bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1526: Circulatory insulin-like growth factor 1 predicts metastasis in triple-negative breast cancer
Authors: Akter Salima , Faruque Mithila , Akhter Hajara , Bhowmik Mousumi , Hasan Mohammad Nazmul , Sultana Lipika , Khan Imran , Mia Mamun , Rahman Md. Mustafizur , Chaudhury Habib Sadat , Rafiqul Islam S. , Nahar Kamrun , Choi Tae Gyu , Kim Sung Soo
Published: June 2022

Background and aims: Aberrant insulin-like growth factor (IGFs) signaling is directly implicated in oncogenesis and metastasis in various solid tumors including breast cancer. Targeting the IGF axis may provide an insight into the mechanism of cancer stemness and therapy resistance. The present study aims to explore the association of the circulatory levels of these proteins in aggressive triple-negative breast cancer (TNBC) in Bangladesh.

Methods: A total of 165 BC patients were recruited in the present study where 42 were TNBC subjects (selected based on their complete hormonal profile). Data were collected from retrospective information. Anthropometric and biochemical parameters were measured by standard methods. Serum IGF1, IGFBP1, and insulin were estimated by ELISA.

Results: TNBC subjects showed significantly higher levels of circulatory IGF1 (185 vs 145, p=0.004), while patients who had distant metastasis showed increased levels of IGF1, IGFBP1, and alkaline phosphatase (ALP) [178 vs 153, p=0.001; 6.44 vs 3.05, and 140 vs 88, p<0.05; metastatic vs non-metastatic BC respectively]. In addition, serum IGF1 was positively associated with serum insulin levels and IGFBP1 was associated with ALP, SGPT, CA15.3, and duration of OCP (p<0.05). In multivariate regression, IGF1 was found to be a significant predictor of breast cancer survival after adjusting confounding variables.

Conclusion: High serum levels of IGF1 may contribute to metastasis in basal breast cancer in a Bangladeshi population. These results need to be confirmed in larger TNBC survivor cohorts.

Citation Format: Salima Akter, Mithila Faruque, Hajara Akhter, Mousumi Bhowmik, Mohammad Nazmul Hasan, Lipika Sultana, Imran Khan, Mamun Mia, Md. Mustafizur Rahman, Habib Sadat Chaudhury, Islam S. Rafiqul, Kamrun Nahar, Tae Gyu Choi, Sung Soo Kim. Circulatory insulin-like growth factor 1 predicts metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1526.
Abstract 153: An unbiased in vitro screen for activating ERBB4 mutations
Authors: Chakroborty Deepankar , Ojala Veera K. , Knittle Anna M. , Kurppa Kari J. , Elenius Klaus
Published: June 2022

Introduction: Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites across different samples. The discovery of most of the currently known driver mutations has been facilitated by their accumulation in mutation hotspots within their respective genes. However, a vast majority of mutations in cancer tissues are rare and their functional significance remains unknown. Several lines of in vitro and clinical evidence also indicate that there is a significant number of, as yet unidentified, activating driver mutations which could serve as predictive markers in oncology. Here, we performed an unbiased functional screen to identify potential activating mutations of ERBB4, a frequently mutated member of the epidermal growth factor receptor family.

Method: To identify functional driver mutations of ERBB4, the previously published pipeline, in vitro screen for activating mutations (iSCREAM) [1], was utilized. iSCREAM is a functional genetics screen based on the expression of random cDNA variants and the ability of driver mutations to promote cellular growth in vitro. The expression library encoding randomly mutated ERBB4 variants was retrovirally introduced into murine lymphoid Ba/F3 cells, that normally require interleukin-3 (IL-3) for survival but can exploit ectopic expression of activated variants of oncogenic kinases to compensate for the deficiency of exogenous IL-3. When expressed under an appropriate promoter, the wild-type ERBB4 receptor failed to promote IL-3-independent survival in the presence of the neuregulin-1 ligand, while the cells expressing activating mutations readily proliferated. The identity and frequency of the activating ERBB4 mutations were subsequently determined from the proliferating cell pool using targeted next-generation sequencing.

Results: Ten candidate activating mutations were identified out of the over 7000 random ERBB4 missense or nonsense mutations present in the original library. The candidate activating mutations were individually characterized using functional assays both in vitro and in vivo, as well as by structural analyses. The activating mutation were sensitive to clinically used pan-ERBB tyrosine kinase inhibitors afatinib, dacomitinib and neratinib.

Conclusions: A subset of ERBB4 missense mutations are activating and sensitive to tyrosine kinase inhibitor drugs.

