bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2404: Role of TPD52L2 in cell migration and triple negative breast cancer progression
Authors: Parizadeh Seyedamirabbas , Geley Stephan , Reiterer-Farhan Veronika , Farhan Hesso
Published: June 2022

The Golgi apparatus plays an important role in cell migration and the establishment of epithelial polarity. A dispersed (or fragmented) Golgi ribbon is often seen in stressed cells and in various pathologies, including neurodegeneration or cancer, but whether and how a fragmented Golgi apparatus affect cellular function is poorly defined. Previously our lab has performed and analyzed published RNAi screens to identify genes required to maintain the Golgi ribbon. These candidate genes were filtered for altered expression patterns in breast cancer patients and for their association with survival data. The selected genes reevaluated by RNAi in BT549 breast cancer cells to assess the effect on Golgi fragmentation. One of the most effective hits was TPD52L2, a poorly characterised gene that encodes a membrane-associated protein that is overexpressed in various types of cancer. The aim of this project is to investigate the role of TPD52L2 in cell migration and cell invasion in breast cancer in order to understand the role of Golgi fragmentation in tumorigenesis. In random cell migration assays in dense and sparse seeding conditions, TPD52L2 depleted cells showed an altered actin cytoskeleton and were found to migrate faster in dense seeding without any change in directional persistence. These findings suggest that TPD52L2 not only is required to maintain an intact Golgi structure but also plays an important role in cellular motility and potentially metastasis.

Citation Format: Seyedamirabbas Parizadeh, Stephan Geley, Veronika Reiterer-Farhan, Hesso Farhan. Role of TPD52L2 in cell migration and triple negative breast cancer progression [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 2404.
Abstract 2450: Investigating the intrinsic and extrinsic factors regulating breast cancer dormancy
Authors: Ren Qihao , Khoo Weng Hua , Ye Jiayu , Faget Douglas V. , Croucher Peter I. , Stewart Sheila A.
Published: June 2022

Unfortunately, breast cancer can recur in patients upwards of 25 years after an initial diagnosis and “cure”. It was once thought that recurrence occurred within patients that harbored dormant breast disseminated tumor cells (DTCs) in distant organs that were not present in patients that did not recur. Interestingly, a significant percent of patients harbor DTCs, yet many do not recur, raising the critical question, what differentiates patients that experience a recurrence versus those who live with dormant DTCs for the rest of their lives? It is likely the answer lies in both cell intrinsic as well as extrinsic factors. Nevertheless, the extreme rarity of dormant DTCs has been the major obstacle to their study. To overcome this challenge, we developed an efficient system to isolate and study rare dormant tumor cells from metastatic organs. Using this system and single cell RNA-sequencing (scRNA-seq), we identified a group of genes differentially expressed in dormant breast cancer cells present in both bone and lung. While modulation of these genes individually had no impact on the metastatic behavior of breast cancer cells, we found that as a group, these genes predicted the dormancy phenotype in murine breast cancer models. Importantly, expression of these genes in primary human breast cancer tumors correlated with disease-free survival, suggesting these genes have predictive value in determining which patients are likely to recur. Moreover, we explored extrinsic mechanisms that impact dormancy and found that the chemotherapy reagent doxorubicin (Doxo) drastically changed the microenvironment in vivo, which allowed dormant breast cancer cells to grow robustly within the visceral fat and this was further exacerbated by a high fat diet. Further, we found that Doxo treatment induces significant adipose tissue dystrophy and macrophage infiltration, which may contribute to dormant metastatic outgrowth. Our study provides novel insight into breast cancer dormancy, and also reveals microenvironmental changes that impact dormant breast cancer cell outgrowth.

Citation Format: Qihao Ren, Weng Hua Khoo, Jiayu Ye, Douglas V. Faget, Peter I. Croucher, Sheila A. Stewart. Investigating the intrinsic and extrinsic factors regulating breast cancer dormancy [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 2450.
Abstract 2588: M1774, a novel potent and selective ATR inhibitor, shows antitumor effects as monotherapy and in combination
Authors: Zimmermann Astrid , Dahmen Heike , Grombacher Thomas , Pehl Ulrich , Blaukat Andree , Zenke Frank T.
Published: June 2022

