bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6807: Cancer suppressive mechanism of IL-6 secreted by cancer-related fibroblasts in biliary tract cancer
Authors: Kimura Kenjiro , Tanaka Ryota , Eguchi Simpei , Hatta Kosuke , Kinoshita Masahiko , Tauchi Jun , Shinkawa HIroji , Nishio Kohei , Ohira Go , Ishizawa Takeaki
Published: March 2024

Background and aims: Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment and can exhibit cancer-promoting functions; however, recent studies indicate that CAFs can also inhibit cancer growth and progression. In this study, we identified CAFs that act in an inhibitory manner in biliary tract cancer and elucidated their mechanisms.

Methods: Three biliary tract cancer cell lines and CAFs isolated from resected specimens from four types of biliary tract cancer were used to evaluate the proliferative potential of cancer cells. Potential suppressors that inhibited the growth of cancer cells were identified using protein arrays from CAFs. We determined the effects of these suppressors on cancer cells.

Results: Among the biliary tract cancer CAFs, we identified CAFs that inhibited cancer growth. Protein arrays of conditioned medium from CAFs revealed that Interleukin-6 (IL-6) was one such growth inhibitor. Treatment of biliary tract cancer cell lines with IL-6 significantly reduced their proliferative potential. SOCS3 expression levels increased and STAT3 expression level decreased, suggesting that SOCS3, a downstream signal of IL-6, may be involved.

Conclusion: Inhibitory CAFs are present among biliary tract cancer CAFs and IL-6 production by CAFs may inhibit the growth of biliary tract cancer cells.

Citation Format: Kenjiro Kimura, Ryota Tanaka, Simpei Eguchi, Kosuke Hatta, Masahiko Kinoshita, Jun Tauchi, HIroji Shinkawa, Kohei Nishio, Go Ohira, Takeaki Ishizawa. Cancer suppressive mechanism of IL-6 secreted by cancer-related fibroblasts in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6807.
Abstract 6837: Prolonged effects of hypoxia in luminal breast cancer cells
Authors: Moriarty Aidan , Iriondo Oihana , Amzaleg Yonatan , Klotz Remi , Yu Min
Published: March 2024

Metastasis is the main cause of tumor-related mortality in breast cancer patients. Improving our understanding of the metastatic cascade is imperative to improve patient outcomes. Prior research indicates cancer cells with increased metastatic potential have acquired adaptive properties induced by factors in the tumor microenvironment (TME), including hypoxia. However, previous hypoxia research has primarily focused on signaling changes that occur when cancer cells are in the hypoxic TME and does not account for long-term changes that may occur once cancer cells re-enter normoxia, such as when they metastasize. Our research in patient-derived circulating tumor cell (CTCs) lines and breast cancer cell lines indicates that certain transcriptional changes are maintained after cells leave hypoxia, and that these post-hypoxic cells maintain a higher metastatic potential compared to cells not exposed to hypoxia. Taking an un-biased approach to define changes in hypoxia-exposed breast cancer cells upon reoxygenation, we performed bulk RNA-Seq on post-hypoxic luminal breast cancer cell lines compared to normoxic controls cultured either in adherent or suspension conditions, the latter to mimic the physiological context of CTCs. We found that a subset of genes that were differentially expressed in hypoxic conditions were maintained as up- or down-regulated after cells were reoxygenated, indicating a “hypoxic memory”. Additionally, a subset of differentially expressed genes were unique to the post-hypoxic cells, demonstrating both prolonged and novel gene expression changes induced by hypoxia exposure. We also performed functional assays and observed that this post-hypoxic state is associated with a more aggressive phenotype. Current research in our lab is on-going to understand the epigenetic changes that may be responsible for this hypoxic memory. Overall, this research will improve our understanding of the prolonged consequences of hypoxia on CTC biology and metastasis.

Citation Format: Aidan Moriarty, Oihana Iriondo, Yonatan Amzaleg, Remi Klotz, Min Yu. Prolonged effects of hypoxia in luminal breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6837.
Abstract 6873: Prognostic impact of squamous cell carcinoma antigen as a mediator of anti-inflammatory macrophage polarization in tumor microenvironment
Authors: Chen Liyun , Shi Victoria , Liu Eric , Yang Jasmine , Singh Abhay , Inkman Matthew , Zhang Jin , Schwarz Julie , Markovina Stephanie
Published: March 2024

