bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2515: Estrogen regulates the immune microenvironment of colorectal cancer liver metastases
Authors: Benslimane Yasmine , Lapin Sarah , Chambon Julien , Leibovitch Matthew , Perrino Stéphanie , Brodt Pnina
Published: June 2022

Liver metastases (LM) remain a major cause of cancer-related death and many cancers preferably metastasize to the liver due to its unique anatomical location, rich blood supply and immune-tolerant microenvironment. Up to 50% of colorectal carcinoma patients develop LM during their disease and this is associated with a poor prognosis. Our laboratory previously identified a sexual dimorphism in the regulation of the immune microenvironment (IME) of LM and showed that estrogen could promote the accumulation of myeloid-derived suppressor cells (MDSCs) and Tregs in the liver in response to invading cancer cells. The objective of this study was to elucidate the role of estrogen in the recruitment and polarization of monocytes/macrophages and in the accumulation and phenotype of natural killer (NK) cells in the IME of colorectal carcinoma LM (CRCLM). Moreover, we sought to determine the therapeutic potential of Selective Estrogen Receptor Degrader (SERD) therapy in tumor bearing female mice. In estrogen-competent female mice bearing CRCLM, we found increased gene and protein expression of the immunosuppressive cytokine TGF-β, and inversely a decrease in the pro-inflammatory cytokine TNF-alpha. Furthermore, we identified a significant increase of newly recruited, immunosuppressive CD68+F4/80+CD163+ M2 macrophages, as compared to estrogen-depleted (ovariectomized) mice. The recruitment of pro-inflammatory CD68+F4/80+TNF-alpha+ M1 macrophages, and the accumulation of the highly cytotoxic NK1.1+ CD49a+ liver NK cells were increased in estrogen-depleted female mice and could be restored upon estrogen supplementation. Moreover, treatment of female mice with the SERD Fulvestrant, markedly reduced the number of CRCLM compared to vehicle-treated mice, and significantly reduced the cell frequencies and counts of recruited myeloid cells and M2 macrophages in the liver. Taken together, our results identify estrogen as a critical regulator of an immunosuppressive tumor ME in the liver and a potential therapeutic target. Funding by the Canadian Institute of Health Research is gratefully acknowledged.

Citation Format: Yasmine Benslimane, Sarah Lapin, Julien Chambon, Matthew Leibovitch, Stéphanie Perrino, Pnina Brodt. Estrogen regulates the immune microenvironment of colorectal cancer liver metastases [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 2515.
Abstract 2546: Targeting MerTK-mediated efferocytosis in the prostate cancer TME
Authors: Myers Kayla V. , de Groot Amber E. , Gonye Anna L. , Loftus Luke V. , Amend Sarah R. , Pienta Kenneth J.
Published: June 2022

The prostate cancer tumor microenvironment (TME) is comprised of many different components and cell types that influence tumor progression and patient outcome. Macrophages are highly abundant immune cells in the prostate cancer TME. Macrophage phenotypes can be modeled on a continuous spectrum of M1-like (anti-tumor macrophages) to M2-like (pro-tumor macrophages) and most macrophages in the prostate cancer TME are M2-like. Efferocytosis, the phagocytosis of apoptotic cells, is a pro-tumor function of M2-like macrophages. We have developed a flow cytometry assay to quantify efferocytosis of the prostate cancer cell line LNCaP. With both cell line-based THP-1 macrophages and human monocyte-derived macrophages (HMDMs), we observe that M2 macrophages efferocytose LNCaP cells more than M1 macrophages. Based on literature from other models and contexts, efferocytosis further supports the M2-like phenotype. Efferocytosis also prevents the apoptotic cell from progressing to secondary necrosis, which would attract an M1-like macrophage phenotype. Due these aspects, we hypothesize efferocytosis in the prostate cancer TME is a tumor-promoting function of macrophages. Following efferocytosis of LNCaP cells by M2 HMDMs, we detect a decrease in the M1-like, anti-tumor marker CD80 and an increase in M2-like, pro-tumor markers CD206 and PDL1. Due to this role in modulating macrophage phenotype, we hypothesize targeting efferocytosis will slow prostate cancer growth and promote an anti-tumor immune infiltrate, including M1-like macrophages. MerTK is a receptor tyrosine kinase that mediates efferocytosis by binding phosphatidylserine on apoptotic cells. At both the protein and mRNA level, we detect higher MerTK expression in M2 than M1 THP-1 macrophages and HMDMs. Upon addition of apoptotic LNCaP cells, we observe an increase in phosphorylated MerTK (active form), suggesting the role of MerTK in prostate cancer cell efferocytosis. Currently, we are targeting MerTK to block prostate cancer cell efferocytosis in vitro and in vivo. We have generated a Mertk-/-, Hi-Myc mouse model on the FVB/N background. This prostate cancer GEMM will be used to assess the role of MerTK across different stages of prostate cancer progression. We will be comparing tumor size and immune infiltration between Mertk+/+ and Mertk-/- Hi-Myc mice aged to 2 months, 6 months and 12 months. We predict that the Mertk-/- mice will have smaller tumors and an overall anti-tumor immune infiltrate compared to Mertk+/+ mice in this model due to lack of efferocytosis.