The protein kinase ataxia telangiectasia- mutated and Rad3-related (ATR) is an important component of the DNA Damage Response (DDR) and a key mediator of the replication stress response (RSR). ATR is recruited to and activated by single-stranded DNA, which commonly forms as a consequence of replication stress (RS). ATR activation and signaling coordinates a multifaceted response to RS, however if left unresolved, replication forks may collapse, form double-strand breaks (DSB), lead to chromosomal rearrangements and eventually cell death. Several chemotherapeutic agents act by increasing RS in tumor cells, by directly damaging the DNA and/or depleting cellular resources required for DNA replication. Increased RS may also result from activation of oncogenes that drive dysregulated replication, a hypoxic environment, or from defects in other repair pathways even in the absence of exogenous DNA damaging insults. Inhibition of ATR activity in cancer cells disables the RSR, potentially enhancing the cytotoxicity of DNA damaging interventions and causing efficacy as monotherapy against tumors with reliance on ATR activity. M1774 is a potent inhibitor of ATR protein kinase with high selectivity towards other protein kinases. A broad range of antiproliferative activities (ranging from ~20 nM to >1 µM) was observed in a cancer cell line panel in vitro. M1774 showed pronounced synergy in combination with several DNA-damaging drugs and inhibitors of the DNA damage response. In vivo efficacy studies confirmed the combination potential suggested from in vitro studies. Pronounced combination benefit with complete tumor growth inhibition/regression was observed in combination with different chemotherapies or targeted DDR pathway inhibitors. Furthermore, M1774 showed pronounced activity as monotherapy in tumor models with gene mutations that result in a reliance on ATR activity. Clinical evaluation is ongoing to investigate M1774 as monotherapy and in combination with niraparib in patients with advanced solid tumors (DDRiver Solid Tumors 301, NCT04170153).

Citation Format: Astrid Zimmermann, Heike Dahmen, Thomas Grombacher, Ulrich Pehl, Andree Blaukat, Frank T. Zenke. M1774, a novel potent and selective ATR inhibitor, shows antitumor effects as monotherapy and in combination [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 2588.
Abstract 2621: Growth hormone receptor antagonist sensitizes melanoma and hepatocarcinoma to drug treatments via downregulation of ABC transporters in vivo
Authors: Qian Yanrong , Basu Reetobrata , Mathes Samuel , Kulkarni Prateek , Davis Emily , Berryman Darlene , List Edward , Duran Silvana , Kopchick John J.
Published: June 2022

Knockdown of the growth hormone receptor (GHR) in melanoma cells downregulates ATP-binding cassette (ABC) transporters and sensitizes them to multiple drug treatments in vitro. The goal for this study is to understand whether a GHR antagonist (GHA) could suppress different types of cancers by sensitizing tumors to drug treatments in vivo through the downregulation of ABC transporters. Sera from GHA transgenic mice inhibited the proliferation of mouse melanoma cells (B16F10) in culture and suppressed expression of multiple ABC transporters. When B16-F10 cells were intradermally inoculated into GHA mice, tumor size was markedly reduced, as was STAT5 activation and ABCG1, ABCG2 levels. We then tested the effect of the GHA on the efficacy of cisplatin in vivo. GHA sensitized melanomas to cisplatin, leading to the smallest tumors among all groups. GHKO mice showed the same effect. We further extended this investigation to hepatocellular carcinoma (HCC). GHA mice were subcutaneously inoculated with mouse HCC (Hepa1 6 cells) and subsequently treated with sorafenib. The HCC tumors in GHA mice were markedly sensitive to sorafenib treatment compared to the same in wild-type mice. RNA analysis showed that when HCC was exposed to the combined treatment, relatively decreased ABC transporters expression was found. Immunohistochemical staining showed that phosphorylation of STAT5 and ABCB1 were downregulated in HCC tissues, suggesting that GHA in vivo downregulates ABC transporters in HCC, and therefore sensitizes them to sorafenib. Clinical data derived from HCC patients using the TCGA database showed that multiple ABC transporters in both types of cancer correlate with GHR levels; that is, when GHR levels are relatively high, patient survival is significantly decreased. Additionally, higher ABCC1 levels also lead to significantly decreased survival rate in HCC patients. Collectively, the results indicate that a GHA is effective in sensitizing both melanoma and HCC to available treatments in vivo and may be used as a therapeutic strategy for higher efficacy of tumor clearance.

Citation Format: Yanrong Qian, Reetobrata Basu, Samuel Mathes, Prateek Kulkarni, Emily Davis, Darlene Berryman, Edward List, Silvana Duran, John J. Kopchick. Growth hormone receptor antagonist sensitizes melanoma and hepatocarcinoma to drug treatments via downregulation of ABC transporters in vivo [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 2621.
Abstract 2640: Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism
Authors: Jones Trace M. , Espitia Claudia M. , Ooi Aikseng , Bauman Julie E. , Carew Jennifer S. , Nawrocki Steffan T.
Published: June 2022

Patients with late-stage and human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) continue to have a very poor prognosis. The development of more effective novel therapies that improve overall survival and overcome drug resistance is an urgent priority. We, and others, have reported that HNSCC tumors significantly overexpress NEDD8 and exhibit high sensitivity to the first-in-class NEDD8 activating enzyme (NAE) inhibitor pevonedistat. Here, we report that disruption of NEDD8-mediated protein turnover with pevonedistat dramatically augments cisplatin-induced DNA damage and apoptosis in HNSCC models. Further analysis revealed that the specific pevonedistat target, CUL4A, played an essential role in driving the synergy of the pevonedistat and cisplatin combination. Targeted inhibition of CUL4A resulted in significant downregulation in Damage Specific DNA binding protein 2 (DDB2), a DNA-damage recognition protein that promotes nucleotide excision repair and resistance to cisplatin. Silencing of CUL4A enhanced cisplatin-induced DNA damage and apoptosis in a manner similar to that of pevonedistat demonstrating that targeted inhibition of CUL4A may be a novel approach to augment cisplatin therapy. Administration of pevonedistat to mice bearing HNSCC tumors significantly decreased DDB2 expression in tumor cells, increased DNA damage and potently enhanced the activity of cisplatin to yield tumor regression and long-term survival of all animals. Our findings provide strong rationale for clinical investigation of CUL4A inhibition with pevonedistat as a novel strategy to augment the efficacy of cisplatin therapy for patients with HNSCC and identify loss of DDB2 as a key pharmacodynamic mediator controlling sensitivity to this regimen.