Squamous cell carcinoma antigen 1 (SCCA1) is a potential biomarker and prognostic indicator for many epithelial cancers including cervix, head and neck, breast, and lung. Cancer patients with high serum levels of SCCA commonly experience treatment resistance and recurrence. We analyze SCCA expression in correlation with tumor microenvironment from 66 human cervical tumor biopsies and find that high serum SCCA is associated with increased macrophage infiltration in tumors and poor overall survival. In a single cell RNAseq analysis of 8 cervical tumors, patients with high SCCA1 show increased immunosuppressive genes (C1QA, C1QC, APOE) and reduced MHC molecules (HLA-DR, HLA-DQA1) in their tumor associated macrophages (TAMs), corresponding to decreased expression of T-cell cytotoxic-associated genes (IFNG, GZMB, PRF1). Thus, we aim to understand the impact of elevated SCCA1 on macrophages and its role in shaping the immune response. Co-culture of peritoneal macrophages isolated from SCCA1-treated mice with T cells from syngeneic wild-type mice, showed significant suppression in T cell proliferation. Downregulation of antigen presentation molecules was also observed SCCA1-treated macrophages and resulted in impaired ability to induce T cell activation by peptide-pulsed macrophages. Compared with treatment-naïve mice, SCCA1-treated macrophages showed increased anti-inflammatory cytokine CCL22, IL10 and immunosuppression regulators ARG1, PD-L1 expression. Moreover, human monocyte-derived M2 macrophages treated with SCCA1 during the differentiation process demonstrated inhibition toward CD3/28-induced T cell proliferation, accompanied by enhanced expression of cytokines, IL10 and CCL18. We further found that targeting SCCA1 by small interfering RNA knockdown significantly reduced tumor growth and macrophage infiltration, resulting in improved T-cell antitumor immunity. Lastly, analysis of cervical cancer patients with chemoradiation therapy showed that patients with high SCCA are associated with increased exhaustion (LAG3, CTLA4) and decreased antitumor gene signature (GZMB, IFNG) during treatment. In summary, SCCA expression promotes immunosuppressive macrophage polarization and function, contributing to T cell exhaustion phenotype and poor treatment response.

Citation Format: Liyun Chen, Victoria Shi, Eric Liu, Jasmine Yang, Abhay Singh, Matthew Inkman, Jin Zhang, Julie Schwarz, Stephanie Markovina. Prognostic impact of squamous cell carcinoma antigen as a mediator of anti-inflammatory macrophage polarization in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6873.
Abstract 6881: Investigating the role of post-transplant bone marrow immune dysregulation in multiple myeloma
Authors: Wang Julia T. , Fiala Mark , Sato Kazuhito , Fortier Julie , Vij Ravi , Ding Li
Published: March 2024

Multiple myeloma (MM) is a cancer of bone marrow plasma cells (BMPCs), which to date remains incurable. Standard of care for qualifying patients involves high-dose chemotherapy followed by autologous stem cell transplant (ASCT), after which minimal residual disease (MRD) testing is used to assess the degree of tumor clearance. Though strongly associated with better survival, MRD-negativity is an imperfect predictor for patient outcomes and fails to account for the pro-tumorigenic BM dysregulation often found in MM patients. As the post-transplant BM milieu in MM is not well understood, we conducted 3' single-cell RNA sequencing (scRNAseq) of 49 newly diagnosed (NDMM) and 79 day-100 post-ASCT whole-BM aspirates to investigate how immunosuppressive microenvironmental signatures may persist or develop following ASCT to influence patient outcomes. 68 of 79 post-ASCT samples come from uniformly-treated clinical trial enrollees with progression-free survival (PFS) ranging from 10 months to over 7 years, with both MRD- and MRD+ cases at either PFS extreme. We observe that although post-ASCT samples have uniformly lower PC count than NDMM samples, only erythroblasts and B cells appear relatively depleted in NDMM samples to compensate for higher PC proportions. T/NK cells and myeloid lineages, though similar in percentage across samples, exhibit clear subtype bias between the two timepoints. Post-ASCT samples are significantly enriched for GZMH+ CD8+ T and NK cells, HIF1A+ hypoxia-responsive monocytes, and depleted for S100A8/9-high CD14+ monocytes. NDMM samples are also enriched for SPINK2+, CDK6+, and AVP+ neutrophils, which are rare in post-ASCT samples. Notably, higher HIF1A expression in myeloid cells post-ASCT is further associated with longer PFS. Regardless of MRD status, NFKBIA, FOS, and GNLY upregulation and GZMK downregulation in post-ASCT T cells are associated with worse PFS. Conversely, enrichment of CD16+ IFN-responsive monocytes and depletion of ARG1+/FUT4+/CEACAM8+ myeloid-derived suppressor cells (MDSCs) are associated with longer PFS. Beyond such general trends, we observe MRD-dependent PFS-association of several CD8+ T cell subsets. Overall, this study enhances our understanding of how post-transplant BM repopulation and immune interaction may impact treatment response in MM.

Citation Format: Julia T. Wang, Mark Fiala, Kazuhito Sato, Julie Fortier, Ravi Vij, Li Ding. Investigating the role of post-transplant bone marrow immune dysregulation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6881.
Abstract 6896: Biomimetic hydrogels at physiological stiffness reprogram patient derived melanoma cells
Authors: Liu Yi L. , Gleba Justyna , Kopecky Chantal , Pawlush Matthew , Copland John A. , Kilian Kristopher A.
Published: March 2024

Melanoma is a uniquely difficult cancer to treat due to complex tumor heterogeneity and resistance, where upon dissemination, survival drops to less than 17%. Drug resistance has arisen for the current standard of care and combinatorial therapies, expediting the need for new efficacious agents. The lack of physiologically relevant preclinical models has impeded the development of these therapeutics.

Potential melanoma drugs are typically assayed using cell lines on tissue culture plastic, then tested for in vivo efficacy and side effects in animals. This paradigm is inherently unsuitable for the generation of effective therapies as typical cell culture platforms fail to replicate the complex microenvironments known to play critical roles in tumor pathology and rodent models are prohibitively expensive for optimizing novel and combination therapies. Thus, our goal is to synthesize tissue-engineered melanoma models using biomimetic polymers that recapitulate patient tumor characteristics to assess combination and novel therapies that attenuate resistance and disrupt metastatic processes.

