bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5226: Exome-wide association study identifies coding variant in IL17RA associated with survival in cancer patients treated with immunotherapy
Authors: Kleeman Sam O. , Chan Michael , Chvasta Matthew , Janowitz Tobias
Published: April 2023

Immune checkpoint inhibitors (ICI) targeting PD-1/PD-L1 and CTLA4 are effective treatments in multiple cancer types, but durable responses are seen only in a minority of patients. Resistance to ICI is likely to reflect a combination of tumor-intrinsic, such as tumor mutational burden, and tumor-extrinsic factors, such as systemic immune suppression and host genetics. With the advent of large-scale germline genotyping studies, there is emerging evidence that host-specific genetic factors can substantially modify both disease risk and responsiveness to therapy. There has been limited characterization to date of the interaction between host genetics and response to ICI. Here, we perform harmonized mutation calling in available normal exome sequencing from 668 metastatic cancer patients treated with ICI (derived from n=7 studies, spanning n=3 cancer types and PD-1/PD-L1/CTLA4-directed therapy) and performed exome-wide association studies in European ancestry patients. For each cancer type (melanoma, renal cell carcinoma, urothelial carcinoma), we conducted exome-wide Cox regression (with PC1-4 and sex as covariates) to identify coding single-nucleotide polymorphisms (SNPs) associated with overall survival (OS). On fixed-effect meta-analysis, we identified a single coding variant (rs140221307, C allele, MAF 0.5% in European populations) that met genome-wide significance (p=1.11 × 10−15, HR=4.86, 95% CI 3.30-7.15). This SNP causes a substitution of Trp to Arg at amino acid 320 at the interface between the transmembrane and extracellular domains of interleukin 17 receptor A (IL17RA). While the secretion mechanism is uncharacterized, soluble IL17RA can be readily detected in plasma and the survival-reducing variant is associated with substantially elevated plasma IL17RA (p=1.90 × 10−9, beta=1.01, Sun et al. 2018) as well as reduced monocyte counts in UK Biobank (p=2.20 × 10−308, beta=-0.60). We have established a prime editing strategy to induce this variant in cultured human monocytes to investigate its biochemical and functional consequences, and are performing validation studies in independent patient cohorts. Soluble IL17RA would be expected to function as a decoy receptor to antagonize IL17/IL17RA signaling, and so these data suggest that activation of IL-17 receptor signaling might improve responsiveness to ICI therapy.

Citation Format: Sam O. Kleeman, Michael Chan, Matthew Chvasta, Tobias Janowitz. Exome-wide association study identifies coding variant in IL17RA associated with survival in cancer patients treated with immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5226.
Abstract 5348: Targeting MEK, alone and in combination with BRAF, in metastatic BRAFK601E-mutated melanoma
Authors: Chan Sze Man , Chow Sin Ting , Yeung Sai Fung , Tsui Kwok Wing Stephen
Published: April 2023

Mitogen-activated protein kinase/ERK signaling pathway has been one of the major mechanisms underlying melanoma haboring BRAF mutation. Of these, K601E mutation belongs to a rare mutation sub-type, comprising only 3% of the BRAF mutations in melanoma. Although patients with BRAF V600E/K mutation respond to MEK inhibitor, whether it also applies to those with BRAF K601E mutation remains elusive. Here, we examined the effects of MEK and BRAF/MEK inhibition on BRAFK601E-expressing melanoma cells. Melanoma cell line (MeWo) was transfected with doxycycline-inducible vector encoding wild-type or K601E-mutant BRAF. Subsequently, cells or xenograft-bearing mice were treated with trametinib, dabrafenib plus trametinib or sorafenib plus trametinib. Compared with BRAFWT, MTT assay demonstrated that BRAFK601E cells were more sensitive towards drug treatment. The effects of combined treatment were more profound. Tumorigenicity assay showed that trametinib treatment inhibited tumor growth of BRAFK601E xenografts. Our preliminary results demonstrated the therapeutic values of MEK and BRAF/MEK inhibitors against BRAFK601E-mutated melanoma. As previous studies report the superior therapeutic effects of BRAF/MEK inhibitors over MEK inhibitor alone, phenotype characterization of the cells, as well as underlying mechanisms behind such response, worth further investigation.

Citation Format: Sze Man Chan, Sin Ting Chow, Sai Fung Yeung, Kwok Wing Stephen Tsui. Targeting MEK, alone and in combination with BRAF, in metastatic BRAFK601E-mutated melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5348.
Abstract 542: Synthetic lethal targeting of AURKA in head and neck squamous cell carcinoma
Authors: da Silva Flaviane N , Nguyen Theodore , Lee Jong Woo , Burtness Barbara , Golemis Erica
Published: April 2023