Citation Format: Trace M. Jones, Claudia M. Espitia, Aikseng Ooi, Julie E. Bauman, Jennifer S. Carew, Steffan T. Nawrocki. Targeted CUL4A inhibition synergizes with cisplatin to yield long-term survival in models of head and neck squamous cell carcinoma through a DDB2-mediated mechanism [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 2640.
Abstract 2653: Synthetic, small molecule glycosaminoglycan mimetics induce novel anti-cancer activity through preferential targeting of a growth factor receptor
Authors: O'Hara Connor , Morla Shravan , Ongolu Ravikumar , Patel Nirmita , Boothello Rio , Patel Bhaumik , Desai Umesh
Published: June 2022

Glycosaminoglycans (GAGs), such as heparan sulfate (HS), have been implicated in tumor initiation, progression and metastasis. Earlier we have shown that a defined GAG sequence of six residues, but not four or eight residues, inhibits colorectal cancer stem cells (CSCs) by inducing activation of p38 MAPK. To transform this sequence into a drug-like molecule, we developed a synthetic mimetic of the GAG sequence, labeled as G2.2, which was also found to selectively target CSCs over bulk adherent tumor cells. Unfortunately, G2.2’s oral bioavailability was low. To improve upon its drug-like properties, we pursued a hypothesis-driven analog design to derive three lipid-modified analogs (LMAs). Across a panel of > 15 patient-derived colorectal cancer (CRC) cell lines, which could be stratified according to their clinically-relevant consensus molecular subtypes, LMAs showed enhanced potency while retaining selectivity against CSCs in spheroid inhibition assays. Both G2.2 and LMAs displayed better inhibition of cell lines with mesenchymal phenotypes and metabolic dysregulation. Microarray-based screening against more than a dozen receptor tyrosine kinases led to identification of IGF1R as a potential receptor of the synthetic GAG mimetics. To ascertain these screening results, biophysical studies were performed to clarify preferred soluble and/or cell surface target receptors. In line with microarray results, G2.2 preferentially bound to IGF1R in comparison to its soluble ligand IGF-1. G2.2 also preferred IGF1R as compared to an alternative receptor FGFR1. Further, LMAs bound to IGF1R with improved affinities as compared to parent mimetic G2.2, which could explain anti-CSC inhibition results. Unexpectedly, detailed fluorescence titrations revealed that IGF1R affinity of LMAs is minimally impacted by salt concentration, suggesting that these GAG mimetics utilize non-ionic forces in binding, which support the selectivity of target engagement. Overall, this work proves a powerful proof of concept that synthetic GAG mimetics, such as G2.2 and LMAs, present a unique class of anti-cancer therapeutics with high potential for selective elimination of the tumor initiating subpopulation of CRC cells in patient tumors.

Citation Format: Connor O'Hara, Shravan Morla, Ravikumar Ongolu, Nirmita Patel, Rio Boothello, Bhaumik Patel, Umesh Desai. Synthetic, small molecule glycosaminoglycan mimetics induce novel anti-cancer activity through preferential targeting of a growth factor receptor [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 2653.
Abstract 2668: Targeting of ERK and HDAC in the treatment of uveal melanoma
Authors: Musi Elgilda , Dao Kim-Hien , Hindley Chris , Schwartz Gary K.
Published: June 2022

Uveal melanoma is one of the most common intraocular malignancies of the adult eye. GNAQ/GNA11 genetic alterations are present in 85% of cases resulting in activation of the MAPK pathway. Single agent approaches targeting the MAPK pathway and histone deacetylases (HDACs) have shown very modest activity in uveal melanoma. We hypothesize that a combination strategy may be an effective, novel therapeutic approach in uveal melanoma. We examined the activity of ASTX029, a novel dual-mechanism ERK inhibitor, which inhibits both the catalytic activity of ERK and its phosphorylation by MEK, and belinostat, a pan-HDAC inhibitor, in one GNAQQ209L- (92.1) and GNAQQ209P- (OMM1.3) and two GNA11Q209L-mutant (MP41, OMM1) uveal melanoma cell lines. The uveal melanoma cell lines were sensitive to ASTX029 with IC50 values between 50 to 100nM and to belinostat with IC50 values between 100 to 250nM. Combination index analysis of ASTX029 and belinostat by the Chou-Talalay method indicated synergism, with CI values ranging from 0.34 to 0.97. We explored other drug candidates for combinatorial approaches including the BCL-2 inhibitor navitoclax, but no combination effect was observed. After 48h of combination treatment with ASTX029 and belinostat, we examined the biochemical effects by western blot which demonstrated a sustained decrease in levels of p-ERK with the combination treatment. Western blot analysis confirmed reduction of downstream markers of MAPK pathway activity including cell cycle markers p-RB (780), cyclin D1 and c-myc with combination treatment. Apoptosis was evident from an increase in the subG1 population by flow cytometry analysis, which correlated with BIM induction and PARP cleavage on western blot. We also observed increased surface expression of immunological markers HLA-A, B, C (MHC Class 1), PD-L1, gp100 and MART-1. These results support that a combination strategy targeting ERK and HDAC in uveal melanoma should be investigated further.