In this study, patient melanoma cells, which were unable to recapitulate drug responses on traditional cell culture platforms, were cultured in a controlled spatial and temporal manner on polyacrylamide hydrogels. The hydrogel scaffolds feature stiffnesses spanning that of physiological tissue, with microcontact printed human fibronectin for biomimetic protein presentation to create a microtumor array. Cells cultured on the hydrogel showed a 2.5-fold increase in expression of the cancer stem cell biomarkers, CD271 and ABCB5, which play a key role in melanoma survival and recurrence, compared to cells cultured on plastic or glass substrates. Further study identified BRAF mutant, drug resistant patient cells, regained resistance to standard of care targeted therapies, trametinib and dabrafenib. Through hydrogel microarrays featuring physiological similarities, the technology has enabled the exploration of heterogeneous cell populations with novel and combinatorial therapeutics.

Citation Format: Yi L. Liu, Justyna Gleba, Chantal Kopecky, Matthew Pawlush, John A. Copland, Kristopher A. Kilian. Biomimetic hydrogels at physiological stiffness reprogram patient derived melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6896.
Abstract 6919: Cell line panel with HIBIT-tagged endogenous proteins to accelerate PROTAC drug discovery
Authors: Zhai Yunpeng , Ma Jiayi , Wang Yu , Liang Meng , Peng Yao , Ning Jinying , Hao Feng
Published: March 2024

Small molecular drugs have been an important part of disease treatment, but there are also some problems along with time and intrinsic disadvantages of small molecular drugs. In recent years, the emergence of proteolysis targeting chimeras (PROTACs) is expected to overcome some defeats of traditional small molecules. PROTACs, also known as hetero-bifunctional compounds, are a new kind of therapeutic, that consists of a specific ligand to bind the target protein, a suitable linker, and an E3 ubiquitin ligase substrate. After binding to target proteins, PROTACs recruit E3 ligase and induce degradation of target proteins through the ubiquitin-proteasome system. Therefore, PROTACs are a good choice for some protein targets that have small molecular binding ligands but poor small molecular efficacy, which expands druggable targets. Meanwhile, the protein degradation induced by PROTACs destroys not only the enzymatic but also the non-enzymatic functions of targets, rather than only inhibiting the protein activity, which reduces drug resistance. In this study, in order to detect the degradation of intracellular target proteins by PROTACs, Kyinno has designed and constructed several HiBiT-tagged cell line models for different target proteins and their mutations, including KRA, CDK2, LDHA, and HPK. HiBiT is a small tag with 11 amino acids that don’t influence the expression, folding, and localization of target proteins, and luminesces with a detection buffer. All these cell lines with endogenous protein mutations and HiBiT knock-ins are constructed by CRISPR-Cas9 technology and validated by siRNA and PROTACs. Compared with overexpressed exogenous HiBiT-tagged target proteins, endogenous proteins can more accurately and stably reflect the efficacy of PROTACs. Our cell models can be used for high-throughput screening not only for PROTAC drugs but also for molecular glues, lysosome-targeting chimeras (LYTACs), and antibody-based PROTACs (AbTACs).

Citation Format: Yunpeng Zhai, Jiayi Ma, Yu Wang, Meng Liang, Yao Peng, Jinying Ning, Feng Hao. Cell line panel with HIBIT-tagged endogenous proteins to accelerate PROTAC drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6919.
Abstract 6981: p38-mediated EZH2 phosphorylation at T367 regulates HER2 pathway in triple negative breast cancer
Authors: Gonzalez Maria E. , Eido Ahmad , Kleer Celina G.
Published: March 2024

Background: Triple negative breast carcinomas (TNBC) are highly aggressive tumors with limited effective treatments. Clinical studies showed that targeting low HER2 levels in TNBC with anti-HER2 agents has survival benefit. Enhancer of Zeste Homolog 2 (EZH2) is a lysine methyltransferase component of the Polycomb Repressive Complex2 that regulates differentiation genes in normal and neoplastic cells. EZH2 phosphorylation at T416 was shown to maintain the TNBC phenotype. We have reported that p38-induced EZH2-T367 phosphorylation enhances metastasis and that high pEZH2-T367 expression is significantly associated with human TNBC tumors compared to other breast cancer subtypes. In this study we tested the hypothesis that pEZH2-T367 may regulate the HER2 pathway in TNBC and that inhibition of pEZH2-T367 may upregulate HER2 signaling, with clinical implications.

Methods: MDA-MB-231 (mesenchymal-like TNBC) and CCN6KO cells derived from MMTV-Cre;Ccn6fl/fl (CCN6KO) TNBC mouse model generated in our lab were transduced with vector or EZH2 shRNA knockdown (KD). MDA-MB-231 EZH2 KD cells were rescued with lentivirus containing EZH2-wild-type (WT) or T367A phosphorylation-deficient mutant. Gene and protein expression was analyzed by RNA sequencing, q-RT-PCR, immunoblot and immunohistochemistry. Vector and EZH2-KD rescue MDA-MB-231 cells were treated with anti-HER2 (trastuzumab or pertuzumab 100 ug/ml) followed by Hoestch proliferation assays. TNBC cells were treated with vehicle, EZH2 inhibitor (EPZ-6438, 20uM), p38 inhibitor (p38i, SB202190, 20uM), EPZ+p38i, anti-HER2 (100 ug/ml), and EPZ+p38i+anti-HER2, followed by Hoestch cell proliferation assays.