The purpose of this study was to identify new combination strategies for improving treatment of head and neck squamous cell carcinomas (HNSCCs). HNSCCs often express high levels of Aurora kinase A (AURKA), most studied as a kinase important for mitotic progression in normal cells. Notably the overexpression of AURKA in many cancers allows it to act throughout the cell cycle, acquiring new cancer-promoting activities. While several AURKA inhibitors are in clinical trials, limited efficacy is observed with single treatment, causing us to evaluate potential AURKA combination therapeutic combinations. Particularly relevant to HNSCC, which often overexpress and depend on EGFR, overexpression of AURKA can promote resistance to EGFR inhibitors by mechanisms variously identified as including activation of the EGFR effectors AKT, ERK, and BIM. Using viability and clonogenic assays, we have found that the AURKA inhibitor VIC-1911 synergizes with the EGFR inhibitors afatinib and erlotinib in multiple HNSCC cell lines and retains activity in HNSCC cell models selected for resistance to these EGFR inhibitors. However, AURKA inhibition increases levels of AURKA protein and its protein-stabilizing partner TPX2, counteracting drug effect. We also combined inhibition of AURKA with inhibition of WEE1, a regulator of the G2/M checkpoint, finding this dual inhibition enhanced levels of apoptosis in EGFR inhibitor resistant HNSCC cell lines. Intriguingly, one recent study reported AURKA can also modulate the glycolytic signaling pathway, increasing the cancer-promoting Warburg effect. We have found that inhibition of AURKA can significantly reduce glycolytic flux in HNSCC cell lines and alter expression and phosphorylation of key glycolytic enzymes. Dual inhibition of AURKA in combination with inhibitors of glycolytic enzymes, or enzymes regulating oxidative phosphorylation, showed marked synergy. These and other results investigating AURKA signaling mechanisms in HNSCC demonstrate that the non-canonical functions of AURKA include viable therapeutic targets that can enhance the efficacy of AURKA inhibition in HNSCC.

Citation Format: Flaviane N da Silva, Theodore Nguyen, Jong Woo Lee, Barbara Burtness, Erica Golemis. Synthetic lethal targeting of AURKA in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 542.
Abstract 5428: Skeletal muscle gauge prediction by a machine learning model in patients with colorectal cancer
Authors: Kang Jeonghyun
Published: April 2023

Background: Skeletal muscle gauge (SMG) was recently introduced as an imaging indicator of sarcopenia for the prediction of clinical outcomes, including chemotherapy toxicity and prognosis, in patients with cancer. Computed tomography (CT) is essential for measuring SMG; thus, the use of SMG is limited to patients who undergo CT.

Objective: We aimed to develop a machine learning algorithm using clinical and inflammatory markers to predict SMG in patients with colorectal cancer (CRC)

Methods: The least absolute shrinkage and selection operator (LASSO) regression model was applied for variable selection and predictive signature building in the training set. The predictive accuracy of the LASSO model, defined as LP-SMG was compared using the area under the receiver operating characteristic (AUROC) and decision curve analysis (DCA) in the test set.

Results: A total of 1,094 patients with CRC were enrolled and randomly categorized into training (n=656) and test (n=438) sets. Low SMG was identified in 142 (21.6%) and 90 (20.5%) patients in the training and test sets, respectively. According to multivariable analysis of the test sets, LP-SMG was identified as an independent predictor of low SMG (OR: 1329.431, CI: 271.684-7667.996, p<.001). Its predictive performance was similar in the training and test sets (AUROC: 0.846 vs. 0.869, p=.427). In the test set, LP-SMG showed better outcomes in predicting SMG than single clinical variables, such as sex, height, weight, and hemoglobin, as measured by AUROC and DCA.

Conclusions: LP-SMG, incorporating clinical variables and serum inflammatory indicators, showed superior performance compared to single variables in predicting low SMG. This machine learning model can be used as a screening tool to detect sarcopenic status without using CT during the treatment period. Applying a machine learning model might be beneficial in reducing the effort, cost, and radiation exposure from conventional CT-based diagnosis.

Citation Format: Jeonghyun Kang. Skeletal muscle gauge prediction by a machine learning model in patients with colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5428.
Abstract 562: Anti-tumor efficacy of GMF-1A3, an MMAE-based antibody drug conjugate targeting cell surface cleaved Amphiregulin in breast cancer
Authors: Lofgren Kristopher A. , Sreekumar Sreeja , Kenny Paraic A.
Published: April 2023

The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Here, we describe the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with monomethyl auristatin E (MMAE) were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We further demonstrate that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection. A pan-cancer tissue microarray analysis indicates that the target is commonly detected (> 50% of cases) in breast, prostate, liver and lung cancer.