Citation Format: Deepankar Chakroborty, Veera K. Ojala, Anna M. Knittle, Kari J. Kurppa, Klaus Elenius. An unbiased in vitro screen for activating ERBB4 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 153.
Abstract 1531: CD146 couples and sequesters regulatory kinases to control breast cancer cell state plasticity
Authors: Balcioglu Ozlen , Gates Brooke L. , Freeman David W. , Hagos Berhane M. , Spike Benjamin T.
Published: June 2022

CD146 (Mcam; Muc18; s-endo; and gicerin) is an integral membrane glycoprotein originally identified in a human malignant melanoma, but mis-expressed in a wider variety of malignancies including breast cancer, where it has been implicated in controlling tumor growth and dissemination. The mechanisms by which CD146 conveys extracellular cues into malignant cellular outputs is not well understood, but its high-level expression in early development and normal mouse mammary stem cells, as well as expression in highly plastic adult pericytes (formerly known as mesenchymal stem cells) suggested to us that it may control cell state plasticity. We tested the role of CD146 in a plastic, stem cell-like mouse mammary tumor cell line derived from the MMTV-PyMT mouse (PY230). We identified domains in the CD146 cytoplasmic tail essential for controlling substrate utilization by upstream kinases in Akt, Mapk, Stat3, and integrin-related signaling pathways that alter cell state plasticity and tumorigenicity. This work suggests that MCAM plays a key role in controlling lineage plasticity in normal and neoplastic mammary cells with relevance for understanding tumor cell plasticity, progression and evasion of targeted cancer therapies.

Citation Format: Ozlen Balcioglu, Brooke L. Gates, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. CD146 couples and sequesters regulatory kinases to control breast cancer cell state plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1531.
Abstract 1539: LKB1 down-modulation by miR-17 biologically and clinically mirrors LKB1-mutated NSCLC
Authors: Borzi Cristina , Ganzinelli Monica , Caiola Elisa , Colombo Marika , Centonze Giovanni , Boeri Mattia , Caleca Laura , Pastorino Ugo , Marabese Mirko , Milione Massimo , Broggini Massimo , Garassino Marina C. , Sozzi Gabriella , Moro Massimo
Published: June 2022

Introduction: In non-small cell lung cancer (NSCLC) LKB1 mutations combined with KRAS hyperactivation define a poor responsive, aggressive and disseminating phenotype. We recently unveiled the vulnerability of KRAS/LKB1 co-mutated (KL) patient-derived xenografts (PDXs) to metabolic stress-based treatments, and a clinical trial to test the efficacy of metformin and fasting-mimicking diet on KL NSCLC is currently ongoing in our Institute. Besides inactivating mutations, LKB1 functionality may be impaired by post-translation regulation. Because miR-17 is a potential epigenetic regulator of LKB1,we hypothesized that wild-type LKB1 (LKB1WT) NSCLC with high miR-17 expression may biologically and clinically mirror LKB1 mutated tumors.

Methods: NSCLC cell lines with different combinations of KRAS mutation and LKB1 deletion were selected, together with a panel of PDXs with high (LKB1WT/miR-17 high) or low (LKB1WT/miR-17 low) miR-17 expression. Taking advantage of series of NSCLC patients and PDXs, we retrospectively evaluated LKB1 and miR-17 expression levels in tumor tissue specimens. In addition, the TCGA dataset was interrogated for miR-17 expression and potential correlation with lung cancer clinical features.

Results: Direct targeting of miR-17 to LKB1 3’UTR was confirmed by luciferase reporter assay. We found that miR-17 overexpression in LKB1WT cell lines functionally impaired the LKB1/AMPK pathway, and prompted apoptotic response to metformin. Of note, LKB1WT/miR-17 high PDXs tumor cells showed a similar behavior upon metformin treatment. A retrospective analysis in patients with NSCLC revealed an inverse correlation between miR-17 and LKB1 expression, and highlighted a prognostic role of miR-17 expression in LKB1WT patients, further confirmed by TCGA data analysis.

Conclusions: We validated miR-17 as epigenetic regulator of LKB1 expression in NSCLC tumors. We propose a miR-17 expression score potentially exploitable to discriminate LKB1WT NSCLC patients with impaired LKB1 expression and poor outcome, eligible for energy-stress-based treatments.

Citation Format: Cristina Borzi, Monica Ganzinelli, Elisa Caiola, Marika Colombo, Giovanni Centonze, Mattia Boeri, Laura Caleca, Ugo Pastorino, Mirko Marabese, Massimo Milione, Massimo Broggini, Marina C. Garassino, Gabriella Sozzi, Massimo Moro. LKB1 down-modulation by miR-17 biologically and clinically mirrors LKB1-mutated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1539.
Abstract 1576: Chemotherapy induced cellular cannibalism is mediated by phosphoinositide species and clathrin
Authors: Frey Wesley D. , Anderson Ashlyn , Nguyen Julie , Cowles Emma , Jackson James
Published: June 2022

Cancer cells can survive chemotherapy and drive lethal relapse if they avoid cell death in conditions of 1) DNA damage and/or mitotic stress caused by treatment; 2) nutrient depletion. We and others have previously shown that the breast cancers most likely to survive chemotherapy are TP53 wild-type, and these are among the most lethal breast cancers. For instance, chemotherapy treated TNBC patients with TP53 wild-type tumors have a median overall survival of 45 months, contrasting with 263 months for TP53 mutant tumors. TP53 wild type cells survive stress by entering a state of arrest and cellular senescence. But to persist in senescence, cells must survive in limited access to vasculature and nutrient sources while also supporting a high metabolic burden that includes production of cytokines and chemokines that drive pro-tumorigenic phenotypes and relapse.