Results: EZH2-T367A deregulates the expression of a 94-gene network of HER2 pathway genes including EREG, SERPINB5, MERTK, IL6R, SOCS3, and FASCN1 compared to EZH2-WT. EZH2-T367A increased protein levels of HER2, EREG, and PP2AB, a major HER2 regulator and was sufficient to sensitize TNBC cells to the anti-proliferative effects of anti-HER2. Supporting the role of pEZH2-T367, we found that the combination of p38i and EPZ significantly sensitizes TNBC cells to anti-HER2 therapies.

Conclusions: We demonstrate that T367 phosphorylation mediates the effect of EZH2 on HER2 signaling in TNBC. Our data support a role for pEZH2-T367 in maintaining the HER2-negative phenotype in and suggest that blocking this phosphorylation event may render TNBC tumors amenable to anti-HER2 treatment strategies.

Citation Format: Maria E. Gonzalez, Ahmad Eido, Celina G. Kleer. p38-mediated EZH2 phosphorylation at T367 regulates HER2 pathway in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6981.
Abstract 6988: TP53RK regulates DNA replication by CDC7-mediated MCM helicase phosphorylation in colorectal cancer
Authors: Choi Younghee , Park Ji-won , Kang Jun-kyu , Kim Eun-ju , Song Sang-Hyun , Yi Eugene C. , Kim Tae-You
Published: March 2024

Background: We identify TP53 regulating kinase (TP53RK), which showed the highest cell growth inhibition efficiency in six colon cancer cell lines. TP53RK is overexpressed in various cancer types, including multiple myeloma and skin cancer, but the mechanism for its tumorigenesis in colon cancer is unknown. Therefore, this study aims to reveal the tumorigenesis mechanism of TP53RK, which is overexpressed in Colorectal Cancer (CRC), through global and phospho-proteomics.

Method: A total 5 CRC cell lines (HT29, SW480, HCT116, H508 and CaCO2), colon normal fibroblast CRL1459, HCT116 p53 null cell and patient-derived normal organoid were used. Immunoblotting, MTT assay, Colony formation assay, Annexin-V assay and cell cycle analysis were performed for evaluation of TP53RK loss-of-function. With kinase-substrate enrichment analysis with phospho-proteome analysis, MCM2 was found to be a substrate for the kinase regulated by TP53RK.

Results: High-throughput genetic screening approach have been widely applied to the study the gene function and molecular mechanism associated with tumorigenesis. By using genome-wide CRISPR/Cas9 library screening, we identify TP53RK, the first committed negative-selected gene, as a tyrosine kinase in the colorectal cancer(CRC) specific biomarker. Through proteome analysis, we found that depletion of TP53RK can hypo-phosphorylate the DNA helicase complex, MCM2 (minichromosome maintenance protein2). According to previous studies, the phosphorylation of MCM2 is regulated by CDC7 (cell division cycle7), and the abnormal states of CDC7 and MCM2 cause DNA replication disorder. Our results show that TP53RK expression is upregulated in CRC and its depletion results in decreased CDC7 expression and hypo-phosphorylation of the MCM complex. Decreased CDC7 expression due to its instability after TP53RK depletion can lead to DNA replication errors. DNA replication errors can lead cancer cells to apoptosis, suggesting that they may be a strategy for cancer treatment. In summary, we provide TP53RK as a novel prognostic and therapeutic target in CRC.

Conclusion: To recapitulate briefly, our findings suggest that the interaction of TP53RK with CDC7 can regulate CDC7 protein stability and stabilized CDC7 can phosphorylate MCM2 to initiate DNA replication. In other words, because overexpression of TP53RK acts as an oncogene that promotes tumor progression in CRC, depletion of TP53RK can degrade CDC7 and induce colon cancer cells to death or regression.

Citation Format: Younghee Choi, Ji-won Park, Jun-kyu Kang, Eun-ju Kim, Sang-Hyun Song, Eugene C. Yi, Tae-You Kim. TP53RK regulates DNA replication by CDC7-mediated MCM helicase phosphorylation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6988.
Abstract 6997: Elucidating the tumorigenic properties of alveolar epithelial type 1 cells in EGFR-mediated LUAD tumorigenesis
Authors: Neely Aaron M. , Lanz Andrea , Wallace William Dean , Kim Anthony W. , Borok Zea , Marconett Crystal N.
Published: March 2024

Originating from within the distal lung, lung adenocarcinoma (LUAD) is the most common pathologic subtype of non-small cell lung carcinoma (NSCLC) and encompasses an assortment of histologic presentations. Colocalized within the distal alveolar epithelium are two alveolar epithelial cell (AEC) subpopulations: alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cells. We previously demonstrated that signatures unique to each AEC subpopulation were detected in LUAD tumors formed by oncogenic KRASG12D activation in mice and were associated with disparate survival outcomes, transcriptomic patterning, and histologic presentation. Epithelial growth factor receptor (EGFR) mutations are among the most frequent oncogenic drivers in thoracic oncology. Therefore, we set out to determine if EGFR oncogenic activation resulted in differential LUAD presentation and outcomes based on the cell of origin in which it was activated. To activate EGFR oncogenic mutations in AT1 or AT2 cells, we employed triple transgenic mouse models (AT1: Gramd2-CreERT2:EF1-LSL-tTA:TetO-EGFRdel20/T790 and AT2: Sftpc-CreERT2:EF1-LSL-tTA:TetO-EGFRdel20/T790), which allowed for EGFR activation in either Gramd2+ AT1 or Sftpc+ AT2 cells. Longitudinal analysis of hematoxylin and eosin-stained lungs over the course of 20 weeks was performed. Blinded pathology review demonstrated that EGFR oncogenic activation in Gramd2+ AT1 cells resulted in histologically defined LUAD with lepidic properties at early time points and solid adenocarcinoma properties at later time points. This differed from EGFR activation in Sftpc+ AT2 cells, which resulted in pre-carcinogenic atypical adenomatous hyperplasia (AAH) formation that did not progress to adenocarcinoma. While preliminary, our results suggest that cell of origin strongly influences EGFR-mediated lung oncogenesis and concomitant pathology and histologic presentation. This work may advance our understanding while informing patient stratification and treatment strategies for LUAD patients.