Citation Format: Kristopher A. Lofgren, Sreeja Sreekumar, Paraic A. Kenny. Anti-tumor efficacy of GMF-1A3, an MMAE-based antibody drug conjugate targeting cell surface cleaved Amphiregulin in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 562.
Abstract 5643: High-spatial-resolution multi-omics analysis of cancer tissues
Authors: Neil Emily , Park Dongju , Lloyd Erica , Dibuono Michael , Hosono Seiyu , Muller Reto , Lafayette Hanna , Smith Hsinyi , Pinard Robert
Published: April 2023

Recent technological breakthroughs in single cell sequencing have revealed that malignant cells and nonmalignant cells are both highly dynamic and in the case of tumor cells, that the intratumoral heterogeneity is quite remarkable. However, several questions remain, especially in a spatial context, and a rapidly growing need has been observed for imaging-based approaches with single-cell resolution to visualize and characterize the tumor microenvironment. In the current study, we used Miltenyi Biotec newly developed spatial gene expression technology and automated imaging platform, to acquire spatial microscopy data for multiple biological analytes and multiple cancer tissues. The approach is based on fluorescence microscopy and the MICS technology principle of cyclic staining (MACSima™ Imaging Cyclic Staining) along with validated fluorochrome-conjugated antibodies and specifically amplified oligonucleotide probes combined with a proprietary signal removal system. FFPE tissue blocks were analyzed and each gene, which display a unique emission spectrum during a single cycle, was decoded. Due to the non-destructive nature of the workflow, H&E staining, immunofluorescence and RNA data can be collected together into a single image and on a single section. Using our antibody and targeted RNA immune-oncology panel coupled with our smart segmentation and flexible gating software suite, we have generated a topographical map of the expression profile level for several cancer tissues i.e.: tonsil, pancreas, kidney and ovary. This technology allows for the analysis of hundreds of protein markers and RNA transcripts on a single tissue, the characterization of the spatial architecture and the expression topography of tumor tissue sections. A better understanding of tissue microenvironment and implementation of spatial biology approaches will help advance the identification of more suitable biomarkers and for cancer therapy.

Citation Format: Emily Neil, Dongju Park, Erica Lloyd, Michael Dibuono, Seiyu Hosono, Reto Muller, Hanna Lafayette, Hsinyi Smith, Robert Pinard. High-spatial-resolution multi-omics analysis of cancer tissues. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5643.
Abstract 5647: Single cell, multiomic spatial phenotyping of immunotherapy responses in head and neck cancer
Authors: Ji HaYeun , Jhaveri Niyati , Ma Ning , Cheikh Bassem B. , Pratapa Aditya , Monkman James , O’Byrne Ken , Hughes Brett , Kulasinghe Arutha , Braubach Oliver
Published: April 2023

Background: Immune checkpoint inhibitors (ICI) have proven to be game-changing treatments for mucosal head and neck squamous cell cancer (HNSCC). Emerging successes with anti-PD-1/PD-L1 therapy have led to durable responses and prolonged survival in both human papillomavirus-positive (HPV+) and negative (HPV-) patients. There is now a need for predictive biomarkers to guide patient selection for highly targeted ICI therapies as currently available diagnostic biomarkers have a limited value. The tumor microenvironment (TME) composition, contexture, and cellular architecture are now recognized as key to understanding immune responsive and resistant phenotypes. Here, we are using a spatial biology approach to explore the TME in metastatic/recurrent HNSCC tumors treated with Pembrolizumab/Nivolumab.

Methods: In this study, we used single cell, multiomic spatial phenotyping utilizing the PhenoCycler-Fusion spatial biology platform to characterize the TME of HNSCC tumors from a cohort of n=40 patients. The discovery cohort consisted of patients who had complete vs. partial vs. stable vs. progressive responses to ICI therapy. We first analyzed tissues using an ultrahigh-plex antibody panel of >60 antibodies that label immune cell lineages, checkpoints, activation markers as well as tissue structure and the stroma. We then conducted whole-slide, single cell resolution RNA detection with complementary markers on serial sections from the same tissue blocks; the combination of these data allowed us to obtain multiomic spatial signatures that offered uniquely comprehensive insight into the TME of our tissue cohorts.

Results: Our study identified highly resolved tissue immune contexture analysis and metabolic tissue signatures associated with resistance and sensitivity to immunotherapy. Most notably, multiomic profiling of HNSCC tumours provided deeper insights into ICI therapy resistance than the single omics based approaches alone.

Conclusions: Our study demonstrates the power of unbiased, multiomic spatial phenotyping with whole-slide imaging to identify biomarkers associated with response to ICI therapy in HNSCC.

Citation Format: HaYeun Ji, Niyati Jhaveri, Ning Ma, Bassem B. Cheikh, Aditya Pratapa, James Monkman, Ken O’Byrne, Brett Hughes, Arutha Kulasinghe, Oliver Braubach. Single cell, multiomic spatial phenotyping of immunotherapy responses in head and neck cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5647.
Abstract 5718: Additivity predicts the clinical efficacy of most approved combination therapies for advanced cancer
Authors: Hwangbo Haeun , Patterson Sarah , Dai Andy , Plana Deborah , Palmer Adam C.
Published: April 2023