Citation Format: Kayla V. Myers, Amber E. de Groot, Anna L. Gonye, Luke V. Loftus, Sarah R. Amend, Kenneth J. Pienta. Targeting MerTK-mediated efferocytosis in the prostate cancer TME [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 2546.
Abstract 2554: Impact of the aged bone marrow on the anti-tumor response to melanoma
Authors: Carey Alexis Erasta , Chhabra Yash , Fane Mitchell , Hueser Laura , Marino Gloria , Zabransky Daniel , Weeraratna Ashani
Published: June 2022

Melanoma is responsible for the most skin cancer-related deaths in the United States. Melanoma occurrence and severity increase with age while the anti-tumor responses decrease. There have been robust responses to immunotherapy treatments, however, there remains a subset of patients who do not benefit from these modalities. Understanding more about immune cell development and age-related changes in the bone marrow can improve patient outcomes. Our hypothesis suggests that during aging, changes in Wnt signaling reshape the aged bone marrow niche, driving precursor cells to become suppressive. This ultimately decreases the anti-tumor response to melanoma due to the skewing of hematopoiesis to yield decreased immune function. In a young and aged setting, we assess the effect of a Wnt inhibitor, sFRP1, on the development of immune cell subtypes by using flow cytometry. We have preliminarily observed in situ that sFRP1 is increased in the aged bone marrow through immunohistochemical staining. We also saw a decrease in CD4 and CD8 T-cells and an increase in CD11b positive myeloid cells in the tumors of young mice treated with recombinant sFRP1 compared to age-matched mice treated with PBS by using immunohistochemical staining. We hypothesize that the increase in sFRP1 in the bone marrow niche will impact immune cell development in the niche and the immune response at the tumor site. As a future direction, we plan to investigate combination treatments of anti-sFRP1 with immunotherapies, such as anti-PD-1. These insights could lead to improve outcomes for elderly patients with melanoma.

Citation Format: Alexis Erasta Carey, Yash Chhabra, Mitchell Fane, Laura Hueser, Gloria Marino, Daniel Zabransky, Ashani Weeraratna. Impact of the aged bone marrow on the anti-tumor response to melanoma [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 2554.
Abstract 2582: Regulatory T cells restrain efficacy of immunotherapy in murine liver tumors
Authors: Green Benjamin L. , Ma Chi , Zhang Qianfei , Ruf Benjamin , Rosato Umberto , Qi Jonathan , Wabitsch Simon , Bauer Kylynda , Myojin Yuta , McCallen Justin , McVey John C. , Subramanyam Varun , Catania Vanessa , Nur Amran , Korangy Firouzeh , Xie Changqing , Greten Tim F.
Published: June 2022

Background: Checkpoint blockade immunotherapy (CBI) can induce a durable response for some patients with hepatic tumors, but many derive no or incomplete oncologic benefit for unclear reasons. Regulatory T cells (Tregs) in the liver play a central role in maintaining the tolerogenic local immune environment, both in homeostasis and disease. Here we sought to explore the impact of CBI on hepatic Tregs to determine if they play a role in CBI resistance of liver tumors in mice.

Methods: High-dimensional flow cytometry was used to examine the ex vivo characteristics of lymphocytes from both tumor-free and orthotopic tumor-bearing livers of mice treated with CBI or other immunomodulatory agents.

Results: All hepatic T cell subsets examined displayed higher proliferative and apoptotic indices compared with those of splenic T lymphocytes. We found that hepatic Tregs intensely proliferate and undergo apoptosis compared with splenic Tregs under homeostatic conditions. Hepatic Treg proliferation was enhanced after administration of CBI treatment. This effect was abrogated by co-treatment with sirolimus. CD8, macrophages, and the gut microbiome were found to be dispensable for the in vivo in response to αPD1. Co-treatment of mice with αPD1 and αCD25 sensitized MC38-bearing liver tumors.

Conclusion: Murine liver Tregs naturally proliferate and undergo apoptosis due to the mTOR rheostat at homeostasis making them highly responsive to CBI. This behavior potentially explains liver-specific CBI-resistance in tumors.

Citation Format: Benjamin L. Green, Chi Ma, Qianfei Zhang, Benjamin Ruf, Umberto Rosato, Jonathan Qi, Simon Wabitsch, Kylynda Bauer, Yuta Myojin, Justin McCallen, John C. McVey, Varun Subramanyam, Vanessa Catania, Amran Nur, Firouzeh Korangy, Changqing Xie, Tim F. Greten. Regulatory T cells restrain efficacy of immunotherapy in murine liver tumors [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 2582.
Abstract 2596: Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response
Authors: Mendoza-Munoz Pamela L. , Gavande Navnath S. , VanderVere-Carozza Pamela S. , Pawelczak Katherine S. , Dynlacht Joseph R. , Garrett Joy E. , Turchi John J.
Published: June 2022

The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double-strand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We have previously reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to DSB generating chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of bleomycin and IR and also induce p53 phosphorylation through the activation of the ATM pathway, which is concomitant with a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 and A549 cell lines, Ku-DBi’s treatment in combination with DNA DSBs-inducing agents such as bleomycin and IR, showed a significant reduction of autophosphorylation events of DNA-PKcs at the S2056 cluster compared to DNA DSBs-inducing agent alone. In addition, analysis of phospho-ATM and phospho-p53 protein levels in these NSCLC cell lines, suggested activation of the ATM pathway as a function of Ku-DBi’s treatment followed by bleomycin, evidenced by an increase of phosphorylation of ATM at Ser1981, and a modest increase of p53 phosphorylation at Ser15. Our findings demonstrate that Ku-DBi’s block DNA-DSB dependent DNA-PKcs autophosphorylation, resulting in potentiating cellular sensitivity to bleomycin and IR, and a likely effect on the ATM-dependent signaling pathway. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs to impact DSB repair and potentially DDR signaling, becoming in a promising approach as part of an anticancer therapeutic strategy in combination with DNA DSBs-inducing agents.