Citation Format: Elgilda Musi, Kim-Hien Dao, Chris Hindley, Gary K. Schwartz. Targeting of ERK and HDAC in the treatment of uveal 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 2668.
Abstract 268: Role of aquaporins in mechanotransduction and extracellular vesicle-mediated cancer cell communication in complex physical microenvironments
Authors: Smith Ian , Moses Allison , Kronstadt Stephanie , Jay Steven , Stroka Kimberly
Published: June 2022

Introduction: Metastasis is driven by the migration of cancer cells to different areas of the body, but the mechanisms that induce cell movement are still poorly understood. Aquaporins (AQPs), transmembrane water transport proteins, have been linked to tumor growth, metastasis, and more invasive phenotypes. However, the exact mechanisms behind AQP dysregulation and its role in mediating cancer migration remain unknown. Physical cues and extracellular signals from the tumor microenvironment have been implicated in the progression of cancer; yet the role AQPs play in sensing and signaling have yet to be elucidated. Recent studies have shown the paracrine signaling within the tumor microenvironment is mediated in part by cell secreted extracellular vesicles (EVs). EVs are membrane-delimited nanoparticles capable of protecting and transporting protein and nucleic acid cargo for intercellular communication. EV mediated paracrine signaling has been shown to participate in various steps of tumor progression. Cells are also able to sense complex mechanical signals from the tumor microenvironment to direct more invasive cellular responses. Understanding the role AQPs play in sensing and signaling within the tumor microenvironment can provide a more holistic view of the way AQPs influence cancer progression.

Materials and Methods: To study the role of AQP containing EVs and AQPs influence on mechanotransduction to facilitate cancer cell migration, this study has employed 3D in vitro models, including microfluidic devices, to recapitulate relevant in vivo conditions in concert with confocal microscopy to understand cell behaviors. EVs were isolated via tangential flow filtration. AQP levels were down regulated with siRNA complex or upregulated via AQP5-GFP plasmids.

Results: Knocking down AQPs with siRNAs, confirmed with western blotting, was shown to reduce cellular migration on 2D surfaces and in confining microenvironments. We have also found that AQPs can enhance the integrin mediated mechanotransduction pathway, which has been implicated in cell proliferation and migration. Effects of integrin mediated mechanotransduction were reversed by suppressing AQP expression. Finally, we have made the exciting discovery that cancer cells secrete extracellular vesicles that contain AQPs. These EVs could be released into the tumor microenvironment to enhance tumor migration. Further work is being conducted to elucidate the AQP-EV relationship.

Citation Format: Ian Smith, Allison Moses, Stephanie Kronstadt, Steven Jay, Kimberly Stroka. Role of aquaporins in mechanotransduction and extracellular vesicle-mediated cancer cell communication in complex physical microenvironments [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 268.
Abstract 2682: Small molecule targeting the lipoic acid post-translational modification impacts proliferation of colorectal and PIK3CA-mutant cell lines
Authors: Doherty Laura , Sangpo Tenzin , Tsvetkov Peter , Davis John , Dianati Navid , Schwede Wolfgang , Zimmermann Katja , Evans Laura , Amatucci Aldo , Seidel Henrik , Kamburov Atanas , Akcay Gizem , Golub Todd , Eheim Ashley , Burkhardt Nils , Eis Knut , Christian Sven , Rees Matt , Roth Jennifer
Published: June 2022

To identify novel therapeutic targets, we utilize the PRISM platform, a multiplexed cell line viability technology of 500 solid tumor cell lines and correlate responses to functional genomic and baseline genetic data. We describe ESD0140656, a small molecule with selective anti-proliferative effect on colorectal and PIK3CA-mutant cell lines. Response to ESD0140656 is correlated to sensitivity to CRISPR/Cas9 KO of components of the protein lipoylation pathway and OGDH complex members, which catalyze a step of the TCA cycle. Lipoylation is a rare post-translational modification attached to just four enzymes in humans, including the OGDH complex. Knockout of the protein that transfers lipoic acid to these four enzymes (LIPT1) sensitizes cells to ESD0140656, and ESD0140656 treatment leads to reduction of lipoic acid in cells. These results suggest ESD0140656 targets the lipoylation pathway and may represent a novel therapeutic angle for colorectal and PIK3CA-mutant tumors.