We previously showed a novel cannibalism phenotype of chemotherapy induced senescent cells. Engulfment occurred after exposure to different chemotherapy drugs in vitro, in 9 different cell lines, and in vivo in syngeneic mouse mammary tumor models. Engulfed prey cells were processed to the lysosomes of predator cells and broken down. While we showed the phenotype was unrelated to entosis, and senescent cells expressed many phagocytosis/macrophage related genes, the basic mechanisms of the engulfment are unknown.

Here, we use biosensors and live cell imaging to delineate the steps of whole cell engulfment in cells that have entered senescence to survive chemotherapy. We show predator cell filamentous actin was localized to the prey cell throughout the process of engulfment. Biosensors to various phosphoinositide (PI) species showed increased concentration and localization of predator PI(4)P and PI(4,5)P2 at the prey cell during early stages of engulfment, followed by a burst of PI(3)P before internalization. PIK3C2B, an enzyme responsible for generating PI(3)P, was required for complete engulfment. Inhibition or knockdown of Clathrin, known to associate with PIK3C2B and PI(4,5)P2, severely impaired engulfment. In sum, these data demonstrate the mechanism of cellular cannibalism used by breast cancer cells to survive chemotherapy.

Citation Format: Wesley D. Frey, Ashlyn Anderson, Julie Nguyen, Emma Cowles, James Jackson. Chemotherapy induced cellular cannibalism is mediated by phosphoinositide species and clathrin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1576.
Abstract 1593: Genetic and pharmacological modulation of DNA mismatch repair promotes immune surveillance in murine colorectal cancer
Authors: Amodio Vito , Germano Giovanni , Mussolin Benedetta , Lamba Simona , Chilà Rosaria , Rospo Giuseppe , Marchiò Caterina , Marsoni Silvia , Mauri Gianluca , Di Nicolantonio Federica , Bardelli Alberto
Published: June 2022

Patients affected by colorectal cancer (CRC) with microsatellite instability (MSI), which is caused by DNA mismatch repair deficiency (MMRd), are eligible for therapies based on immune checkpoint inhibitors (CPI), while microsatellite stable (MSS) tumors are not. However, a subset of MSS CRCs contains variable fractions of MMRd cells. How the presence of MMRd cells in tumors classified as MSS impacts cancer immune surveillance is largely unknown. It is also unclear whether the pharmacological modulation of MMRd cells percentage in MMR heterogeneous tumor can occur and if this might result in the improvement of tumor immune control.To shed light on these aspects we studied isogenic mismatch repair proficient and deficient mouse tumor cells, generated by genetic inactivation of MLH1, a key component of the MMR machinery. We mixed MLH1+/+and MLH1-/-cells at different ratios, injecting the resulting heterogeneous populations in mice. In the presence of a competent immune system, the tumorigenic potential and immune surveillance of MSS/MSI (MLH1+/+/MLH1-/-) heterogeneous tumors was dependent upon the MMRd fraction. Tumor rejection was observed when at least 50% of the cells were MMRd, but tumor growth delay was also evident when as low as 20% of MMRd cells were present in the mixed population and this was paralleled by immune infiltration of the tumor. Molecular profiles of samples from MSS/MSI heterogeneous tumors that evaded immune control, showed enrichment of the MSS fraction. Treatment of MSS/MSI mixed populations with the antimetabolite 6-Thioguanine (6TG) greatly enriched the MMRd fraction and improved immune response. Overall, these results suggest that genetic and/or pharmacological modulation of the DNA mismatch repair machinery can foster immune surveillance of MMR heterogeneous tumors and modulate the cancer immune environment.

Citation Format: Vito Amodio, Giovanni Germano, Benedetta Mussolin, Simona Lamba, Rosaria Chilà, Giuseppe Rospo, Caterina Marchiò, Silvia Marsoni, Gianluca Mauri, Federica Di Nicolantonio, Alberto Bardelli. Genetic and pharmacological modulation of DNA mismatch repair promotes immune surveillance in murine colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1593.
Abstract 1601: CD47 related to intratumor heterogeneity in alectinib-resistant ALK-rearranged lung cancer cell lines
Authors: Funazo Tomoko Y. , Ozasa Hiroaki , Hashimoto Kentaro , Yoshida Hiroshi , Ogimoto Tatsuya , Hosoya Kazutaka , Ajimizu Hitomi , Tsuji Takahiro , Yoshida Hironori , Itotani Ryo , Sakamori Yuichi , Hirai Toyohiro
Published: June 2022