Citation Format: Pamela L. Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy E. Garrett, John J. Turchi. Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response [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 2596.
Abstract 265: Inhibiting proteasome in extracellular matrix (ECM) detached cells promotes anoikis
Authors: Adeshakin Funmilayo , Zhang Guizhong , Adeshakin Adeleye , Wan Xiaochun
Published: June 2022

The underlying mediators for altered cell-extracellular matrix (ECM) communication in tumors remain to be understood and are likely to be multifactorial. Detached cells from the ECM are often cleared by a death process called ‘anoikis’. Anoikis is critical for health and homeostasis maintenance. However, aggressive cancer cells acquire anoikis resistance to promote invasion and metastasis in new sites. We previously identified metabolic reprogramming and upregulation of Vacuolar-ATPase members responsible for anoikis resistance while the manipulation of these pathways induced anoikis. However, the mechanisms underlying anoikis resistance and subsequent metastasis are yet to be fully elucidated, thus limiting anoikis-targeting drug exploration for metastasis control. Herein, our RNA-seq data revealed upregulated genes associated with proteasome activity (such as PSMA, PSMB, PSMD, and POMP) in ECM detached cancer cells compared to adherent cancer cells. Targeting this, using proteasome inhibitors (MG132 or bortezomib) in breast cancer, cervical, and melanoma cell lines sensitized cancer cells to anoikis through increased cellular, mitochondrial stress, and misfolded protein accumulation. Moreover, Bortezomib treatment in vivo in melanoma tumor model repressed metastasis. These data demonstrate proteasome activity is hijacked by ECM detached cancer cells to maintain proteostasis thus promoting anoikis resistance and metastasis. Regardless of the impressive outcome in treating hematological malignancies, proteasome inhibitors show little effect against solid tumors in several clinical trials that utilized selective and more potent inhibitors either as a single or in combinatory therapy. In this sense, our finding suggests proteasome inhibitors may be more attractive for metastasis control rather than local tumor treatment. Altogether, our findings demonstrate enhanced proteasome activity within the tumor cells is critical for anoikis resistance, and targeting this can markedly overcome resistance and control tumor metastasis.

Citation Format: Funmilayo Adeshakin, Guizhong Zhang, Adeleye Adeshakin, Xiaochun Wan. Inhibiting proteasome in extracellular matrix (ECM) detached cells promotes anoikis [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 265.
Abstract 2671: ERAS-601, a potent allosteric inhibitor of SHP2, demonstrates anti-tumor activity in RAS/MAPK-driven tumor models
Authors: Martin Leenus , Patel Roopal , Zhang Jingchuan , Yang Jennifer , Nevarez Robin , Congdon Taylor , Brail Les , Shoemaker Robert
Published: June 2022

SHP2 is a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. SHP2 transduces upstream receptor tyrosine kinase (RTK) signaling to the RAS/MAPK pathway via its phosphatase-mediated regulation of guanine nucleotide exchange factors (GEFs). The modulation of GEF activity impacts the rate at which KRAS cycles from the inactive GDP-bound state to the active GTP-bound state. ERAS-601 is a potent, selective small molecule allosteric inhibitor of SHP2. ERAS-601 potently inhibits the wild type SHP2 protein with a biochemical IC50 of 4.6 nM. ERAS-601 is a selective SHP2 inhibitor and demonstrates no appreciable inhibition against any off-target kinase or phosphatase across panels of 300 kinases and 12 phosphatases. ERAS-601 inhibits the loading of active GTP-bound oncogenic RAS and inhibits RAS/MAPK pathway signaling as measured by pERK1/2 inhibition and DUSP6 mRNA. ERAS-601 demonstrates anti-proliferative activity across a panel of human cancer cell line models with oncogenic alterations in the RAS/MAPK pathway. In a mouse in vivo study, ERAS-601 achieves substantial systemic exposure and demonstrates inhibition of ERK1/2 phosphorylation and DUSP6 mRNA levels in the NCI-H358 xenograft model. ERAS-601 also inhibits tumor growth in multiple RAS/MAPK-driven CDX and PDX models that harbor EGFR, KRAS, BRAF Class III, and NF1LOF mutations. ERAS-601 is a potent and selective allosteric SHP2 inhibitor that demonstrates anti-tumor activity in vitro and in vivo and is currently being studied as a monotherapy in an ongoing Phase 1 clinical study in patients with advanced or metastatic solid tumors (FLAGSHP-1, NCT04670679).