Citation Format: Laura Doherty, Tenzin Sangpo, Peter Tsvetkov, John Davis, Navid Dianati, Wolfgang Schwede, Katja Zimmermann, Laura Evans, Aldo Amatucci, Henrik Seidel, Atanas Kamburov, Gizem Akcay, Todd Golub, Ashley Eheim, Nils Burkhardt, Knut Eis, Sven Christian, Matt Rees, Jennifer Roth. Small molecule targeting the lipoic acid post-translational modification impacts proliferation of colorectal and PIK3CA-mutant 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 2682.
Abstract 2692: Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene
Authors: Van Goubergen Jasper , Handle Florian , Cronauer Marcus V. , Santer Frédéric R.
Published: June 2022

Castration resistant prostate cancer (CRPC) involves the upregulation of androgen receptor variants (AR-Vs), of which AR-V7 is clinically the most relevant. By lacking the ligand-binding domain, AR-V7 is constitutively active and may therefore bypass AR signaling inhibitors. Treating CRPC involves improved AR signaling inhibitors or taxane-based chemotherapy. However, the optimal treating sequence may differ from patient to patient. The presence of AR-V7 in the nucleus of circulating PCa cells, which is associated with AR targeted therapy resistance, is used as a therapy-guiding biomarker. This stratification has a high predictive power and specificity, but its sensitivity is unsatisfying and can only be done by highly specialized laboratories. A PCR/sequencing based test would improve the sensitivity of this biomarker, but it requires a better understanding of AR-V7’s functional regulation. Therefore, we aim here to analyze single nucleotide polymorphisms (SNPs) in the 3’ untranslated region (UTR) of AR-V7 that might have an impact on AR-V7’s expression and/or functional activity.

The LD trait tool of NIH was used to search for trait association with AR cryptic exon (CE3) SNPs. Four RNA binding motif databases (oRNAment, ATtRACT, RBP map, and RBPDB) and SpliceAid v2 were used for evaluating SNPs in their capacity to alter motifs. By CRISPR/CAS9 mediated non-homologous end joining (NHEJ) these predictions were assessed experimentally using qPCR and Westen Blot.

SNP rs5918762 was selected as it is in linkage disequilibrium with several PCa tagSNPs (rs5919393, rs5919432). rs5918762T/C is characterized by an unequal minor allele frequency (MAF) distribution among different populations. Using an in silico approach, this SNP is predicted to destroy/create a binding site for the splicing machinery involved protein SRSF9. Disrupting this SNP region via NHEJ resulted respectively in an increased and decreased expression of AR-FL and AR-V7 mRNA in 22RV1 cells (C allele). Additionally, analysis of the SU2C dataset (CRPC patients) correlated SRSF9 expression with AR-V7 expression. Subsequently, SRSF9 knock out (KO) resulted in reduced AR-V7 mRNA expression.

Our data suggests that SNP rs5918762T/C is located in a region important for CE3 inclusion during AR splicing, which might involve SRSF9. Further validation of these results is in need and encompasses splicing assays by an AR-V7 mini-gene assay, protein-RNA interaction assays and an analysis of a lack of effect in cell lines expressing the T allele.

Citation Format: Jasper Van Goubergen, Florian Handle, Marcus V. Cronauer, Frédéric R. Santer. Identification of functional single nucleotide polymorphisms in cryptic exon 3 of the androgen receptor gene [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 2692.
Abstract 2711: Repurposing CAR targets in solid tumors from pan-cancer single-cell RNA-seq data
Authors: Madan Sanna , Schäffer Alejandro , Ruppin Eytan
Published: June 2022

Chimeric antigen receptor (CAR) T cell therapy is a powerful and promising tool for unleashing lasting antitumor immunity, which is currently studied in numerous blood and solid cancers. Here, mining single cell transcriptomics datasets of solid tumors, we survey many existing CAR targets, aiming to identify new solid tumor types in which these targets are differentially expressed in tumor cells and not on non-tumor cells within the tumor microenvironment, thus providing new repurposing opportunities. Quite surprisingly, we find that many existing solid cancer CAR targets have a better discriminatory power in uncharted cancer types than in the ones they have been developed or being tested for. Furthermore, we show that several existing liquid cancer CAR targets may also hold promise in specific solid tumors. Taken together, this analysis uncovers a large potential of repurposing current CARs that is currently yet untapped.