Overcoming the treatment resistance of cancer is a problem to be solved in improving the prognosis of cancer patients. Recently, genome analysis has revealed that multiple clusters exist in one tumor, and it has been reported that intratumor heterogeneity causes treatment resistance even in lung cancer. However, the mechanism of intratumor heterogeneity has not yet been clarified. Previous studies of intratumor heterogeneity have mainly focused on analyzing cancer cells from patients, classifying clusters of different properties, and identifying factors that define their characteristics. Multiple clusters are present in the tumor tissue of a single patient, and analysis of multiple patients classifies them into even more clusters, but it is difficult to research all of them. To solve this problem, an early model of intratumor heterogeneity in which homogeneous cells become heterogeneous is needed.We hypothesized that intratumor heterogeneity cell model could be found in resistant strains.Alectinib was exposed in vitro to an ALK-positive lung cancer cell line (H2228) to create an alectinib-resistant cell line (H2228-AR1S). H2228-AR1S has two subpopulations that look different. Of the two subpopulations, the smaller cells have high CD47 expression (CD47 high subpopulation), and the scattered spindle-shaped cells have low CD47 expression (CD47 low subpopulation). Using flow cytometry, each subpopulation was isolated, and its properties were investigated. There was no difference in sensitivity to alectinib between the CD47 high subpopulation and the CD47 low subpopulation. In the low subpopulation of CD47, epithelial markers were decreased, and mesenchymal markers were increased using immunoblotting. It suggests that CD47 low subpopulation has undergone epithelial-mesenchymal transition (EMT). The CD47 high subpopulation had high sphere formation ability in vitro, and high tumorigenicity using Xenograft model. CD47 gene inhibition using siRNA reduced the sphere formation ability of the CD47 high subpopulation. This suggests that CD47 is involved in sphere formation.There have been no reports of CD47 being involved in the characteristics of intratumor heterogeneity. Furthermore, using intratumor heterogeneity cell model, we are exploring the mechanism that regulate division into two subpopulations.

Citation Format: Tomoko Y. Funazo, Hiroaki Ozasa, Kentaro Hashimoto, Hiroshi Yoshida, Tatsuya Ogimoto, Kazutaka Hosoya, Hitomi Ajimizu, Takahiro Tsuji, Hironori Yoshida, Ryo Itotani, Yuichi Sakamori, Toyohiro Hirai. CD47 related to intratumor heterogeneity in alectinib-resistant ALK-rearranged lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1601.
Abstract 1625: c-Myc inhibition in prostate cancer cell lines
Authors: Riveros Carlos , Chalfant Victor , Shukla Sanjeev , Osumi Teruko , Balaji KC
Published: June 2022

The proto-oncogene c-Myc is a transcription factor that is upregulated in several human cancers. Despite its key role, therapeutic targeting of c-Myc remains a challenge because of a disordered protein tertiary structure. The basic helical structure and zipper protein of c-Myc forms an obligate heterodimer with its partner MAX to function as a transcription factor. An attractive strategy is to inhibit MYC/MAX dimerization to decrease c-Myc transcriptional function. Several methods have been described to inhibit MYC/MAX dimerization including small molecular inhibitors and proteomimetics. We used NOD SCID gamma mice (NGS mice) that are immunodeficient, non-obese diabetic laboratory mice (Jackson Laboratories, Cat Number J000077451) carrying patient-derived xenograft (PDX) following implantation of grade IV adenocarcinoma of the prostate from a patient into the flanks of each mouse. For maximum tolerable dose studies, we used C57BL/6 wild type mice (Jackson Laboratories, Cat Number J000664). We performed a maximum dose tolerability study with the drug 3JC48-3 up to 100 mg/kg to determine limiting toxicity. We monitored the rate of growth for 2 weeks and measured the final volume of the tumors. Immunoprecipitation studies were performed using the prostate cancer cell line, PC-3, that were treated with the drug 3JC48-3 at different doses (0, 5, 10, and 20uM) for 24 hours and subsequently lysed. We found 3JC48-3 decreases prostate cancer growth and viability in a dose-dependent fashion in vitro. We confirmed the inhibition of MYC/MAX dimerization by 3JC48-3 using immunoprecipitation experiments. Normal mice and mice with patient-derived prostate cancer xenografts (PDX) tolerated intraperitoneal injections of 3JC48-3 up to 100 mg/kg body weight without dose-limiting toxicity. Preliminary results in these PDX mouse models suggest that 3JC48-3 may be effective in decreasing the rate of tumor growth. Our study demonstrates that 3JC48-3 is a potent MYC/MAX heterodimerization inhibitor that decreases prostate cancer growth and viability in association with upregulation of both PrKD1 expression and kinase activity. In addition, 3JC48-3 is well tolerated in prostate cancer PDX mice models and may be a candidate drug for further preclinical development.

Citation Format: Carlos Riveros, Victor Chalfant, Sanjeev Shukla, Teruko Osumi, KC Balaji. c-Myc inhibition in prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1625.
Abstract 1646: Humanized TROP2 mice provide a preclinical tool for the evaluation of therapeutic drugs targeting TROP2
Authors: Huang Dexuan , Shen Zhiyuan , Ma Limei , Li Chong , Jin Tianquan
Published: June 2022

Human TROP2, also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a type I cell surface glycoprotein that mediates intracellular calcium signals. It plays a crucial role in cell proliferation, survival, self-renewal, and cell adhesion. Several ligands are found to interact with TROP2, including claudin-1, claudin-7, cyclin D1, and IGF-1. TROP2 is expressed on the surface of many stem and progenitor cells as well as other tissues. Importantly, TROP2 is highly expressed in a variety of solid tumors, making it a new target for the development of antibody-conjugated drugs. To evaluate the efficacy of anti-TROP2 antibodies, we generated a humanized B-hTROP2 mouse at Biocytogen. In this mouse model, the murine TROP2 gene was replaced by the full length human TROP2 gene. Western blot analysis showed that human TROP2 protein expression was detected in skin and kidney of homozygous B-hTROP2 mice. These results suggest that B-hTROP2 mice is an ideal tool for in vivo efficacy evaluation of therapeutic drugs targeting human TROP2.