Most advanced cancers are treated with drug combinations. Rational designs aim to identify synergistic drug interactions to produce superior treatments. However, metrics of drug interaction (i.e., synergy, additivity, antagonism) apply to pre-clinical experiments, and there has been no established method to quantify synergy versus additivity in clinical settings. Here, we propose and apply a model of drug additivity for progression-free survival (PFS) to assess if the clinical efficacies of approved drug combinations are more than, or equal to, the sum of their parts. This model accounts for the benefit from patient-to-patient variability in the best single drug response, plus the added benefit of the weaker drug per patient. Among all FDA approvals of combination therapies for advanced cancer between 1995 and 2020, we identified 37 combinations across 13 cancer types (24,723 patients) where the efficacies of monotherapies and combination therapies could be analyzed at matched doses. 95% of combination therapies exhibited progression-free survival times that were additive, or less than additive. Across all trials and follow-up times, the coefficient of determination of the additivity model was R2=0.90, with no fitting or training, only the operation of addition. While there were deviations from additivity, with several combinations being less than additive, 100% of the approved combinations studied would have been predicted to succeed by the additivity model. This study has two key findings. First, a synergistic effect (more than additive) is neither a necessary nor even common property of clinically effective drug combinations. Second, the predictable efficacy of many of the best drug combinations established over the past 25 years suggests that additivity can be used as a design principle for novel drug combinations and clinical trials, which could be helpful as the number of possible therapies grows and as cancers are increasingly divided into subtypes with distinct drug sensitivities.

Citation Format: Haeun Hwangbo, Sarah Patterson, Andy Dai, Deborah Plana, Adam C. Palmer. Additivity predicts the clinical efficacy of most approved combination therapies for advanced cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5718.
Abstract 5855: Spatial transcriptomics deciphers the cellular society of advanced colorectal cancer
Authors: Hashimoto Masahiro , Ozato Yuki , Nakano Yusuke , Abe Tadashi , Hisamastu Yuichi , Toshima Takeo , Yonemura Yusuke , Uemura Mamoru , Masuda Takaaki , Yamamoto Hirofumi , Mori Masaki , Eguchi Hidetoshi , Doki Yuichiro , Mimori Koshi
Published: April 2023

Introduction: In recent years, single-cell RNA sequencing (scRNA-seq) and high-dimensional spatial transcriptomics (ST-seq) have contributed significantly to our understanding of the tumor microenvironment (TME) in many cancers. This is essential for understanding the tumor-stromal crosstalk. Thus, integrating scRNA-seq data with ST-seq data will facilitate understanding intercellular communication in TMEs.

Material and Method: We performed and combined ST-seq in CRC with public scRNA-seq. And, immunohistochemistry (IHC) (HLA-G, CD68, and SPP1) was performed on 20 CRC surgical specimens collected in our hospital. To verify this, we knocked out H2-M3 (HLA-G in humans) in mouse CRC cell lines (MC-38 cells) using the CRISPR/Cas9 system and examined H2-M3 KO tumor volume in xenograft mouse models.

Results: We identified co-localized cells with CRC cells at the invasive front and those in the center based on the spatial distribution estimated by an analytical pipeline, Cell2location. At the invasive front, CRC cells co-localized more frequently with SPP1+ macrophages than other cells. To dissect the molecular machinery and cell types inducing SPP1+ macrophages, we found ligand activity by another pipeline, NicheNet. The prominent signal to SPP1+ macrophages was mediated by several ligand-receptor pairs including HLA-G- immunoglobulin-like transcript (ILT)2/ILT4. Next, we disclosed the effect on CD8+ cells, and SPP1+ macrophages secrete IL-10 suppressing the immune activity of CD8+ T cells. And the ratio of HLA-G-positive cancer cells was predominantly higher in areas with more SPP1+ macrophages by IHC in CRC. In vivo analysis, H2-M3 KO reduced CRC tumor volume and tumor tissue from H2-M3 KO cells exhibited higher SPP1 and CD68 staining compared with wild type cells.

Conclusion: SPP1+ macrophages and cancer cells secreting high levels of HLA-G may represent targets to improve the prognosis of patients with CRC. Further analysis is currently underway with a focus on early-stage cancer in carcinoma in adenoma.

Citation Format: Masahiro Hashimoto, Yuki Ozato, Yusuke Nakano, Tadashi Abe, Yuichi Hisamastu, Takeo Toshima, Yusuke Yonemura, Mamoru Uemura, Takaaki Masuda, Hirofumi Yamamoto, Masaki Mori, Hidetoshi Eguchi, Yuichiro Doki, Koshi Mimori. Spatial transcriptomics deciphers the cellular society of advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5855.
Abstract 5879: Defining the cancer Immunogram using multimodal analysis to guide immunotherapy in diffuse large B cell lymphoma
Authors: Mattie Michael D. , Perbost Regis , Turcan Sarah , Danan Corinne , Locke Frederick L. , Neelapu Sattva S. , Miklos David B. , Jacobson Caron A. , Lekakis Lazaros J. , Lin Yi , Ghobadi Armin , Kim Jenny J. , Wang Zixing , Xue Allen , Filosto Simone , Scholler Nathalie , Galon Jerome
Published: April 2023

(a) Expansion of therapeutic strategies against diffuse large B-cell lymphoma (DLBCL) offers new opportunities to fight aggressive non-Hodgkin's lymphoma. Therapy approaches, such as R-CHOP and stem cell transplant, and CAR-T cell therapies are now available for the treatment of DLBCL. While the role of the tumor microenvironment (TME) during the therapeutic response is now recognized, the spatial organization and the communication between the diverse factors that compose the TME remains to be fully understood. In this study, we describe a new immunogram tool to help clinical researchers measure the tumor microenvironment contribution to drug efficacy or toxicity in DLBCL context.