Citation Format: Leenus Martin, Roopal Patel, Jingchuan Zhang, Jennifer Yang, Robin Nevarez, Taylor Congdon, Les Brail, Robert Shoemaker. ERAS-601, a potent allosteric inhibitor of SHP2, demonstrates anti-tumor activity in RAS/MAPK-driven tumor 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 2671.
Abstract 2672: ERAS-007 is a selective ERK1/2 inhibitor with preclinical activity across RAS/MAPK pathway-driven CRC models
Authors: Lew Erin D. , Oh Joanne , Congdon Taylor , Zhang Jingchuan , Shoemaker Robert F.
Published: June 2022

Colorectal cancer (CRC) remains an unmet medical need where more than 50% off patients have tumors that are driven by somatic mutations in the RAS/MAPK signaling pathway. Targeted therapies, such as anti-EGFR inhibitors for patients with BRAFwt/RASwt CRC tumors and encorafenib + cetuximab for patients with BRAFV600E CRC tumors, are available treatment options but demonstrate both limited overall response rates and duration of response (Yaeger, 2018). In particular, in the previously treated setting, overall survival is typically less than a year (Grothey, 2013; Mayer, 2015; Kopetz, 2019). In addition, targeted treatment options are limited for patients harboring KRAS or NRAS mutations as emergence of these mutations is a predictive biomarker to anti-EGFR therapy resistance in metastatic CRC.

ERAS-007 is a selective ERK1/2 inhibitor targeting the terminal node of the RAS/MAPK signaling pathway. In addition, ERAS-007 demonstrates single digit nanomolar biochemical ERK1/2 inhibition as well as a durable target residence time, thus making it a promising combination partner in RAS/MAPK pathway activated or altered CRC. To this end, ERAS-007 was evaluated in preclinical models in combination with encorafenib + cetuximab in BRAFV600E CRC and with palbociclib in KRASmut CRC. ERAS-007 demonstrated monotherapy activity and combination benefit in cell-based assays as well as superior combination efficacy in vivo relative to respective monotherapy control arms.

In summary, ERAS-007 demonstrates promising preclinical activity across a wide range of RAS/MAPK pathway-driven CRC models both as a monotherapy and in combination that support further exploration in the clinic. Accordingly, ERAS-007 combinations with encorafenib + cetuximab in BRAFV600E CRC and with palbociclib in KRASmut CRC are currently being evaluated in the HERKULES-3 phase 1b/2 master protocol (NCT05039177).

Citation Format: Erin D. Lew, Joanne Oh, Taylor Congdon, Jingchuan Zhang, Robert F. Shoemaker. ERAS-007 is a selective ERK1/2 inhibitor with preclinical activity across RAS/MAPK pathway-driven CRC 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 2672.
Abstract 2690: Loss of E-cadherin induces IGF1R activation revealing a targetable pathway in invasive lobular breast carcinoma
Authors: Elangovan Ashuvinee , Savariau Laura , Yates Megan E. , Hooda Jagmohan , Nagle Alison M. , Oesterreich Steffi , Atkinson Jennifer M. , Lee Adrian V.
Published: June 2022

Invasive Ductal Carcinoma (IDC) and Invasive Lobular Carcinoma (ILC) are two major subtypes of breast cancer with significant differences in their histological and molecular underpinnings. ILC has a unique loss of E-cadherin (CDH1) which we have previously demonstrated as a negative regulator of the Insulin-like Growth Factor 1 receptor (IGF1R) through a comprehensive analysis of cell line models and tumor samples. We propose that loss of E-cadherin in ILC sensitizes cells to growth factor signaling and thus alters their sensitivity to growth factor signaling inhibitors. To investigate this, we used CRISPR to generate CDH1 knockout (KO) IDC cell lines (MCF7, T47D, ZR75.1) to uncover the mechanism by which E-cadherin loss activates the IGF pathway while also assessing its targetability. CDH1 KO cells exhibited anchorage independent growth in suspension culture and altered p120 catenin localization as observed in ILC tumors. Through in vitro studies, we show increased signaling sensitivity to IGF/insulin ligands and enhanced signaling duration in CDH1 KO cells. In addition, we observed a higher migratory potential of CDH1 KO cells compared to wild type (WT) cells, which was further enhanced as a chemotactic response to IGF1 or serum. Further, this phenotype could be reversed with an IGF1R inhibitor, BMS-754807. We additionally identified an increase in Collagen I haptotaxis in the CDH1 KO cells, which was also translated into a novel invasive phenotype towards serum in the T47D CDH1 KO cells. Despite no significant differences in membranous IGF1R levels between WT and CDH1 KO cells, higher ligand-receptor interaction was observed with CDH1 KO cells, demonstrating an increased ligand-receptor complex formation upon stimulation. Our results suggest that loss of CDH1 results in an increase in IGF1 receptor availability for ligand binding which in turn allows for an enhanced downstream signaling activation. Interestingly, a physical repression of E-cadherin on IGF1R could not be demonstrated, suggesting spatial changes on the membrane following E-cadherin loss may control ligand binding. Critically, increased sensitivity to IGF1R, PI3K, AKT and MEK inhibitors was observed in CDH1 KO cells suggesting that these targets should be further explored in ILC and that CDH1 loss may be exploited as a biomarker of response, or for patient stratification to inhibitors targeting these pathways.