Citation Format: Dexuan Huang, Zhiyuan Shen, Limei Ma, Chong Li, Tianquan Jin. Humanized TROP2 mice provide a preclinical tool for the evaluation of therapeutic drugs targeting TROP2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1646.
Abstract 1653: Toward developing a humanized mouse model of the microenvironment associated with the malignant cells of Hodgkin lymphoma
Authors: Bilotta Maria Teresa , Antignani Antonella , Fitzgerald David J.
Published: June 2022

The tumor microenvironment (TME) represents a highly complex compartment that includes cancer cells, non-hematopoietic stromal cells, extracellular matrix, lymphocytes, and myeloid cells. Interactions between these populations and the factors they secrete play critical roles in determining the host response to cancer. Conventional mouse xenograft models of human cancer lack a competent immune system. Indeed, the use of xenograft models often fail to translate into effective human treatments. Classical Hodgkin Lymphoma (HL) is characterized by the presence in the lymphonodes of abnormal Reed-Sternberg cells, and many cell types beyond them, in particular Treg. To reconstitute a more complete TME, human PBMCs were injected into NSG mice bearing L540 HL cells. Then over time the presence of various human CD45+ cell populations were monitored within the tumor mass, in the spleen and in circulation. The goal of our study is to gain a better understanding of the interaction of the immune cell infiltrates into the HL microenvironment, leading ultimately to improved treatment outcomes. Lymphocyte infiltration into the tumor showed a predominance of human CD3+ cells (T lymphocytes), even if in the spleen we found both human T and B cells, indicating that this model might help us design more effective treatments. To reflect the immune reconstitution of these animals and its interaction with tumor and to better understand the capacity of T lymphocytes to kill the cancer cells, we evaluated the production of human IFN- gamma. We found an increase of IFN- gamma in L540-bearing mice reconstituted with hPBMCs compared to the mice without them, indicating a certain functionality of the T cells in these tumors. Moreover, in this model, the immune reconstitution did not decrease tumor growth, rendering this model useful for the study of biotherapeutic and immunotherapeutic studies. The model might be useful to study the anticancer therapy at a preclinical level because a remodeling of the anti-tumor immune response can be evaluated.

Citation Format: Maria Teresa Bilotta, Antonella Antignani, David J. Fitzgerald. Toward developing a humanized mouse model of the microenvironment associated with the malignant cells of Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1653.
Abstract 1674: FGF8 as a modulator of Wilms' tumor in vitro growth and longevity
Authors: Cooper Garrett , Lee Benjamin , Smyth Nate , Mittal Karuna , Lawrence Taylor , Jonus Hunter , Shim Jenny , Goldsmith Kelly , Hong Andrew
Published: June 2022

Wilms’ tumor is the most common pediatric renal tumor accounting for 6-7% of all childhood cancers. Development of faithful Wilms’ tumor cell lines is needed to identify novel therapeutics. Currently, established Wilms’ tumor cell lines that are suitable for high throughput studies only represent anaplastic Wilms, a rare subtype. In contrast, cell lines representing the more common favorable histology Wilms’ tumor (FHWT) often have poor proliferative capacity and are unable to passage long-term (e.g. >20 passages). For this reason, there is a need to establish robust cell lines representing FHWT. We hypothesize that there are core factors missing from the cell culture medium which prevent proper growth signaling and proliferation. We first performed bulk RNA-sequencing from patients and associated cell lines at Aflac Cancer and Blood Disorders Center with an underlying diagnosis of FHWT. Transcriptomic analyses showed that the reduced growth and proliferation in these FHWT cell lines may be due to reduced presence of the mitogenic fibroblast growth factor 8 (FGF8) in the cell culture environment as compared to the tumor environment. Wilms’ tumor has notably high FGF8 expression levels compared to all other pediatric tumors, implicating FGF8 as a potential driving factor in tumor maintenance. To characterize the phenotypic effects of FGF8 reintroduction in short term FHWT cell lines, we both endogenously overexpressed FGF8 via a lentiviral vector and ectopically added recombinant FGF8 to the cell culture medium. Qualitatively, endogenous overexpression of FGF8 induced distinct morphological changes in cells, converting the enlarged and flattened cells into more focal cells resembling proliferative early passage cells. Quantitatively, overexpression of FGF8 in one favorable histology cell line led to an increased rate of cellular proliferation and extended the cellular longevity by seven passages (47% above baseline). RNA-sequencing analysis of these FGF8 overexpressing cells implicated the anti-apoptotic gene, NTSR1, and an extracellular matrix protein, SNED1, as potential downstream targets of FGF8 overexpression. However, ectopic addition of recombinant FGF8 protein to the cellular medium had less pronounced effects, with seemingly cell-line specific effects. These data demonstrate FGF8 as possibly being required for survival in certain Wilms’ tumor cell lines that can be modulated to influence in vitro proliferation and longevity.