(b) In a previous work, using a high-quality testing and innovative multimodal and integrative approach, we constructed the first Veracyte Biopharma Atlas for DLBCL lesions. Based on this knowledge and the understanding of the immune contexture of tumors, we constructed an immunogram using the scoring of clinically relevant biomarkers. Our immunogram scoring approach is composed of key features driving computational derivation of DLBCL Atlas clusters, which include immune gene signatures, immune cell phenotyping combined with spatial information, and genomic and clinical parameters.

(c) Patterns that strongly associated with CAR-T treatment response were determined. In addition, promising results have been obtained in the prediction to CAR-T toxicity related to T-regulatory cells or R-CHOP response with transcriptomic signature.

(d) Retrospective analysis of CAR-T clinical trial data and tumor biopsies highlighted the Veracyte Immunogram as a powerful tool to score the patient probability of response to CAR-T. This work provides the framework for identification of predictors of response to therapy, potential targets of interest or biomarkers that could be applied in future clinical trials.

Citation Format: Michael D. Mattie, Regis Perbost, Sarah Turcan, Corinne Danan, Frederick L. Locke, Sattva S. Neelapu, David B. Miklos, Caron A. Jacobson, Lazaros J. Lekakis, Yi Lin, Armin Ghobadi, Jenny J. Kim, Zixing Wang, Allen Xue, Simone Filosto, Nathalie Scholler, Jerome Galon. Defining the cancer Immunogram using multimodal analysis to guide immunotherapy in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5879.
Abstract 5992: Cells rapidly adapt to CDK2 inhibitors via plasticity of the CDK2/4/6-Rb-E2F axis
Authors: Arora Mansi , Moser Justin , Hoffman Timothy E. , Watts Lotte P. , Min Mingwei , Musteanu Monica , Rong Yao , Schneider Jordan , Ill C Ryland , Nangia Varuna , Sanclemente Manuel , Lapek John , Nguyen Lisa , Niessen Sherry , Dann Stephen , van Arsdale Todd , Barbacid Mariano , Miller Nichol , Spencer Sabrina L.
Published: April 2023

CDK2 is a core cell-cycle enzyme that phosphorylates a variety of substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, application of a novel small molecule CDK2/4/6 inhibitor, PF-06873600, enabled in-depth interrogation of CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. CDK2-specific inhibition leads to rapid loss of substrate phosphorylation, but unexpectedly, CDK2 substrate phosphorylation rebounds within several hours. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, here we demonstrate an early dependence on CDK4/6 activity. CDK2 inhibition reveals latent CDK4/6 activity throughout S phase that backstops the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and Cyclin A2 expression, enabling re-activation of CDK2. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 is required to suppress adaptation to CDK2 inhibitors.

Citation Format: Mansi Arora, Justin Moser, Timothy E. Hoffman, Lotte P. Watts, Mingwei Min, Monica Musteanu, Yao Rong, Jordan Schneider, C Ryland Ill, Varuna Nangia, Manuel Sanclemente, John Lapek, Lisa Nguyen, Sherry Niessen, Stephen Dann, Todd van Arsdale, Mariano Barbacid, Nichol Miller, Sabrina L. Spencer. Cells rapidly adapt to CDK2 inhibitors via plasticity of the CDK2/4/6-Rb-E2F axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5992.
Abstract 6020: The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment
Authors: Omar Khadra K. , Grundy Erin E. , Cox Olivia , Hadley Melissa , Chiappinelli Katherine B.
Published: April 2023

The purpose of this study was to understand the effect of p53 mutation status in ovarian cancer cells on the expression of repetitive elements (REs) following epigenetic treatment. High-grade serous ovarian carcinoma is the most aggressive subtype of ovarian cancer and the 5 year survival rate has remained unchanged for over two decades. Over 90% of high-grade serous ovarian carcinoma (OC) patients have a mutation in the TP53 gene, which encodes the p53 protein. P53 is a transcription factor primarily involved in maintaining genome integrity, but it can also regulate the expression of REs in the genome. REs account for more than half of the genome and these repetitive genomic elements are epigenetically silenced in healthy cells to prevent genome instability. The expression of some REs, such as long interspersed nuclear elements and endogenous retroviruses, are upregulated in human OC patients and cell lines. The mechanism by which p53 regulates expression of REs is not well-understood; the literature documents some instances of p53 activating RE transcription and others of p53 repressing RE transcription, depending on the target elements. We have previously shown that RE expression can be induced by treating OC cells with low doses of DNA methyltransferase inhibitors (DNMTis). Recently, we showed that OC cell lines with mutant p53 have higher baseline expression of REs compared to wild type (WT) cell lines. Treatment of TP53 mutant OC cell lines with DNMTis had a muted effect on RE expression compared to treatment of WT OC cell lines. To explore the relationship between TP53 mutation status and RE expression after DNMTi treatment, OC cell lines with different p53 mutation statuses were treated with 500 nM of the DNMTi 5-azacytidine. We then analyzed the expression of several REs via RT-qPCR and western blot. We hypothesized that TP53 mutant cell lines would have lower DNMTi upregulation of REs than TP53 WT cell lines. While at the RNA level RE expression was variable, at the protein level, we found that DNMTi treatment increased protein levels of several REs in TP53 WT and mutant, but not null, OC cell lines. Future work will examine the feasibility of utilizing these RE proteins as immunotherapy targets in OC.