Citation Format: Sanna Madan, Alejandro Schäffer, Eytan Ruppin. Repurposing CAR targets in solid tumors from pan-cancer single-cell RNA-seq data [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 2711.
Abstract 2743: Tumor growth modeling and its applications in preclinical pharmacology studies to improve translatability of animal models
Authors: Zhou Huajun , Mao Binchen , Guo Sheng
Published: June 2022

Successful oncology drug development hinges on proper use of preclinical xenograft and allograft mouse models that faithfully reflect histopathology and genomic features of patient tumors and exhibit clinically similar drug response. For in vivo pharmacology studies, tumor-bearing mice are treated by drugs and efficacy is monitored by continuingly measuring tumor volumes over weeks or even months, resulting in mouse-specific tumor growth data. In our previous study, we used linear mixed model (LMM) to fit tumor growth data and demonstrated its superiority to simple readouts, such as tumor growth inhibition (TGI), in discovering biomarkers and exploring mechanism of action for drugs. Here we extended the study to more parametric statistical models on an expanded dataset with over 100 thousand tumor growth curves. Six parametric models (exponential, exponential square, Gompertz, Logistic, Monomolecular, and von Bertalanffy) were fit to tumor growth curves within a single-treatment group, by the least square method after variance stabilization transformation. Best models were selected by Akaike information criterion (AIC)-based confidence set. In the Irinotecan case study, logarithmic transformation of tumor volume converts exponential or exponential square models to mixed-effects linear models that integrate random effect of mouse difference within a treatment group, and are used to identify predictive biomarkers for drug efficacy. We compared three variance stabilization transformations and found that log transformation of tumor volume is appropriate for majority of tumor growth curves. In log transformed data, exponential or exponential square model can satisfactorily fit nearly all tumor growth curves, as 96.1% of best model confidence sets contain exponential or exponential square model, followed by von Bertalanffy (92.5%), Gompertz (92.1%), Logistic (90.5%), and Monomolecular (58.9%). The percentage of studies, in which all treatment groups can be best fit by exponential or exponential square model, is 91.3% (82.4% for von Bertalanffy, 81.4% for Gompertz, 79.1% for Logistic, 37.3% for Monomolecular). Thus, exponential or exponential square model can be applied to most in vivo mouse studies, and should be preferred for their mathematical simplicity and interpretability. We further demonstrated the utility of the modeling method on a 16-PDX-model study for Irinotecan to identify biological processes and pathways related to its mechanism of action.

Citation Format: Huajun Zhou, Binchen Mao, Sheng Guo. Tumor growth modeling and its applications in preclinical pharmacology studies to improve translatability of animal 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 2743.
Abstract 2855: Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia
Authors: Luo Yan , Lui Kaihin , To Tommy T. , liu Andrew , Yassine Farah , Dabaja Mohamed A. Kharfan , Gadd Martha E. , Qin Hong
Published: June 2022

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults with CD19-targeting CAR T-cell therapy showing an excellent initial curative response. However, 10-20% of patients who receive anti-C19 CAR T-cell treatment experience relapse due to CD19 antigen loss. B-cell activating factor receptor (BAFF-R) that is expressed on the surface of normal and neoplastic B cells mediates the survival of mature peripheral B cells along with the B-cell receptor. Since B-CLL is characterized by apoptotic disruption and subsequent accumulation of mature B cells, BAFF-R is a logical, novel target for CAR T-cell therapy directed against B-CLL, especially if CD19 antigen loss occurs. In this study, we first manufactured BAFF-R CAR T-cells by infecting healthy donor Tnaïve/memory (Tn/mem) cells with BAFF-R CAR lentivirus before evaluating the antitumor activity of BAFF-R CAR T-cells on wild type MEC-1 (a B-CLL cell line), modified MEC-1, and primary cell lines. Antigen-specific cytotoxicity and activation of the BAFF-R CAR T-cells or control CD19 CAR T-cells were determined upon incubation with B-CLL cell lines and patient-derived primary B-CLL by measuring degranulation and IFN-γ release, respectively. BAFF-R CAR T-cells demonstrated substantial cytotoxicity against MEC-1, CD19-deficient MEC-1, and primary B-CLL cell lines; CD19 CAR T-cells showed no effect on CD19-deficient MEC-1 cells but did show effects with primary B-CLL and MEC-1 cells. Similarly, the release of IFN-γ was greater when BAFF-R CAR T-cells were co-cultured with CD19-deficient MEC-1 or primary B-CLL compared to CD19 CAR T-cells co-cultured with CD19-deficient MEC-1. In summary, BAFF-R CAR T-cells exert significant antitumor properties not only on patient-derived primary B-CLL cells but also on a CD19-deficient B-CLL cell line. We, therefore, assert BAFF-R-targeting CAR T-cells to be a novel therapeutic avenue for patients newly diagnosed with B-CLL and patients experiencing relapsed B-CLL after anti-CD19 therapy.

