation in either the pancreas or tumor.

In conclusion, we report the development of a clinically relevant, patient-derived orthotopic tumor model and demonstrate its use in the investigation of tissue specificity of two novel fluorescent compounds.

Keywords: PDX, fluorescent dyes, orthotopic pancreatic model, MRI

Citation Format: Olivier Duchamp, Cyril Berthet, Peggy Provent, Elodie Marie Dit Chatel, Jordan Longin, Stanislas Urbain, François Scherninski. Development of an orthotopic murine model of a patient-derived pancreatic adenocarcinoma to evaluate the tissue specificity of two novel fluorescent compounds [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 1659.
Abstract 1661: Temozolomide suppresses a doxorubicin resistant follicular dendritic cell sarcoma in a patient derived orthotopic xenograft mouse model
Authors: Oshiro Hiromichi , Tome Yasunori , Hoffman Robert M. , Nishida Kotaro
Published: September 2020

Introduction: Follicular dendritic cell sarcoma (FDCS) is a very rare and highly refractory soft tissue sarcoma. The CHOP chemotherapy has been used as systemic therapy for FDCS, which includes cyclophosphamide, doxorubicin (DOX), vincristine, and prednisolone. However, FDCS has poor clinical outcome even though CHOP therapy was administered. A FDCS patient tumor was previously implanted orthotopically in the biceps femoris of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model in our laboratory at AntiCancer Inc. The FDCS patient had resistance to DOX in clinical setting. We previously found the FDCS PDOX model had resistance to DOX as well as clinical settings. In the present study, we evaluated the efficacy of temozolomide (TMZ), trabectedin (TRAB), and pazopanib (PAZ) on the DOX resistant FDCS PDOX model.

Methods: The FDCS PDOX mouse model was established and randomized into five groups of eight mice, respectively. Group 1, Control (Ctrl) treated with PBS, i.p.; Group 2, DOX, 2.4 mg/kg, i.p., weekly; Group 3, PAZ, 50 mg/kg, oral gavage, daily; Group 4, TRAB, 0.15 mg/kg, i.v., weekly; Group 5, TMZ, 25 mg/kg, oral gavage, daily. Treatment term was for 2 weeks. Treatment efficacy was evaluated based on tumor volume ratio (tumor volume after treatment relative to tumor volume at the beginning of treatment) and histopathology. Adverse event was evaluated based on body weight ratio. Tumor volume and body weight were assessed 2 times per week.

Results: Tumor volume ratio was the Ctrl group: 11.35±4.50, DOX: 8.24±1.87, PAZ: 9.67±4.66, TRAB: 6.97±3.23, TMZ: 4.28±1.41, respectively. TMZ significantly arrested the FDCS PDOX model compared to the control group (p<0.05). PAZ and TRAB did not have significant efficacy compared to the Ctrl group (p=0.93, p=0.41). In H&E-staining, partial necrosis was detected only in PDOX tumors treated with TMZ. There was no significant difference of body weight among all groups.

Conclusion: The FDCS PDOX model was resistant to DOX as well as in the clinical settings demonstrating concordance of the FDCS model and clinic. TMZ was the most effective for the FDCS PDOX mouse model established from a patient who failed DOX treatment. There is possibility that TMZ has effective to FDCS patients who has resistant to the first line chemotherapy.

Citation Format: Hiromichi Oshiro, Yasunori Tome, Robert M. Hoffman, Kotaro Nishida. Temozolomide suppresses a doxorubicin resistant follicular dendritic cell sarcoma in a patient derived orthotopic xenograft mouse 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 1661.
Abstract 1682: Epithelial-mesenchymal heterogeneity of high-grade serous ovarian carcinoma samples correlates with let-7 levels and predicts tumor growth and metastasis
Authors: Unternaehrer Juli , Chirshev Evgeny , Hojo Nozomi , Bertucci Antonella , Sanderman Linda , Nguyen Anthony , Wang Hanmin , Suzuki Tise , Brito Emmanuel , Martine Shannalee , Castañón Christine , Mirshahidi Saied , Vazquez Marcelo , Oberg Kerby C. , Ioffe Yevgeniya J.
Published: September 2020

Objective: Patient-derived samples present an advantage over current cell line models of high grade serous ovarian cancer (HGSOC), which are flawed in terms of being reliable and phenotypically faithful models of in vivo HGSOC. To improve upon cell line models of HGSOC, we characterized a panel of patient-derived cells to determine their epithelial and mesenchymal characteristics, invasiveness, proliferation, stemness, and in vivo growth.