Citation Format: Khadra K. Omar, Erin E. Grundy, Olivia Cox, Melissa Hadley, Katherine B. Chiappinelli. The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6020.
Abstract 6039: ELOVL5 affects prostate cancer cell proliferation and modulates the AR pathway
Authors: Rodrigues Silvia D. , Ribeiro Caroline , Teixeira Isadora , Pakula Hubert , Fanelli Giuseppe N. , Socciarelli Fabio , Butler Lisa , Swinnen Johannes , Loda Massimo
Published: April 2023

Prostate cancer (PCa) is characterized by alterations in lipid metabolism, exemplified by increased rates of de novo lipogenesis and elongation by fatty acid elongase enzymes known as ELOVLs (elongation of very long chain fatty acids). We have previously shown that the ELOVL family members ELOVL2, 5, and 7 are Androgen Receptor (AR)-regulated proteins; and that ELOVL5 is overexpressed in both primary and castration-resistant prostate cancer. To confirm the role of ELOVL5 activity in prostate carcinogenesis, we generated genetically engineered mouse models (GEMM) with deletion of Elovl5, validated by immunohistochemistry, in combination with the overexpression of c-Myc, an oncogene known to affect lipid metabolism in cancer cells. As expected, we observed alterations in the lipid profile of the murine prostate with the partial deletion of Elovl5, as determined by a significant reduction in levels of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) with long-chain fatty acids (22 carbons) and increased levels of PE with short-chain fatty acids (14 carbons), suggesting an overall shortening on acyl chains of the most common phospholipid species. Interestingly, we also observed that the heterozygous deletion of Elovl5 reduced the expression levels of the AR on the anterior lobe of the murine prostate, in comparison to tissue from Elovl5 wild type mice. Preliminary data also indicates a modulation in prostate volume when Elovl5 is genetically deleted in comparison to tissue from wild type mice. Heterozygous Elovl5 deletion did not affect healthy tissue.To explore biological effects, we generated a 3D organoid line derived from the prostate of mice with homozygous deletion of Elovl5 and overexpression of c-Myc. We found that Elovl5 knock-out decreased organoid diameter and proliferation of c-Myc-driven cells compared to Elovl5 WT. This effect was potentiated when organoids were treated with Enzalutamide, suggesting that targeting Elovl5 may sensitize prostate cells to anti-androgen therapy.Our findings suggest lipid elongation via ELOVL5 as a potential therapeutic target for PCa.

Citation Format: Silvia D. Rodrigues, Caroline Ribeiro, Isadora Teixeira, Hubert Pakula, Giuseppe N. Fanelli, Fabio Socciarelli, Lisa Butler, Johannes Swinnen, Massimo Loda. ELOVL5 affects prostate cancer cell proliferation and modulates the AR pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6039.
Abstract 604: Hybrid quantification of SILAC-barcoded interacting tumor and T cells
Authors: Molero Sofía Ibáñez , Atmopawiro Alisha , Terry Alex , Altelaar Maarten , Peeper Daniel , Stecker Kelly
Published: April 2023

Therapeutic interference of cell-cell communication mechanisms has allowed the clinical development of multiple cancer treatments. For example, the therapeutic intervention of T cell-tumor cell interactions of PD-1 and PD-L1, respectively, by immune checkpoint blockade (ICB) has improved the clinical outcome of cancer patients. However, only a minority of patients are currently benefiting from ICB. Thus, there is an urgent need to improve our understanding of the T cell-tumor cell interplay. Recently developed algorithms serve to deconvolute gene expression of physically interacting cells (PIC). However, such approaches are limited for proteomics (MS) and phosphoproteomics (pMS), which are required to get molecular insight into protein functionality. Here, we present a new method: Hybrid quantification of SILAC-barcoded interacting cells (HySic). Briefly, we use Stable Isotope Labelling by Amino acids in Cell culture (SILAC) to differentiate between two cell types of interest: T cells and tumor cells. We co-cultured the cells allowing to engage in functional interactions and without the need to separate them, and subsequently performed a label-free quantification (LFQ) LC-MS analysis. After initial bioinformatic analysis and validation with independent techniques, we used HySic to unravel protein mechanisms contributing to functional T cell-tumor cell interactions. We used established interaction mechanisms like those mediated by IFNγ signaling proteins to benchmark HySic, while this analysis also brought new protein pathways to light, which will be presented. In conclusion, we have established and validated HySIC as a new method to study protein signaling dynamics in physically interacting cells. Our data shows the effectiveness and applicability of HySIC to study cell-cell interactions, which could be easily extended to other biological systems.