Citation Format: Yan Luo, Kaihin Lui, Tommy T. To, Andrew liu, Farah Yassine, Mohamed A. Kharfan Dabaja, Martha E. Gadd, Hong Qin. Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia [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 2855.
Abstract 2886: Antigen internalization and its prevention during treatment with bispecific antibodies
Authors: Klimovich Maria , Zekri Latifa , Jung Gundram , Salih Helmut R.
Published: June 2022

Antigen-mediated antibody internalization is an important factor influencing kinetics and efficacy of antibody-based therapeutics. Antibodies directed against HER2, CD20, FLT3, EGFR, CD10 and CD22 have been shown to undergo internalization upon binding to their target antigens. Although antibody-induced internalization may be desirable under some circumstances, for example in case of antibody-drug conjugates, it can subvert the therapeutic efficacy of ADCC-inducing antibodies and bispecific antibodies (bsAbs) aiming to mobilize T cells against cancer. Here we examined the turnover of PSMA, a target antigen widely used for diagnostic and therapeutic interventions in prostate cancer. As a model system, we used prostate cancer cell lines expressing varying levels of PSMA in functional analyses with our PSMAxCD3 bsAb CC-1 which is currently being evaluated in two clinical trials (NCT04104607 and NCT04496674). Antigen internalization kinetics was analyzed by flow cytometric-internalization assays and live-cell imaging of antibody internalization. These analyses revealed that surface PSMA expression is rapidly downregulated after binding of PSMAxCD3 bsAb. Internalization occured in a dose-dependent manner, with complete negativity observed at concentrations ≥0.1 µg/ml. After withdrawal of the bsAb, antigen surface levels were fully restored within 24 hours. PSMA internalization was found to be partially prevented by inhibiting clathrin-dependent endocytosis. Currently, we are screening various clinically approved clathrin-dependent endocytosis inhibitors (e.g. statins and chloroquine) to identify compounds for combinatorial approaches to prevent undesired PSMA internalization in patients receiving PSMAxCD3 bsAbs. Altogether, our results document the involvement of clathrin-dependent endocytosis in bsAbs-driven PSMA internalization and provide a basis for combinatorial approaches to increase the efficacy of PSMA-directed therapeutics.

Citation Format: Maria Klimovich, Latifa Zekri, Gundram Jung, Helmut R. Salih. Antigen internalization and its prevention during treatment with bispecific antibodies [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 2886.
Abstract 2887: Three dimensional colon cancer organoids model the response to CEA CD3 T cell engagers
Authors: Teijeira Alvaro , Migueliz Itziar , Garasa Saray , Bacac Marina , Klein Christian , Melero Ignacio
Published: June 2022

Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy.

Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials.

Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing.

Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.

Citation Format: Alvaro Teijeira, Itziar Migueliz, Saray Garasa, Marina Bacac, Christian Klein, Ignacio Melero. Three dimensional colon cancer organoids model the response to CEA CD3 T cell engagers [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 2887.
Abstract 2929: High-Definition Digital PCR (HDPCR™) enables sensitive measurement of DNA and RNA variants in non-small cell lung cancer samples
Authors: Leatham Bryan , McNall Katie , Subramanian Hari K. , Alvarado John , Jacky Lucien , Wang Mimi , Rajagopal Aditya , Schwartz Jerrod
Published: June 2022

Digital PCR (dPCR) is an emerging platform for detecting genomic variants in cancer genomes due to its high sensitivity and fast time to result compared to massively parallel sequencing. However, translational oncology applications often require the measurement of more biomarkers than there are color channels available on dPCR platforms. One approach to address this is to split a sample across many wells and profile a subset of variants in each well. For input-limited samples, however, this results in fewer molecules being profiled in each well, resulting in a reduction in sensitivity and fewer samples processed per instrument run.

ChromaCode has developed a research use only (RUO) high-definition digital PCR assay, for multiplexed detection of 14 DNA variants and 15 RNA fusion variants relevant in non-small cell lung cancer (NSCLC) samples. The assay is constructed using both amplitude modulation and multi-channel resilient signal encoding methods. Amplitude modulation enables different variants to generate a signal at different intensity levels in single color channel, allowing for greater than n targets in n color channels. In contrast, resilient encoding generates a signal in more than one color channel to create a form of error detecting code.

DNA and RNA samples were obtained using a combination of synthetic templates, cell line nucleic acids, and commercially available reference materials. Samples were run with the HDPCR NSCLC RUO assay on the Thermo Fisher Absolute Q digital PCR system, and data analysis was performed with custom analysis algorithms. The HDPCR NSCLC RUO assay demonstrated a limit of detection as low as 10 variant copies in a 10,000 haploid human genome copy DNA background. By employing HDPCR technology on digital PCR systems, it is possible to build a comprehensive and sensitive research assay that quickly detects many genomic alterations relevant to NSCLC samples.