Citation Format: Ashuvinee Elangovan, Laura Savariau, Megan E. Yates, Jagmohan Hooda, Alison M. Nagle, Steffi Oesterreich, Jennifer M. Atkinson, Adrian V. Lee. Loss of E-cadherin induces IGF1R activation revealing a targetable pathway in invasive lobular breast carcinoma [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 2690.
Abstract 2741: A communication theory framework for modeling cytokine-mediated signaling in healthy and breast cancer derived peripheral blood immune cells
Authors: Matache Adina , Karolak Aleksandra , Branciamore Sergio , Rodin Andrei , Lee Peter P. , Rockne Russell C.
Published: June 2022

Previous studies have shown that approximately 40% of estrogen receptor positive (ER+) breast cancer (BC) patients harbor immune signaling defects in their blood at diagnosis that predicts overall survival. The aim of this work is to apply communication theory and signal processing methods to model cytokine-mediated signaling errors in immune cells in the peripheral blood. Our approach uses communication theory concepts to develop a communication system model consisting of a transmitter, receiver, and channel, applied to the JAK/STAT signaling pathway and tested using multi-color flow cytometry analysis of peripheral blood mononuclear cells stimulated with IFNγ, IL-2, IL-6, IL-4, IL-10, IL-12, or TGFβ in samples from 2 BC patients and 4 healthy controls. We use our model to estimate the detection error rate when one of the 7 cytokines is being transduced through the JAK/STAT signaling pathway, but its signal is being received on multiple phosphorylated signal transducers and activators of transcription (pSTAT) molecules. In addition, we extend the measured error rate value to an error rate curve by varying the signal-to-noise ratio available to the receiver in our system model. Our results show a trend towards higher detection error rates in immune cells from BC patients as compared to healthy controls, which may indicate altered immune signaling and incorrect cell decisions in these patients. Also, considering different cell subtypes, our results show two orders of magnitude increase in error rate in CD4+ memory T cells compared to CD4+ naïve T cells in BC patients. With further development, we believe that our communication theory model and quantification of optimal signal detection in cells will provide a better understanding into how immune cells can accurately “decode” information to respond properly to various signals and how the immune signaling is altered in BC patients, causing better or worse outcomes. The benefit of our novel approach is that it integrates all the transmitted (cytokines) and received signals (pSTATs) into a single communications framework that can be efficiently analyzed together.

Citation Format: Adina Matache, Aleksandra Karolak, Sergio Branciamore, Andrei Rodin, Peter P. Lee, Russell C. Rockne. A communication theory framework for modeling cytokine-mediated signaling in healthy and breast cancer derived peripheral blood immune cells [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 2741.
Abstract 2751: The modular Tmod dual_receptor system exploits tumor deletions to achieve robust tumor-selective cytotoxicity
Authors: Xu Han , Hamburger Agnes , Ando Yuta , Asuelime Grace , Bolanos Kristian , Daris Mark , DiAndreth Breanna , Imun Maria C. , Lee Wen-Hua , Li Chuck Z. , Luna Breanna , Manry Diane , Martin Aaron D. , McElvain Michele E. , Mock Jee-Young , Naradikian Martin , Sandberg Mark L. , Oh Julyun , Shafaattalab Sanam , Sharma Shruti , Tokatlian Talar , Wang Xueyin , Wong Lu Min , Kamb Alexander
Published: June 2022

We set out to examine the behavior in preclinical models of a dual-receptor cell therapy approach (Tmod࣪ system) designed to exploit instances of somatic LOH in cancer that can be readily identified through molecular diagnostics. We utilized quantitative pharmacologic assays in vitro and dual-flank efficacy/selectivity models in vivo to test the activity of a variety of constructs comprised of: (i) CAR “activators” that target tumor-associated antigens (TAAs) such as CEA, MSLN and others; and, (ii) LIR-1-based “blockers” that target HLA class I proteins. We generated substantial datasets around multiple targets, including well known tumor-associated antigens (TAAs). In the presence of the HLA class I blocker antigen, Tmod cells were OFF within a wide range of activator antigen expression levels on the “normal” target cells. They were ON when tumor cells expressed the TAA without the HLA blocker antigen, and killed tumor cells selectively even in mixed tumor-normal cell populations. The Tmod system transformed imperfect TAAs into true tumor-selective targets, where the activity of the engineered T cells is controlled by the presence/absence of the HLA blocker antigen. The system was sufficiently modular to use with autologous and allogeneic products. Several of these Tmod constructs are advancing to the clinic where a robust genetic selection tool is being used to screen patients for treatment and match them with a suitable Tmod therapy. In summary, Tmod is a robust system with quantitative pharmacologic behavior in preclinical models consistent with efficacy and safety across a range of targets.

Citation Format: Han Xu, Agnes Hamburger, Yuta Ando, Grace Asuelime, Kristian Bolanos, Mark Daris, Breanna DiAndreth, Maria C. Imun, Wen-Hua Lee, Chuck Z. Li, Breanna Luna, Diane Manry, Aaron D. Martin, Michele E. McElvain, Jee-Young Mock, Martin Naradikian, Mark L. Sandberg, Julyun Oh, Sanam Shafaattalab, Shruti Sharma, Talar Tokatlian, Xueyin Wang, Lu Min Wong, Alexander Kamb. The modular Tmod dual_receptor system exploits tumor deletions to achieve robust tumor-selective cytotoxicity [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 2751.
Abstract 2783: Increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression in normal colonic mucosa with imipridone ONC201/TIC10 treatment: A potential biomarker for chemoprevention studies
Authors: Raufi Alexander G. , De La Cruz Arielle , George Andrew , Hacking Sean , Madka Venkateshwar , Prabhu Varun , Safran Howard , Zhou Lanlan , Brenner Dean , Rao Chinthalapally V. , El-Deiry Wafik S.
Published: June 2022