Citation Format: Sofía Ibáñez Molero, Alisha Atmopawiro, Alex Terry, Maarten Altelaar, Daniel Peeper, Kelly Stecker. Hybrid quantification of SILAC-barcoded interacting tumor and T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 604.
Abstract 6049: Exogenous serine promotes cancer stem cells in oral squamous cell carcinoma
Authors: Jankowski Stacy Ann , Hardy Nina C. , Kukuruzinska Maria A.
Published: April 2023

Cancer development and progression is associated with metabolic reprogramming of tumor cells required to meet their proliferative, bioenergetic and survival challenges. Some metabolites, including a non-essential amino acid, serine, have been shown to play a role in tumorigenesis by promoting aggressive cell states, such as cancer stem cells (CSCs). Previous studies from our laboratory showed that the nuclear branch of the Wnt/β-catenin signaling pathway, the β-catenin/CBP/MLL1 axis, enhanced CSC states during oral squamous cell carcinoma (OSCC) evolution through H3K4 trimethylation (H3K4me3) and epigenetic remodeling of the chromatin landscape. Given that metabolism has also been shown to modulate cell plasticity via epigenomic modifications, we have aimed to decode the relationship between serine synthesis and cell identity in OSCC.

We have shown that under serine starvation conditions, OSCC cells upregulate steady-state mRNA and protein levels of serine synthesis pathway enzymes. The latter is associated with an increase in the by-product, alpha-ketoglutarate (αKG), a co-substrate for nuclear αKG-dependent dioxygenases, which demethylate histone marker H3K27me3 and de-repress differentiation-associated genes. Furthermore, increased production of αKG is associated with downregulation of the β-catenin/CBP/MLl1 complex and CSC-associated H3K4me3.

In this study, we used human OSCC cell lines CAL27 and HSC3, derived from a primary tongue tumor and a metastatic site, respectively. Each cell line was cultured in either complete medium or serine starvation medium. Serine starvation conditions led to a downregulation of H3K27me3 along with H3K4me3, promoting a switch from cancer stem cell to differentiation-enhancing chromatin landscape. The observed changes in cell plasticity were further investigated through tumorsphere formation, a surrogate assay for cancer stem cells. We found that OSCC CAL27 and HSC3 cells readily formed tumorspheres replete with stem cell markers BMI1, KRT14 and SOX2, under non-adhesive conditions when grown in complete media, supporting our findings that utilization of exogenous serine enables OSCC cells to maintain stemness. Our findings suggest that a switch from exogenous serine uptake to the endogenous serine biosynthesis promotes OSCC cell differentiation concomitant with the loss of CSC identity.

Citation Format: Stacy Ann Jankowski, Nina C. Hardy, Maria A. Kukuruzinska. Exogenous serine promotes cancer stem cells in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6049.
Abstract 605: Single cell RNA sequencing reveals tumor immune microenvironment and T cell receptor diversity in epidermal growth factor receptor (EGFR) mutated lung cancer
Authors: Chakrabarti Turja , Wu Wei , Kerr Daniel L. , Allegakoen Paul , Bivona Trever G. , Blakely Collin M.
Published: April 2023

Background:

Somatic alterations in the Epidermal Growth Factor Receptor (EGFR) gene represent a common subset of oncogene-driven lung cancer. Patients who receive EGFR-targeted tyrosine kinase inhibitor (TKI) therapy inevitably develop treatment resistance with limited options after disease progression, with immune checkpoint inhibitor therapy (ICI) typically ineffective. Tumor Immune Microenvironment (TME) characteristics, including T-cell receptor (TCR) sequencing and clonality have been incompletely explored in this population.

Methods:

We further analyzed our group’s available data from a previous study on therapy-induced evolution of human lung cancer (Maynard et. al, PMID: 32822576). Tumor samples were sorted and subjected to smartseq-based single-cell RNA sequencing. A total of 7495 cells were derived from 17 samples from 10 patients with tumors that harbored EGFR driver gene mutations. This included 1322 gene marker-annotated T-cells. Samples were grouped into three treatment conditions: treatment naïve (TN), residual disease (RD), and progressive disease (PD). We then used the TraCeR tool to infer the TCR α, β, and the complementarity-determining region 3 (CDR3) sequences.

Results:

T-cells were enriched in RD (55.2%) relative to TN (12.8%) and PD (31.9%) (p<0.0001, Chi-square test) with cytotoxic T-cells as the predominant phenotype across treatment conditions. Increased expression of CTLA4, and LAG3 occurred in PD (p<0.0001, Kruskal-Wallis test). Single-cell TCR analysis revealed increased TCR clonal diversity in PD (Shannon Diversity Index: 0.261 PD > 0.255 TN > 0.205 RD) while increased clonal expansion occurred in RD (32 clones from 4 patients) as compared to TN (17 clones from 1 patient) and PD (14 clones from 1 patient) (p<0.0001, Chi-square test).