Citation Format: Aaron M. Neely, Andrea Lanz, William Dean Wallace, Anthony W. Kim, Zea Borok, Crystal N. Marconett. Elucidating the tumorigenic properties of alveolar epithelial type 1 cells in EGFR-mediated LUAD tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6997.
Abstract 704: Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance
Authors: Beckers Claire , Vasilikos Lazaros , Sanchez-Fernandez Alba , Moor Lorena , Pruschy Martin
Published: March 2024

Tumor cells undergo biological adaptations in order to survive stress conditions, such as hypoxia and nutrient deprivation. These biological adaptations include the activation of hypoxic survival pathways that play an important role in acquiring radioresistance which becomes most challenging with treatment regimens using high doses of radiation/fraction (e.g., hypofractionated radiotherapy). Therefore, inhibiting key regulators within these pathways and thereby exploiting these survival mechanisms, gives rise to new potential drug targets for combined treatment modalities. This novel strategy investigated in our laboratory relies on the concept of biological cooperation: while ionizing radiation kills primarily well-oxygenated cells, such inhibitors of survival kinases will drive quiescent tumor cells under hypoxia and/or nutrient deprivation either directly into cell death or into re-entry into a proliferative and thereby more ionizing radiation-sensitive state. One of the survival kinases we are investigating in combination with ionizing radiation (IR) is DYRK1B (dual-specificity tyrosine regulated kinase 1B). We demonstrated that the expression of DYRK1B was upregulated under serum-starvation and hypoxia, but not in response to IR. The small molecule DYRK1B inhibitor AZ191 and shRNA-mediated DYRK1B knockdown significantly reduced proliferative activity and clonogenicity of SW620 tumor cells alone and in combination with IR under serum-starved conditions, which correlated with increased ROS levels and DNA damage. Furthermore, AZ191 successfully targeted the hypoxic core of tumor spheroids while IR preferentially targeted normoxic cells in the rim of the spheroids. A combined treatment effect was also observed in patient-derived colorectal carcinoma organoids but not in healthy tissue organoids. This data shows that inhibition of DYRK1B in quiescent tumor cells could drive tumor cells on their own into crisis or into a more radiosensitive cell cycle phase and thus supports our strategy of biological cooperation. In the continuation of this project, we have performed a drug-screen with approx. 3’000 clinically relevant compounds in colorectal tumor cells lines under normoxic and hypoxic conditions in order to explore additional hypoxia-mediated survival pathways contributing to radioresistance. Thereby we aim to identify novel combined treatment modalities to overcome radiotherapy-induced hypoxia and hypoxia-related resistance mechanisms.

Citation Format: Claire Beckers, Lazaros Vasilikos, Alba Sanchez-Fernandez, Lorena Moor, Martin Pruschy. Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 704.
Abstract 7043: Genomic profiling of DLL3-positive and negative CTCs in small cell lung cancer
Authors: Simandi Zoltan , Bui Thai , Ferrarini Alberto , Hepburn Steven , Del Mastro Richard G.
Published: March 2024

BACKGROUND: Delta-like ligand 3 (DLL3), an atypical Notch family protein implicated in neuroendocrine tumorigenesis, has emerged as a promising therapeutic target in small cell lung cancer (SCLC), prompting numerous clinical trials investigating DLL3-targeted treatments. Exploring DLL3 in circulating tumor cells (CTCs) through liquid biopsy, coupled with comprehensive genomic analysis, presents an invaluable and minimally-invasive avenue for monitoring disease progression. This approach holds the potential to tailor personalized treatment strategies, emphasizing the critical need for in-depth investigations into both DLL3-positive and negative cell genomic compositions.

METHODS: This study examined blood collected in CellSave preservative tubes from stage I (n=17) and stage IV (n=12) SCLC patients, as well as healthy donors (n=10). The CELLSEARCH® platform was employed to count EpCAM-enriched CTCs and evaluate DLL3 protein expression. Subsequently, individual CTCs were isolated based on the presence and absence of DLL3 expression using the DEPArray™ PLUS. The isolated DLL3-negative and positive CTCs were subjected to low-pass sequencing using the Ampli1™ LowPass Kit to uncover genomic aberrations.

RESULTS: CTCs were absent in the healthy control group. In stage I patients, CTCs were identified in 5.8% of blood samples, while in stage IV patients, they were present in 50% of samples, with an average CTC count of 148 in stage IV. Among a total of 2074 detected CTCs, 460 were DLL3-positive, accounting for 6-28% of EpCAM-enriched CTCs in individual patients. Analysis of >300 copy number alteration (CNA) profiles of sorted single cells revealed considerable inter-patient and intra-patient heterogeneity, delineating recurrent clonality and specific genomic alterations in DLL3-negative CTCs. In contrast, DLL3-positive cells showed more stochastic CNA profiles, suggestive of apoptosis-related DNA fragmentation.