Citation Format: Garrett Cooper, Benjamin Lee, Nate Smyth, Karuna Mittal, Taylor Lawrence, Hunter Jonus, Jenny Shim, Kelly Goldsmith, Andrew Hong. FGF8 as a modulator of Wilms' tumor in vitro growth and longevity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1674.
Abstract 1685: Deep learning approaches to deciphering intra-tumoural heterogeneity in glioblastoma
Authors: Dent Anglin J. , Faust Kevin , Lam Brian , Leon Alberto J. , Tsang Queenie , Diamandis Phedias
Published: June 2022

BACKGROUND: Emerging evidence strongly implicates intra-tumoral heterogeneous biology in treatment resistance and disease progression across many cancer types. Thus, there is a need for workflows capable of systematically resolving and targeting distinct tumor subpopulations1. Using glioblastoma (GBM) as a prototype, I have aimed to leverage the computational power of Artificial Intelligence (AI) and deep learning to develop an autonomous workflow for the objective definition of biologically distinct tumor subpopulations2.

OBJECTIVES: I hypothesize that AI may be leveraged as a tool to resolve spatial heterogeneity, by identifying tumoral subpopulations with unique molecular profiles and therapeutic targets. To highlight the need for routine analysis of tumor heterogeneity, I will address if:

METHODS: I apply our developed image clustering workflows to quantify AI-defined subregions within a clinical cohort of 10 GBM patient tumors2,3. Laser capture microdissection and mass spectrometry-based proteomics are leveraged to address if AI-defined subregions show intra-tumoral molecular variation. Further, existing pharmacogenomic databases are utilized to carry out drug sensitivity and transcriptional clustering to define AI-defined region-specific therapeutic sensitivities and resistances across my clinical GBM cohort4.

RESULTS: Preliminary data shows that region to region heterogeneity can be found in IDH wild-type GBM using our unbiased omics approach, in addition to predicting different pharmacogenomic sensitivities.

CONCLUSIONS: This project aims to develop the first AI-driven tool to guide the routine and systematic molecular analysis of spatial morphogenomic heterogeneity. Further, this tool may have the potential to provide novel approaches for personalized care by selecting drug combinations that target a larger fraction of a tumor’s true biology.

1. Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science (80-. ). 344, 1396-1401 (2014).

2. Faust, K. et al. Unsupervised Resolution of Histomorphologic Heterogeneity in Renal Cell Carcinoma Using a Brain Tumor-Educated Neural Network. JCO Clin. Cancer Informatics 811-821 (2020) doi:10.1200/cci.20.00035.

3. Roohi, A., Faust, K., Djuric, U. & Diamandis, P. Unsupervised Machine Learning in Pathology: The Next Frontier. Surgical Pathology Clinics vol. 13 349-358 (2020).

4. Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity. Nat. 2012 4837391 483, 603-607 (2012).

Citation Format: Anglin J. Dent, Kevin Faust, Brian Lam, Alberto J. Leon, Queenie Tsang, Phedias Diamandis. Deep learning approaches to deciphering intra-tumoural heterogeneity in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1685.
Abstract 1718: Characterization of infiltrating immune cells after irradiation using 10x genomics single cell sequencing
Authors: Ponthan Frida , Ubertini Valentina , Beale Gary
Published: June 2022

Background: Radiation therapy is delivered to approximately 50% of all cancer patients. It has long been known that radiation causes not only DNA strand breaks, apoptosis, and necrosis, but also immunogenic modulation. Lately, there has been renewed interest in the potential of irradiation in colorectal cancer, not only as an adjuvant and palliative treatment, but also as an immune stimulant.

Materials and Methods: CT26 murine colorectal carcinoma cells were subcutaneously implanted on the flank of BALB/c mice. Tumors were exposed to sham, single dose or fractionated radiotherapy regimens via a 300kV, 10mA X-ray with additional filtration to give a radiation quality of 2.3mm Cu half-value layer (HVL). Mice were anaesthetized, restrained and delivered a single uniform dose of radiation at a rate of 0.696Gy/min with the whole mouse being lead shielded except for a 1cm hole in the lead above the tumor site. Tumors were harvested at different timepoints post irradiation and dissociated into single cell suspensions. The tumor microenvironment and immune response within the tumors were analyzed with flow cytometry comparing the different treatment regimens. The individual CD45+ cell profile was investigated using 10x gene expression. 10x sequencing was demultiplexed using CellRanger and data analyzed using Seurat and ACTINN.

Results: Following the initial cytotoxicity, flow cytometry showed a clear immune stimulatory effect of radiotherapy with immune cells infiltrating the irradiated tumors. The level of infiltration was dependent on the treatment scheduling. For single cell assessments, we observed approximately 8000 reads/cell and T-SNE cluster analysis using Seurat identified 16 clusters in control and treated samples. Identification of clusters by ACTINN demonstrated that CD8+ T-cells were increased 8-fold in irradiated samples whereas monocytes numbers decreased 3-fold. In irradiated tumors, the main population of CD8+ T-cells with increased numbers also expressed NKG7.