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States and currently the most effective method at reducing risk of death is through screening. Although these strategies have reduced the incidence of CRC, they are invasive, costly, and have low rates of compliance. Chemoprevention aims to prevent or delay the onset of CRC through eradication or prevention of precursor colonic adenomas, ideally with affordable and nontoxic drugs. The imipridone ONC201, which induces apoptosis, in part, through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), is currently undergoing preclinical evaluation as a potential chemopreventive agent. In preclinical models, we found that treatment with ONC201 is capable of increasing TRAIL expression in normal cells and can improve tumor suppression through immune surveillance by the innate immune system. To further assess potential biomarkers of response to ONC201, we evaluated expression of TRAIL and death receptor 5 (DR5), as well as markers related to cellular stemness such as LGR5 in colonic mucosa obtained from mice treated with ONC201 and vehicle controls. In this study, 16-week-old female C57BL/6 mice were administered vehicle or ONC201 50mg/kg (n=11) body weight by oral gavage twice a week for a total of five doses. Both immunohistochemistry and western blotting were used to evaluate differential expression of various potential biomarkers in both fresh frozen and FFPE colonic tissue. Using western blotting we found a significant increase in TRAIL expression in normal colonic mucosa obtained from mice treated with ONC201 as compared to vehicle controls. This finding was further validated using immunohistochemistry. Other markers, such as DR5 appeared unaffected with treatment. As efforts are currently underway to develop a CRC chemoprevention trial using ONC201, TRAIL may represent a biomarker of interest. Further analysis of stemness markers, as well as serum analyses are currently in process and may reveal other potential biomarkers for use in chemoprevention trial development.

Citation Format: Alexander G. Raufi, Arielle De La Cruz, Andrew George, Sean Hacking, Venkateshwar Madka, Varun Prabhu, Howard Safran, Lanlan Zhou, Dean Brenner, Chinthalapally V. Rao, Wafik S. El-Deiry. Increased tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression in normal colonic mucosa with imipridone ONC201/TIC10 treatment: A potential biomarker for chemoprevention studies [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 2783.
Abstract 2791: Integrating biospecimen science approaches into clinical assay development-PAR 22-049
Authors: Agrawal Lokesh , Rao Abhi , Guan Ping , Ossandon Miguel , Moore Helen
Published: June 2022

Introduction to the problem: Biospecimens are the essential starting materials for the biomarker assays that enable precision medicine. Biospecimen preanalytical factors can directly influence molecular results from assays conducted for basic research, biomarker discovery, and biomarker validation, and can also influence the development of validated clinical assays. The U.S. National Cancer Institute (NCI) Biorepositories and Biospecimen Research Branch supports extramural research as a part of this FOA to investigate and mitigate preanalytical challenges facing clinical assay development and analytical validation in a variety of biospecimen types. The overall goal of this initiative is to expedite biomarker clinical assay development through evidence based standardization of biospecimen handling practices. The work is centered on emerging and clinically relevant biomarker assays conducted on a variety of molecular platforms.

Proposed solution and methods: Researchers may investigate tissue and fluid biospecimens including core biopsies, fine needle aspirates, tissue exudates, blood, urine, saliva, breast milk, feces, sweat, CSF, and pleural and esophageal aspirates. Investigator designed experiments explore how different biospecimen preanalytical conditions affect biomarkers in a clinical setting. To date, ten grants have been funded. The grants address important areas in biospecimen science in both liquid and solid biopsy biopsies in breast cancer, glioblastomas, lung cancer, bladder cancer, prostate cancer, cutaneous and peripheral T cell lymphoma. Investigators can collaborate with each other and with the NCI, FDA and NIST through the U01 funding mechanism.

Conclusions: The results from this research program will elucidate how analytical quantification of clinically relevant biomarkers is affected by patient context factors, variation in biospecimen collection, processing, and storage procedures, and how such effects can be mitigated to improve the development, performance and accuracy of clinical biomarker assays utilized in clinical research and patient care. The receipt dates for this program, PAR 22 049 in 2022, are January 11, 2022, June 7th, 2022, and September 13th, 2022.

Citation Format: Lokesh Agrawal, Abhi Rao, Ping Guan, Miguel Ossandon, Helen Moore. Integrating biospecimen science approaches into clinical assay development-PAR 22-049 [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 2791.
Abstract 2793: Minimal residual disease (MRD) as a predictive biomarker/endpoint for novel drug development
Authors: Jones Nicholas , Ngo Brian , Davis Floyd , Fancke Benjamin
Published: June 2022