CONCLUSIONS: Integrating CellSearch, DEPArray, and low-pass sequencing provides a minimally-invasive approach to compare genomic characteristics in DLL3-positive and negative CTCs from SCLC patients. Extension of this approach to a larger patient cohort and employing machine learning algorithms will facilitate identification of patterns within diverse CNA profiles, improving patient stratification and predicting treatment responses for personalized therapeutic strategies in SCLC.

Citation Format: Zoltan Simandi, Thai Bui, Alberto Ferrarini, Steven Hepburn, Richard G. Del Mastro. Genomic profiling of DLL3-positive and negative CTCs in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7043.
Abstract 7062: CIP2A enhances mitochondrial respiratory electron transport chain complexes and induces lung cancer in vivo
Authors: Yang Fu-Ying , Wang Di , liang Li-Jun , Wang Gui-Zhen , Zhou Guang-Biao
Published: March 2024

Identification of oncogenes that can induce cancer in vivo is critical to elucidation of tumorigenesis and development of targeted therapies. Here, we reported for the first time that forced ectopic expression of CIP2A, the cancerous inhibitor of protein phosphatase 2A (PP2A), in lung tissues induced lung adenocarcinoma in mice. CIP2A upregulated the transcription and translation of mitochondrial DNA (mtDNA), and increases the enzymatic activity of mitochondrial electron transport chain (ETC) complex IV and V, thus enhancing the function of mitochondria and promoting the level of oxidative phosphorylation in tumors. CIP2A inhibited the binding of PP2A-B56α to FOXO3a, leading to the enhancement of the phosphorylation level of FOXO3a. The phosphorylated FOXO3a then translocated into the mitochondrion, and acted as a transcription factor to regulate CIP2A-mediated metabolic reprogramming in cancer cells. We found that oxidative phosphorylation inhibitors effectively inhibited CIP2A-induced cell proliferation. In non-small cell lung cancer, CIP2A level in tumor tissues was positively associated with the level of mtDNA. Our results indicated that CIP2A is able to induce lung adenocarcinoma in mice and has a critical role in mitochondrial oxidative phosphorylation, and represents a promising therapeutic target for non-small cell lung cancer.

Competing interests: The authors declare no competing interests.

Citation Format: Fu-Ying Yang, Di Wang, Li-Jun liang, Gui-Zhen Wang, Guang-Biao Zhou. CIP2A enhances mitochondrial respiratory electron transport chain complexes and induces lung cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7062.
Abstract 7064: BRCA 1/2 haploinsufficiency-associated metabolic dysfunction in benign fallopian tube epithelia
Authors: Ravix Jovanka , Paudel Iru , Pulivendala Gauthami , Sowamber Ramlogan , Smith Elizabeth , Schlumbrecht Matthew , Lombard David , George Sophia
Published: March 2024

In 3-17% of women with BRCA1/2 mutations who undergo risk-reducing surgery, pre-neoplastic and neoplastic lesions are found in their fallopian tubes that display differential loss of heterozygosity of BRCA1/2. Precisely how mutations in BRCA1/2 predispose women to develop high grade serous ovarian carcinoma (HGSC) is not completely understood. Although linked to various processes localized in the nucleus, BRCA1/2 may also have essential roles in the cytoplasm, specifically in the regulation of key processes associated with mitochondrial homeostasis and metabolism. Understanding how haploinsufficiency of BRCA1/2 contributes to the potential neoplastic transformation of fallopian tube epithelial cells should reveal new insights into BRCA1/2 function, and ultimately provide novel opportunities for preventative interventions in individuals with this mutation and new treatment opportunities for HGSC associated with BRCA1/2 heterozygosity. We have identified distinct metabolic and mitochondrial deficits in BRCA2 mutation carriers compared to BRCA1 carriers and non-carriers. Fallopian tube epithelial cell lines derived from BRCA2 mutation carriers showed increased levels of mitochondrial respiration and activity followed by BRCA1 mutation carriers and non-carriers. Western blot analysis of mitochondrial proteins regulating glycolysis and oxidative phosphorylation showed differential expression based on mutation status. Transmission electron microscopy of BRCA2 mutation carriers showed increased disrupted mitochondrial networks when compared to wild type, suggesting that mutation status may contribute to increased mitochondrial fission. These observations reveal an entirely new aspect of BRCA1 and BRCA2 biology and further studies will disaggregate the effects of BRCA1 and BRCA2 on mitochondrial function, versus DNA damage repair. Ultimately, this work will provide new insight into the adaptive changes within FT cells which promote neoplastic transformation and inform how high-grade serous carcinoma (HGSC) cells distinctively respond to microenvironmental stimuli.