Experimental procedures: Patient-derived xenograft (PDX) models of HGSOC were analyzed in vitro for phenotypic (RNA, let-7 miRNA, and protein expression, flow cytometry) and functional aspects including growth in spheroids, wound healing assays, invasion assays and in vivo (orthotopic PDX) growth characteristics.

Results: Samples fell along the spectrum from epithelial to mesenchymal, and all had hybrid characteristics. Those toward the more epithelial end of the spectrum were most active in self-renewal assays, and grew most robustly in orthotopic xenograft models. Chemoresistance correlated both with the mesenchymal state and with BRCA2 wild type status. Loss of microRNA let-7 (lethal-7) is an important component of the cancer stem cell phenotype, and we observed an inverse association between let-7 expression and the epithelial state. We observed lower levels of let-7, more efficient spheroid and tumor formation, and increased sensitivity to platinum-based chemotherapy in cells with the most epithelial phenotype.

Conclusions: Surprisingly, in these HGSOC cells, stemness could be dissociated from invasiveness: epithelial cells (those with least let-7 expression) were less migratory, but more tumorigenic, than the mesenchymal cells with higher let-7 expression. We conclude that epithelial/mesenchymal state and let-7 expression are valuable predictors of HGSOC proliferation, in vitro self-renewal, and tumor burden in vivo.

Citation Format: Juli Unternaehrer, Evgeny Chirshev, Nozomi Hojo, Antonella Bertucci, Linda Sanderman, Anthony Nguyen, Hanmin Wang, Tise Suzuki, Emmanuel Brito, Shannalee Martine, Christine Castañón, Saied Mirshahidi, Marcelo Vazquez, Kerby C. Oberg, Yevgeniya J. Ioffe. Epithelial-mesenchymal heterogeneity of high-grade serous ovarian carcinoma samples correlates with let-7 levels and predicts tumor growth and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1682.
Abstract 1702: Cancer stem cell marker DCLK1 correlates with tumorigenic immune infiltrates in the colon and gastric adenocarcinoma microenvironments
Authors: Qu Dongfeng , Wu Xiangyan , Weygant Nathaniel , Peng Jun , Houchen Courtney
Published: September 2020

Immunotherapy that has proved efficacy in several solid cancers plays a partial role in improving clinical outcomes of advanced gastrointestinal (GI) cancers. There is an unmet need to find new immune-related therapeutic targets. Doublecortin-like kinase 1 (DCLK1) marks tuft cells which are recognized as cancer initiating cells and regulators of the type II immune response, and has been studied for its role in many cancers including colon and gastric cancers, but its role in tumor immunity remains unexplored. In the current study, we analyzed colon and gastric cancer RNA sequencing data from 283 and 415 patients respectively from The Cancer Genome Atlas (TCGA). High DCLK1 expression predicted the worse clinical outcomes in colon and gastric cancer patients and correlated with increased immune and stromal components. Further analysis indicated that DCLK1was strongly linked to infiltration of multiple immune cell types, especially TAMs and Tregs, and strongly correlated with increased CD8+ T cell inhibitors TGFB1 and CXCL12 and their receptors, suggesting it may contribute to TAM-mediated inhibition of CD8+ T cells. Interestingly, we found that DCLK1 was a prognostic biomarker in left-sided colon cancer which has worse outcomes and demonstrates a reduced response to existing immunotherapies. In conclusion, our results demonstrate that DCLK1 is linked with functional regulation of the tumor microenvironment and may have potential as a prognostic biomarker and adjuvant target to promote immunotherapy sensitivity in colon and gastric cancer patients.

Citation Format: Dongfeng Qu, Xiangyan Wu, Nathaniel Weygant, Jun Peng, Courtney Houchen. Cancer stem cell marker DCLK1 correlates with tumorigenic immune infiltrates in the colon and gastric adenocarcinoma microenvironments [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 1702.