MRD is defined as the continual presence of cancer cells in patients post therapy that is below the threshold of detection by conventional morphologic or radiologic assessments. Monitoring patient status clinically as MRD negative or MRD positive has become the basis for predicting outcomes, remission or reoccurrence, as well as determining therapeutic options. The three primary methods to assess MRD status in patients include Flow Cytometry (FC), Polymerase Chain Reaction (PCR) and Next-Generation Sequencing (NGS). All three are highly quantitative measurements with a high degree of sensitivity to detect “new” circulating tumor cells or ctDNA that can potentially indicate disease recurrence before it happens. The choice of MRD methodology usually depends on the tumor type, liquid or solid. Flow Cytometry has been widely used to monitor MRD status in patients with hematologic malignancies. Specifically designed MRD panels have been used clinically in CLL, MM, CML and AML patients. In addition to its prognostic/diagnostic relevance, recent guidance by the FDA is now driving the use of MRD testing as a predictive biomarker/endpoint for novel cancer drug development. NeoGenomics Pharma Services has provided flow cytometry MRD expertise to multiple sponsors conducting clinical trials to evaluate the effectiveness of novel cancer therapy. In a CLL study, NeoGenomics employed the ERIC MRD assessment to monitor patient status during therapeutic intervention at various points throughout treatment. Representative patient data show progression from full, active disease prior to therapy to non-detectable disease levels below 0.01%MRD. These examples show patient progression through multiple cycles of treatment to full remission. Clearly, this is an example of how MRD can serve as the primary “biomarker” to evaluate a patient’s response to novel drug therapy in clinical trials. This presentation will further discuss the data and the relevance of MRD testing to support new drug development.

Citation Format: Nicholas Jones, Brian Ngo, Floyd Davis, Benjamin Fancke. Minimal residual disease (MRD) as a predictive biomarker/endpoint for novel 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 2793.
Abstract 281: Culture of circulating tumor cells isolated from cancer patients with metastatic disease
Authors: Sultanpuram Nikhila Reddy , Kim Isaiah , Sendi Hossein , Kanpe Nicole , Hagen Tilden , Wagner Kyle , Wang Andrew
Published: June 2022

OBJECTIVE: The purpose of this study is to investigate the feasibility of using decellularized tissue ECM to culture circulating tumor cells (CTCs) collected from patients with metastatic solid tumor malignancies.

BACKGROUND: Circulating tumor cells (CTCs) have been shown to recapitulate the heterogeneity and biology of patients’ cancers. However, there is a lack of technologies that can culture patient CTCs. Our group has previously shown that tumor cells cultured on decellularized tissue closely mimic the in vivo condition. Therefore, we hypothesized that we can utilized decellularized tissue as strata to culture CTCs.

METHODS: Peripheral blood samples were collected from patients with metastatic disease who had not received any systemic therapy in the last six months or are progressing on systemic therapy. PMBC layer, which contains lymphocytes, monocytes, dendritic cells and CTCs (in metastatic patients) is separated from whole blood by Ficoll gradient. The cells are then mixed with decellularized lung or liver material and cultured using stemcell media.

RESULTS: 26 subjects were enrolled in this study. We were able to generate cell colonies from 12 patients’ samples (either colonies on liver or lung strata). Of the patient samples that did not develop tumor colonies, 9/14 subjects had no metastatic tumors in either lung or liver. Colonies from the cultures were characterized by histological staining and SEM imaging. Of the samples that the tumor colonies were large enough for H&E, we observed phenotypes that are indicative of tumor cell colony. Ongoing work aim to further validate the CTC cultures using genomic sequencing and RNAse

CONCLUSION: We showed decellularizd organ technology can be utilized to generate CTC cultures.

Citation Format: Nikhila Reddy Sultanpuram, Isaiah Kim, Hossein Sendi, Nicole Kanpe, Tilden Hagen, Kyle Wagner, Andrew Wang. Culture of circulating tumor cells isolated from cancer patients with metastatic disease [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 281.
Abstract 2857: DR30303, a SMART-VHHBody powered anti-CLDN18.2 VHH-Fc with enhanced ADCC activity for the treatment of gastric and pancreatic cancers
Authors: Fang Yongliang , Yao Gaofeng , Zhong Weixiang , Duan Wenwen , Zhou Zhenxing , Wen Xiaofang , Chen Yonglu , Fang Jingjin , Wang Yongwei , Jiang Weiqin , Xu Junfang , Huang Yanshan
Published: June 2022

Claudin 18.2 (CLDN18.2) is aberrantly overexpressed in gastric cancer, pancreatic adenocarcinomas, and a fraction of non-small cell lung cancer. Its tumor specificity allows CLDN18.2 to be an attractive target for developing biotherapeutics for the treatment of gastric and pancreatic cancers. DR30303 is a humanized ant-CLDN18.2 heavy chain antibody Fc fusion protein (VHH-Fc) generated via Doer Biologics’ proprietary SMART-VHHBody platform. DR30303 exhibits strong and highly selective binding to CLDN18.2, but not its close splicing variant CLDN18.1. In addition, it exhibits enhanced ADCC activity as a result of Fc engineering and low immunogenicity risk validated via in-silico T-cell epitope predictor. Its smaller size (78 kDa vs 150 kDa for conventional full-length IgG) allows DR30303 to penetrate solid tumors faster and deeper, resulting in a strong competitive advantage and differentiation compared with other anti-CLDN18.2 antibodies in the market. Single-agent therapeutic potency of DR30303 was demonstrated in various gastric and pancreatic cell-derived xenograft (CDX) mouse models with a minimum effective dose of 0.3 mg/kg dose of DR30303 led to TGI≥90%, which is significantly more potent (p<0.05) than the benchmark reference Zolbetuximab analog in same molar ratio. Moreover, DR30303 showed strong synergistic anti-tumor activities with paclitaxel, gemcitabine, and EOF (epirubicin, oxaliplatin, and fluorouracil) in patient-derived xenograft (PDX) mouse models (gastric cancer) and CDX model (pancreatic cancer). DR30303 also demonstrated favorable pharmacokinetic properties (t1/2~ 120 hours) and tolerable toxicities (MTD≥ 748.5 mg) in both SD rats and cynomolgus monkeys. Doer Biologics submitted IND to the NMPA in November 2021 and plans to initiate the Phase 1 clinical trial in H1 2022 upon IND approval.