Citation Format: Jovanka Ravix, Iru Paudel, Gauthami Pulivendala, Ramlogan Sowamber, Elizabeth Smith, Matthew Schlumbrecht, David Lombard, Sophia George. BRCA 1/2 haploinsufficiency-associated metabolic dysfunction in benign fallopian tube epithelia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7064.
Abstract 7070: MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition
Authors: Droll Stephenie , Zhang Benny , Xue Celia , Levine Maxwell , Bao Xiaomin
Published: March 2024

Recent studies have connected dysregulation of H2A.Z deposition to glioblastoma as well as lung, pancreatic, and metastatic breast cancers. The H2AZ1 and H2AZ2 isoforms, collectively known as H2A.Z, are histone variants that regulate gene expression, stem cell identity, and differentiation during development and in homeostatic tissue. Most tumors originate from epithelial tissues. We utilize skin epidermis, an accessible model of epithelial tissue, to understand the regulatory mechanisms controlling proliferation and differentiation. Primary human epidermal keratinocytes retain their abilities to proliferate, differentiate, and stratify in vitro. First, we observed that progenitor keratinocytes express high levels of H2A.Z mRNA and protein, and that H2A.Z levels significantly and progressively decrease during four days of calcium-induced differentiation. Using ChIP-sequencing, we find a significant, genome-wide decrease in H2A.Z chromatin occupancy in differentiated keratinocytes. Therefore, we hypothesized that H2A.Z is critical for progenitor function. Knock down of either H2AZ1 or H2AZ2 significantly impaired keratinocyte proliferation in holoclone assays. Furthermore, H2AZ1 or H2AZ2 knockdown inhibited epidermal stratification in organotypic culture. Canonically, two chromatin remodeling complexes, SRCAP and EP400, deposit H2A.Z into nucleosomes. While both SRCAP and EP400 knockdown diminish keratinocyte proliferation, only SRCAP knockdown decreases H2A.Z deposition in epidermal keratinocytes. Finally, we find that MEK and ERK signaling is required to sustain expression of SRCAP, H2AZ1, and H2AZ2. MEK or ERK inhibition is sufficient to significantly decrease H2A.Z occupancy genome-wide in progenitor keratinocytes.

Citation Format: Stephenie Droll, Benny Zhang, Celia Xue, Maxwell Levine, Xiaomin Bao. MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7070.
Abstract 7080: Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells
Authors: Aceituno Veronica Castro , Cui Tiantian , Feng Haihua , Yang Linlin , Nair Sindhu , Williams Terence M.
Published: March 2024

Background and Purpose: Caveolin-1 (Cav-1), a scaffolding protein implicated in cellular signaling and membrane dynamics, undergoes intricate regulation at multiple levels. Recent evidence has suggested a potential association between oncogenic mutations and alterations in Cav-1 expression. Our study aimed to comprehensively explore how RAS/RAF mutations influence Cav-1 expression in cancer cells, as well as the underlying molecular mechanisms, using tetracycline (tet)-inducible cell lines and cancer cell lines harboring endogenous BRAF or KRAS mutations.

Experimental Design: We assessed the regulation of Cav-1 expression using tet-inducible cell lines with BRAFV600E, KRASG12D, or KRASG12C mutations. We measured the half-life of Cav-1 at protein levels by using cycloheximide. We monitored Cav-1 localization and degradation dynamics by SNAP-based pulse-chase assay. Additionally, we performed protein and gene expression analyses by immunoblotting and qRT-PCR, luciferase reporter assay, and immunofluorescence to dissect the impact of BRAF and KRAS mutations on Cav-1 regulation at various stages of its life cycle.

Results: We found a significant downregulation of Cav-1 at both mRNA and protein levels upon induction of BRAF or KRAS mutations. The use of KRASG12C, BRAFV600E, ERK, and/or MEK inhibitors in tet-inducible cell lines rescued the effects observed upon induction. The rescue effects have also been observed in H23, MiaPaCa-2 and 8505-C cancer cell lines bearing either BRAF or KRAS mutations. Notably, BRAFV600E exhibited a more pronounced reduction in Cav-1 expression than other mutations. We observed shortened half-life of Cav-1 at protein levels after induction of RAS/RAF mutations by using cycloheximide, and SNAP-based pulse-chase. These mutations accelerated lysosomal-mediated degradation of Cav-1 as determined by immunoblotting and immunofluorescence, which was reversed by lysosomal inhibitors.

Conclusions: Our study establishes a novel regulatory paradigm wherein RAS/RAF mutations exert a multi-faceted influence on Cav-1 dynamics in cancer cells. The transcriptional and translational downregulation, coupled with accelerated lysosomal degradation, collectively contributes to the decreased half-life of Cav-1 following the induction of BRAF or KRAS mutations. These findings provide insights into the intricate molecular mechanisms underpinning the dysregulation of Cav-1 in the context of RAS/RAF mutations, offering potential avenues for therapeutic interventions.

Citation Format: Veronica Castro Aceituno, Tiantian Cui, Haihua Feng, Linlin Yang, Sindhu Nair, Terence M. Williams. Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7080.
Abstract 7122: A novel USP1 inhibitor coupled with a novel proteomic response biomarker for precision oncology therapy
Authors: Alchanati Iris , Morley Andrew , Hay-Koren Avital , Fierro Fabrizio , Otonin Galina , Herman Shay , Shtrikman Alon , Katzenelenbogen Yonatan , Arad Gali , Tivon Barr , Pevzner Kirill , Seger Eran
Published: March 2024