Conclusion: These data provide evidence that radiotherapy can enhance immune cell trafficking to locally treated CT26 tumors. Furthermore, 10x genomics single cell sequencing revealed increased levels of cytotoxic T-cells expressing NKG7, a protein implicated in enhanced killing of extracellular targets. This response could prime the tumors such that they may be exploited by immune modulating drugs.

Citation Format: Frida Ponthan, Valentina Ubertini, Gary Beale. Characterization of infiltrating immune cells after irradiation using 10x genomics single cell sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1718.
Abstract 1759: PRO1160, a novel CD70-directed antibody-drug conjugate, demonstrates robust anti-tumor activity in mouse models of renal cell carcinoma and non-Hodgkin lymphoma
Authors: Wang Lei , Liu Haidong , Shang Xiao , Han Tae , Zhao Baiteng
Published: June 2022

PRO1160 is an antibody-drug conjugate directed toward CD70, an antigen that is aberrantly overexpressed in multiple hematologic malignancies and carcinomas with limited normal tissue expression. PRO1160 is comprised of a 1) human monoclonal antibody that selectively binds to CD70, 2) a cleavable, hydrophilic linker, and 3) exatecan, a topoisomerase 1 inhibitor. Upon binding to CD70 on the surface of malignant cells, PRO1160 is internalized and exatecan released through enzymatic cleavage of the linker. Exatecan blocks the ligation step of the cell cycle and generates DNA single- and double-strand breaks, which leads to cell death. PRO1160 demonstrated robust anti-tumor activity, in vitro and in vivo. In multiple cell-derived xenograft mouse models, PRO1160 demonstrated more potent tumor growth inhibition compared to an MMAE-conjugated ADC with the same parent antibody. PRO1160 was well tolerated in mice with no to minimal impact on body weight gain. The in vivo pharmacokinetics in rats showed that PRO1160 is stable in circulation. These nonclinical data suggest the potential for a broad therapeutic window and further development of PRO1160 is warranted. PRO1160 has the potential for an expanded therapeutic index. PRO1160 is being developed for the potential treatment of patients with CD70-expressing hematologic and solid tumor cancers.

Citation Format: Lei Wang, Haidong Liu, Xiao Shang, Tae Han, Baiteng Zhao. PRO1160, a novel CD70-directed antibody-drug conjugate, demonstrates robust anti-tumor activity in mouse models of renal cell carcinoma and non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1759.
Abstract 1817: EG-011 is a first-in-class Wiskott-Aldrich syndrome protein (WASp) activator with anti-tumor activity
Authors: Spriano Filippo , Barnabei Laura , Carrasco Del Amor Ana Maria , Tomasso Meagan R. , Tarantelli Chiara , Riveiro Eugenia , Pazzi Natalina , Padrick Shae B. , Cristobal Susana , Cavalli Franco , Gaudio Eugenio , Bertoni Francesco
Published: June 2022

Background. Novel therapeutic targets are needed to improve the outcome of individuals with cancer. EG-011 is a small molecule with in vitro and in vivo anti-tumor activity in lymphoma and acute leukemias, but, at least so far, no activity in solid tumor models (Gaudio et al AACR 2019). Since no information was available on its target, we have now performed experiments showing that EG-011 targets WASp.

Methods. Target identification: kinase screens with DiscoverX KINOMEscan, ProQinase Wildtype-Profiler; thermal proteomic profiling. Target validation: pyrene actin polymerization assay. In vitro drug treatment of cell lines followed for changes in actin filaments (F-actin) by confocal imaging with Alexa Fluor 488 Phalloidin.

Results. Extensive kinome screens excluded kinases as targets of EG-011. We then applied thermal proteomic profiling to identify new protein targets interacting with EG-011. Over 3,300 proteins from the soluble proteome from the EG-011 sensitive mantle cell lymphoma cell line REC1 were analyzed and 48 possible protein targets were initially identified. Among the proteins undergoing a thermal shift, WASp was among the most highly destabilized by EG-011. Due to the pattern of expression of WASp, compatible with the anti-tumor activity observed only in hematological cancers (Gaudio et al AACR 2019), we performed further experiments to confirm the possibility that EG-011 targets WASp. One of the main functions of WASp is the regulation of actin filaments formation. Pyrene actin polymerization assays demonstrated that EG-011 activated the auto-inhibited form of WASP with strong actin polymerization. Further confirmation was obtained using confocal imaging of cell lines exposed to DMSO or EG-011 (500 nM, 5 μM) and stained for F-actin. An increase in actine polymerization was seen in EG-011 sensitive (VL51) and not in resistant (Z138) cell lines at 4, 8 and 24h with both concentrations.

Conclusions. These data demonstrate that EG-011 is the “first-in-class” activator of the auto-inhibited form of WASp with selective anti-tumor activity in lymphomas.