Citation Format: Yongliang Fang, Gaofeng Yao, Weixiang Zhong, Wenwen Duan, Zhenxing Zhou, Xiaofang Wen, Yonglu Chen, Jingjin Fang, Yongwei Wang, Weiqin Jiang, Junfang Xu, Yanshan Huang. DR30303, a SMART-VHHBody powered anti-CLDN18.2 VHH-Fc with enhanced ADCC activity for the treatment of gastric and pancreatic cancers [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 2857.
Abstract 2885: Generation of TCR-like fully human antibodies against HLA-antigen-peptide complexes using HLA transgenic RenMice
Authors: Du Jun , Wang Nannan , Hui Li , Wang Ruixue , Liu Taolin , Lin Qingcong
Published: June 2022

With the development of immunotherapy in recent years, therapeutic antibodies have become a major player combating cancer with the limitation of recognizing cell surface or secreted molecules. In contrast, the human T cell receptor (TCR) recognizes its cognate peptide antigen when presented on human leukocyte antigen (HLA) molecules, and thus detects a broader range of targets including intracellular proteins. Here we established an innovative strategy of generating fully human TCR mimic (TCRm) antibodies that can recognize antigen with a higher specificity and affinity. Previously, Biocytogen established RenMab™ and Renlite࣪ mice (RenMice). These humanized immunoglobulin mouse models generate fully human antibodies that carry human Ig heavy chain and human kappa light chain variable domains. Here, HLA genes were introduced into RenMice to build tolerance towards HLA. When immunized with HLA- peptide complexes, these mice generate antibodies that specifically target the HLA-Peptide complex, with no reactivity to HLA alone nor other peptide-HLA complexes. Compared to natural TCRs, these TCRm antibodies show higher affinity and specificity. Using optimized immunization methods and high-throughput screening, our antibody discovery platform generated fully human TCRm antibodies from HLA/RenMab™ and HLA/Renlite™ mice. Producing TCR-like binders with this unique TCRm technology can become a very powerful tool in the immunotherapy field.

Citation Format: Jun Du, Nannan Wang, Li Hui, Ruixue Wang, Taolin Liu, Qingcong Lin. Generation of TCR-like fully human antibodies against HLA-antigen-peptide complexes using HLA transgenic RenMice [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 2885.
Abstract 2907: 1C3, a Novel non-blocking anti-human TNFR2 antibody generated from RenMice, exhibits promising anti-tumor activity and safety in syngeneic tumor models in humanized TNFR2 mice
Authors: Yang Yongfei , Cao Shuzhen , Zhang Jing , Shao Zhe , Jiang Xueyuan , Duan Qingya , Tian Maopeng , Mei Sen , Yu Luke (Zhaoxue)
Published: June 2022

Regulatory T Cells (Treg) inhibit effector T cell (Teff) activity in the tumor microenvironment (TME). Therefore, depletion of regulatory T cells (Treg cells) is a promising therapeutic strategy for tumor immunotherapy. Tumor necrosis factor receptor-2 (TNFR2) is highly expressed on Treg cells and is a potential target for anti-tumor therapy. A TNFR2 non-blocking antibody (1C3) was generated from humanized IgG mice (RenMice), which is an effective platform for human antibody generation. 1C3 was selected from a large panel of antibody candidates through an unbiased, high-throughput in vivo efficacy screen in syngeneic tumor mouse models in humanized TNFR2 mice. Compared with other TNFR2 antagonistic antibodies, 1C3 effectively promoted the proliferation of CD8+ cytotoxic T cells in vitro, a population that is inhibited by Treg cells. To evaluate 1C3 efficacy and safety in vivo, syngeneic mouse tumor models were established. 1C3 monotherapy significantly inhibited tumor growth in a dose-dependent manner in syngeneic tumor mouse models in TNFR2 humanized mice. In combination with anti-human PD-1 or PD-L1 antibody, 1C3 also greatly increased antitumor activity compared to the monotherapy of each therapeutic agent. Mechanistically, 1C3 treatment significantly increased the ratio of Teff/Treg in the TME. In addition, 1C3 was well tolerated in TNFR2 humanized mice, and no side effects were observed even when the dose was increased to 100 mg/kg. Taken together, our data demonstrates that 1C3 is a novel anti-TNFR2 antibody for anti-tumor therapy and provides a great potential for the next generation of immunotherapy.

Citation Format: Yongfei Yang, Shuzhen Cao, Jing Zhang, Zhe Shao, Xueyuan Jiang, Qingya Duan, Maopeng Tian, Sen Mei, Luke (Zhaoxue) Yu. 1C3, a Novel non-blocking anti-human TNFR2 antibody generated from RenMice, exhibits promising anti-tumor activity and safety in syngeneic tumor models in humanized TNFR2 mice [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 2907.