Ubiquitin-specific protease 1 (USP1) is a well-characterized deubiquitinating enzyme (DUB) that plays a critical role in DNA damage repair (DDR). It was shown to regulate various key repair processes, most notably proliferating cell nuclear antigen (PCNA) in the Translesion Repair (TLS) pathway and FANCD2/FANCI in the Fanconi Anemia (FA) repair pathway. Research indicates that USP1 deficiency leads to a decrease in cell survival and disruption of genomic stability, suggesting that USP1 inhibitors (USP1i) may hold promise in treating DDR deficient tumors. Through the integration of computational and medicinal chemistry approaches, we discovered a novel, potent inhibitor of USP1, showing anti-USP1 activity in both cell-free enzymatic assay and in measurement of PCNA-ubiquitination change upon USP1 inhibition in cells. We demonstrate anti tumor activity in DDR-related and other indications. By utilizing our platform to analyze the basal proteomics of resistant and sensitive cell lines treated with USP1i, we revealed a distinctive proteomic biomarker signature for USP1i response. This data demonstrates the potential of our novel USP1 inhibitor to serve as a potent therapeutic agent for DDR-deficient and other biomarker-positive cancer patients.

Citation Format: Iris Alchanati, Andrew Morley, Avital Hay-Koren, Fabrizio Fierro, Galina Otonin, Shay Herman, Alon Shtrikman, Yonatan Katzenelenbogen, Gali Arad, Barr Tivon, Kirill Pevzner, Eran Seger. A novel USP1 inhibitor coupled with a novel proteomic response biomarker for precision oncology therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7122.
Abstract 7139: Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer
Authors: Lee Jeong-Won , Lee Kwangho , Koh Byumseok , Lee Yoo-Young , Bae Duk-Soo
Published: March 2024

Ovarian cancer, a leading cause of cancer-related deaths in women, remains a formidable challenge, especially in the context of platinum-resistant disease. This study investigated the potential of the benzimidazole derivative BNZ-111 as a novel treatment strategy for platinum-resistant ovarian cancer. With improved pharmacokinetic parameters, BNZ-111 effectively disrupts tubulin polymerization through hydrogen bonds and hydrophobic interactions. It demonstrated strong cytotoxicity in both chemo-sensitive and chemo-resistant epithelial ovarian cancer cell lines, inducing apoptosis and G2/M cell cycle arrest. In vivo experiments using orthotopic and patient-derived xenograft models showed significant tumor growth inhibition without apparent toxicity to vital organs. Unlike paclitaxel, BNZ-111 proved effective in paclitaxel-resistant cells, potentially by bypassing interaction with MDR1 and modulating β-3 tubulin expression to suppress microtubule dynamics. This study suggests that BNZ-111, with favorable drug-like properties, holds promise as a therapeutic option for platinum-resistant ovarian cancer, addressing a critical clinical need in gynecologic oncology

Citation Format: Jeong-Won Lee, Kwangho Lee, Byumseok Koh, Yoo-Young Lee, Duk-Soo Bae. Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7139.
Abstract 7150: Molecular insights into the oncogenic influence between mutant HER3, mutant KRAS, and their synergistic interplay in colorectal cancer pathogenesis
Authors: Kilroy Mary K. , Park Briley , Mishra Rosalin , Feroz Wasim , Wischmeier Cecilia , Garrett Joan T.
Published: March 2024

Colorectal cancer (CRC) is a disease of the colon and rectum that will claim about 52,550 American lives in 2023. In recent years, more attention has been paid to HER2 and HER3’s roles in colorectal cancers, as they may cause resistance to targeted therapies. We are investigating the role of naturally co-occurring HER3 and KRAS mutations in colorectal cancer, as about 6% of all colorectal cancers contain a HER3 mutation and 41% contain a KRAS mutation. We have observed that there is a statistically significant co-occurrence of HER3 and KRAS mutations in CRCs while there is mutual exclusivity with mutations in EGFR or HER4 and mutant KRAS in CRC examining 6791 samples from 17 studies. There is a tendency for co-occurrence with HER2 and KRAS mutations that is not statistically significant in these CRC tumor samples. We have found that genetic knockdown of both KRAS and HER3 with siRNA targeting KRAS and HER3 in the CRC cell line SNU-407 (HER3V104M, KRASG12D) results in a statistical reduction in cell proliferation in comparison to genetic knockdown with only KRAS or HER3. Using the SW620 CRC cell line (HER3WT, KRASG12V) as a comparison, we have observed that knocking down HER3 using siRNA had no effect on cell proliferation whereas KRAS knockdown reduced proliferation and the combination showed no statistical difference compared to knocking down only KRAS. Additionally, we have found that there is a striking increase in total HER3 levels in SNU-407 cells when treated with the KRASG12D inhibitor MRTX1133. This feedback loop could limit the efficacy of the KRAS inhibitor. We wished to delineate mutant HER3’s binding partner(s) as HER3 is kinase impaired. Strikingly, we have found that EGFR immunoprecipitates with HER3 in SNU-407 cells. We are expanding our studies to CRC patient derived xenografts (PDXs) with co-occurring HER3 and KRAS mutations and have generated patient derived organoids from these PDXs. Ongoing studies are determining if there is a synergistic relationship with KRAS inhibitors and HER family inhibitors including a HER3 antibody-drug conjugate. Our findings may present a new paradigm for targeted combination therapies in colorectal cancer with the eventual goal of increased overall patient survival.

Citation Format: Mary K. Kilroy, Briley Park, Rosalin Mishra, Wasim Feroz, Cecilia Wischmeier, Joan T. Garrett. Molecular insights into the oncogenic influence between mutant HER3, mutant KRAS, and their synergistic interplay in colorectal cancer pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7150.