Conclusion:

T-cell infiltration is greatest in RD, while immunosuppressive genes were significantly expressed in PD. Increased TCR clonal expansion with less clonal diversity in RD suggests the presence of tumor-specific TCR clones and that this may be the optimal time to initiate ICI treatment. Future directions will include the identification of clonally expanded TCR and matched neoantigens to provide potential tailored immunotherapies for patients with TKI-resistant tumors.

Citation Format: Turja Chakrabarti, Wei Wu, Daniel L. Kerr, Paul Allegakoen, Trever G. Bivona, Collin M. Blakely. Single cell RNA sequencing reveals tumor immune microenvironment and T cell receptor diversity in epidermal growth factor receptor (EGFR) mutated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 605.
Abstract 6134: Exploiting the oncogenic state of colorectal cancer for therapeutic development
Authors: de Sousa e Melo Felipe
Published: April 2023

Over the past two decades, only incremental progresses have been made in the therapeutic management of colorectal cancer (CRC). This is in part attributed to the fact that the Wnt pathway, the oncogenic pathway that initiates the transformation of CRC and sustains tumor maintenance, has been largely undruggable. While most efforts targeting Wnt signaling have revolved around interfering with Wnt target genes that regulate cell growth, we turned our attention to a subset of genes encoding for negative regulators of the pathway. Of particular interest are the cell-surface E3 ubiquitin ligases, ring finger protein 43 (RNF43) and ZNRF3, two known negative regulators of Wnt signaling that promote the turnover of Wnt receptors Frizzled/low-density lipoprotein receptor-related proteins (FZD/LRPs) via ubiquitination-mediated degradation. Given their exposed extracellular domains (ECDs), we set out to explore whether this intrinsic cellular degradative machinery could be repurposed for an antibody-based targeted protein degradation platform. More specifically, we have devised a technology that exploits the elevated expression of ZNRF3 and RNF43 in colon cancer by generating bispecific antibodies that can tether these E3 ubiquitin ligases to a variety of cell surface receptors to promote tumor receptor degradation. Most notably, these antibody degraders are highly active in vivo and can degrade receptors in a tumor specific manner while sparing normal tissues. This platform, that we have been dubbed Proteolysis Targeting Antibodies (PROTABs) describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Citation Format: Felipe de Sousa e Melo. Exploiting the oncogenic state of colorectal cancer for therapeutic development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6134.
Abstract 6155: Lurbinectedin exhibits combinatorial activity with BCL2/BCL2L1 inhibitors in vitro and in vivo by modulation of MCL1 expression
Authors: Vaidya Kedar S. , Gupta Aparna , Hauptschein Robert , Humphreys Robin C.
Published: April 2023

Lurbinectedin (Zepzelca) is a marine-derived anti-tumor agent currently approved for metastatic small cell lung cancer (SCLC). Lurbinectedin has been previously shown to exert its anti-tumor effect by binding to the minor groove of DNA and inhibition of nucleotide excision repair. In addition, lurbinectedin inhibits transcription by inducing degradation of RNA polymerase II. While these activities hint towards a general mechanism, specific molecular targets for lurbinectedin remain obscure. We demonstrate that in vitro, Lurbinectedin specifically induced downregulation of MCL1 protein, a member of the BCL2 family of pro-survival proteins, in a dose- (0-30 nM) and time-dependent manner (0-24 h) by Western analysis. Expression of other pro-survival proteins BCL2 and BCL2L1 were not affected. The downregulation of MCL1 was consistent in all nine tumor cell lines tested regardless of indication. Increased activity or expression of MCL1 is a known compensatory mechanism to offset loss of BCL2 or BCL2L1. We therefore hypothesized that lurbinectedin induced MCL1 loss, along with inhibition of the pro-survival proteins BCL2 and BCL2L1 would lead to an enhancement in cell killing. Consistent with the dominant pro-survival role of BCL2 in SCLC, the combination of lurbinectedin with a specific BCL2 inhibitor (venetoclax) in vitro induced synergistic cytotoxicity (measured by Bliss Sum analysis) in a panel of small cell lung cancer lines. In solid tumor lines other than SCLC, the combination of lurbinectedin with a specific BCL2L1 inhibitor (A-1331852) was also synergistic. Correspondingly in vivo, enhancement in the efficacy of lurbinectedin was observed in combination with venetoclax in SCLC models, while in solid tumor models other than SCLC the combination with a specific BCL2L1 inhibitor enhanced efficacy. Collectively, these data identify MCL1 as a specific lurbinectedin target and suggest that combinations that target other pro-survival proteins may represent a viable strategy to enhance the anti-tumor activity of lurbinectedin.

Citation Format: Kedar S. Vaidya, Aparna Gupta, Robert Hauptschein, Robin C. Humphreys. Lurbinectedin exhibits combinatorial activity with BCL2/BCL2L1 inhibitors in vitro and in vivo by modulation of MCL1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6155.