Citation Format: Bryan Leatham, Katie McNall, Hari K. Subramanian, John Alvarado, Lucien Jacky, Mimi Wang, Aditya Rajagopal, Jerrod Schwartz. High-Definition Digital PCR (HDPCR™) enables sensitive measurement of DNA and RNA variants in non-small cell lung cancer samples [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 2929.
Abstract 297: Prophylaxis and treatment of metastatic breast cancer and melanoma using S100A9-targeted cowpea mosaic virus nanoparticles
Authors: Chung Young Hun , Park Jooneon , Cai Hui , Steinmetz Nicole F.
Published: June 2022

The current study investigates the use of an immunostimulatory plant virus nanoparticle called the cowpea mosaic virus (CPMV) in both the prophylaxis and treatment of metastatic breast cancer and melanoma. Metastatic cancer remains to this day one of the most difficult areas in oncology to treat and diagnose. Current standardized treatments consist of systemically administered chemotherapeutics, which are not targeted to the diseased areas and oftentimes cause significant side effects. Therefore, novel treatments such as targeted immunotherapies are under development. The S100A9 protein is upregulated in the tumor microenvironment and it plays an important role in tumor metastasis, aggressiveness, and tumor immunosuppression. It is present not only in the pre-metastatic tumor environment, but is also heavily secreted and expressed after metastatic tumor establishment has occurred. This makes S100A9 a prime target in both prophylactic and therapeutic settings; yet, targeted immunotherapies against S100A9 have never previously been explored. CPMV has been shown in the past to be immunostimulatory when injected directly into the tumor with the ability to recruit immune cells for tumor cell killing. However, metastatic tumors cannot be treated through intratumoral injections; therefore, the current work utilizes systemically-administered S100A9-targeted CPMV nanoparticles. Biodistribution studies indicate that S100A9-targeted CPMV homes to the lung while confocal imaging of S100A9 and S100A9-targeted CPMV within the lungs of tumor-inoculated mice demonstrates co-localization. We further demonstrate using flow cytometry that after CPMV homes to the lungs, immune cells such as neutrophils and dendritic cells are recruited to the lungs, and macrophages are polarized towards their antitumor M1 phenotype. Prophylaxis efficacy studies where CPMV is injected before the tumors show that the S100A9-targeted CPMV causes tumor rejection in both metastatic breast cancer and melanoma but that native CPMV does not produce a significant effect. When used as a treatment, the targeted CPMV, similarly shows efficacy against these two cancer models with native CPMV again showing ineffectiveness. The S100A9-targeted CPMV holds great potential as a targeted immunotherapy both prophylactically (e.g. after primary tumor debulking to reduce the onset of metastases) and therapeutically after tumor establishment has occurred.

Citation Format: Young Hun Chung, Jooneon Park, Hui Cai, Nicole F. Steinmetz. Prophylaxis and treatment of metastatic breast cancer and melanoma using S100A9-targeted cowpea mosaic virus nanoparticles [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 297.
Abstract 3008: Induction of necroptosis through caspase-independent mechanisms in MCF-7 breast cancer cells following HDAC inhibition
Authors: Natarajan Umamaheswari , Waldron Grace , Venkatesan Thiagarajan , Rathinavelu Appu
Published: June 2022

Cell proliferation and cell death are essential for maintaining homeostatic balance in eukaryotic organisms. Many diseases develop when cellular homeostasis is disturbed. Currently, several anti-cancer drugs are known to induce apoptosis through activation of intrinsic or extrinsic pathways. However, many types of cancer cells have acquired resistance due to quick adaptation to resist pro-apoptotic mechanisms. Therefore, simultaneous induction of other forms of cell deaths such as necroptosis, autophagy, etc., in addition to apoptosis, is becoming an attractive strategy for controlling tumor growth and metastasis. For this purpose, we have explored the role of XIAP in caspase-independent mechanisms of cell death during HDAC (Histone Deacetylase) inhibition. Our experiments with HDAC inhibitor SAHA (Suberoylanilide hydroxamic acid) have revealed that the anti-apoptotic biomarkers, including survivin and XIAP, exhibit important regulatory functions for inducing necroptosis in cancer cells. Interestingly, many of the signal adaptors of apoptosis and necroptosis are known to show dual functions during drug-induced cancer cell death. For example, the formation of the survivin-XIAP complex prevents XIAP from undergoing proteasomal degradation and polyubiquitination, thereby stabilizing XIAP in cancer cells. When the survivin level is increased, it is also known to stop the inhibition of XIAP by Smac. In addition, since XIAP can inhibit caspase -9 and the executioner caspases -3 and -7, the apoptosis mechanisms will be blocked, and cell proliferation will continue to promote cell survival and tumorigenesis. Our western blot data revealed that the caspase -9, -3, -7, and -8 levels were significantly downregulated in MCF-7 breast cancer cells following SAHA treatment. Furthermore, SAHA treatment also increased the levels of necroptosis inducers such as phospho-RIP3 and MLKL in MCF-7 cells. However, treatment of the same MCF-7 cells with other drugs such as RG-7388, an MDM2 inhibitor, was able to induce apoptosis through activation of caspases. Our current results show that MCF-7 cells may switch to the necroptosis pathway due to the inhibition of caspases and increased levels of survivin and XIAP. (This research was supported by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida).

Citation Format: Umamaheswari Natarajan, Grace Waldron, Thiagarajan Venkatesan, Appu Rathinavelu. Induction of necroptosis through caspase-independent mechanisms in MCF-7 breast cancer cells following HDAC inhibition [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 3008.