Citation Format: Filippo Spriano, Laura Barnabei, Ana Maria Carrasco Del Amor, Meagan R. Tomasso, Chiara Tarantelli, Eugenia Riveiro, Natalina Pazzi, Shae B. Padrick, Susana Cristobal, Franco Cavalli, Eugenio Gaudio, Francesco Bertoni. EG-011 is a first-in-class Wiskott-Aldrich syndrome protein (WASp) activator with anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1817.
Abstract 1819: CBP-1008 shows excellent efficacy and desirable drug safety profile in preclinical models
Authors: Tan Wei , Wang Wei , Bu Tingting , Liu Xuelian , Teng Yan , Huang Baohua
Published: June 2022

Folate Receptor Alpha (FRα) is the high affinity folate transporter which is highly expressed in ovarian, lung and breast cancer. Transient Receptor Potential Vanilloid 6(TRPV6), a calcium channel characterized by its high calcium selectivity. It plays an important role in calcium absorption in small intestine and calcium uptake in other epithelial tissues. TRPV6 is also upregulated in breast, ovarian and prostate cancer.

CBP-1008, A first-in-class drug conjugate which targets both FRα and TRPV6 with low molecular-weight ligands has been developed. CBP-1008 binds to FRα with high affinity and TRPV6 with low affinity. In presence of TRPV6, CBP-1008 shows faster FRα-mediated internalization and cytotoxicity than it does in absence of TRPV6. Systemic clearance CBP-1008 is observed within 6 hrs. However, the drug retained and accumulated in tumors with a half-life over 48 hrs. As a result, CBP-1008 shows excellent efficacy on tumors with target expression and the response is durable. MTD of CBP-1008 reaches 4 mg/kg in mice, 2 mg/kg in rat and 1 mg/kg in monkey.

During the development of molecularly-targeted therapeutics, biomarker assays which can stratify patients and enrich those who respond well to the therapy, play a very critical role. We generated an anti-TRPV6 antibody and developed a first-in-class IHC-based TRPV6 assay. This assay along with an FRα IHC assay have been validated per CAP/CLIA guidelines for patient selection in our clinical trials. Intended indications including ovarian cancer, endometrial cancer, breast, NSCLC and pancreatic cancer. We have evaluated a panel of ovarian PDX models for their biomarker expression and response to CBP-1008 to determine their correlation. It is found that FRα+/TRPV6+ tumor responds better than tumors which have at least one marker negative. Within the FRα+/TRPV6+ tumors, tumor growth inhibition well correlates with IHC score of FRα (R2=0.89).

It is indicated that clinical trials in selected indications using biomarkers assay to screening patients will expedite the clinical development of CBP-1008. Initial clinical response has been observed in our Phase I trial.

Citation Format: Wei Tan, Wei Wang, Tingting Bu, Xuelian Liu, Yan Teng, Baohua Huang. CBP-1008 shows excellent efficacy and desirable drug safety profile in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1819.
Abstract 183: Ex vivo 3D drug response profiling of XPDX-derived tumor cells for acceleration of preclinical drug development
Authors: Carlson Aaron L. , Elrod Ashley K. , Williams Natalie A. , Moriarty Alyssa D. , Wick Michael J. , DesRochers Teresa M.
Published: June 2022

Patient-Derived Xenografts (PDX) represent a versatile tool for preclinical drug development because they recapitulate many key features of the parent tumors, including molecular and histopathological profiles, tumor microenvironment, and tumor heterogeneity. The clinical relevance of PDX thus offer several advantages over other commonly used tools such as cell lines and genetically modified mice. Many large collections of PDX have been developed across a wide range of tumor types that profile the genetic diversity of each disease and the ability to bank PDX material allows repeated generation of mice for in vivo drug screens. As a result, PDX are used in several parts of the drug development pipeline, including early cancer biology studies, biomarker development, evaluation of therapeutic efficacy, and assessing/overcoming drug resistance. However, there remain drawbacks to this approach, most notably the large number of mice required for comprehensive drug screens and the associated time and costs. One approach to streamline PDX model selection and in vivo study execution would be to predict in vivo drug responses by assessing responses to the same drugs in an ex vivo platform utilizing PDX-derived dissociated tumor cells. KIYATEC’s KIYA-PREDICT™ PDX assay is a 3D spheroid-based ex vivo platform that has been used to screen a wide range of drugs and tumor types to predict drug responses in primary patient-derived tumors and PDX-derived tumors, including several PDX from XenoSTART’s extensive library of XPDX models. Here, we dissociated XPDX-derived tumors from a panel of 20 breast, ovarian, and lung cancer models provided by XenoSTART and evaluated their ex vivo responses to a panel of chemotherapy agents in our 3D KIYA-PREDICT™ assay. After exposure to drugs for 3-7 days, viability was assessed and both IC50s and percent survival values were calculated. The percent survival was then compared to in vivo drug response data provided by XenoSTART, and correlations between in vivo and ex vivo data were assessed. Drug responses were highly correlative between ex vivo and in vivo models, including the ability to recapitulate palbociclib resistance and platinum and taxane response. These results indicate that the KIYA-PREDICT™ PDX assay is a valuable tool to incorporate into drug development pipelines to accelerate the screening of new drug compounds on a wide range of clinically relevant samples and to guide selection of PDX models for in vivo studies.

Citation Format: Aaron L. Carlson, Ashley K. Elrod, Natalie A. Williams, Alyssa D. Moriarty, Michael J. Wick, Teresa M. DesRochers. Ex vivo 3D drug response profiling of XPDX-derived tumor cells for acceleration of preclinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 183.