bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6029: Multi-omic landscape of squamous cell lung cancer
Authors: Stewart Paul , Lui Ashley , Welsh Eric , Ercan Dalia , Rubio Vanessa , Ackerman Hayley , Li Guohui , Fang Bin , Eschrich Steven , Koomen John , Flores Elsa , Haura Eric , DeNicola Gina
Published: April 2023

Patients with squamous cell lung cancer (SCC) have high unmet medical need. Knowledge of these tumors is limited, and a lack of targetable genomic drivers means patients have few treatment options. To provide a detailed analysis on the influence of genomic alterations to proteome-level changes in SCC, we previously integrated DNA copy number, somatic mutations, RNA-sequencing, and expression proteomics in a cohort of 108 SCC patients. A major finding was identification of three proteomic subtypes, two of which made up the majority (87%) of tumors: the “Inflamed” subtype was enriched for B-cell rich tertiary lymphoid structures (TLS), and the “Redox” subtype was enriched for redox pathways and NFE2L2/KEAP1 alterations but had significantly less immune infiltration. We hypothesized these proteomic subtypes would give rise to distinct metabolic signatures. Therefore, we performed untargeted metabolomics on 87 tumors from the same cohort using chromatographic separation on a HILIC column, followed by analysis on a Q Exactive HF mass spectrometer. This analysis yielded 7,344 features corresponding to 7,072 unannotated metabolites and 272 identified metabolites. Glutathione, a key redox metabolite, was anticorrelated with immune score (R = -0.44, padj = 0.004) calculated from our transcriptomic data with the ESTIMATE algorithm, and glutathione was elevated in the Redox proteomic subtype (0.58 log2 ratio, padj = 9.87E-04). Consensus clustering was next used to identify novel metabolomic subtypes of SCC. Surprisingly, none of the five metabolomic subtypes we identified corresponded to proteomic subtype or NFE2L2/KEAP1 alteration (Fisher’s Exact test p-values > 0.05). The fifth subtype had 332 metabolites (26 identified) differentially expressed (> 1.5 fold-change, padj < 0.05) with ascorbate and aldarate metabolism as the top enriched pathway (padj = 3.36E-04). Interestingly, this fifth metabolomic subtype had significantly higher DNp63-alpha (p = 2.40E-05), a primary transcript of delta-N p63 that is known to promote non-small cell lung cancer. Ongoing integrative analyses across omic types will determine how p53, p63, and p73 transcripts influence these metabolomic subtypes, how these transcripts relate to the poor immune infiltration in some SCC tumors, and if these transcripts relate to novel metabolic vulnerabilities in SCC.

Citation Format: Paul Stewart, Ashley Lui, Eric Welsh, Dalia Ercan, Vanessa Rubio, Hayley Ackerman, Guohui Li, Bin Fang, Steven Eschrich, John Koomen, Elsa Flores, Eric Haura, Gina DeNicola. Multi-omic landscape of squamous cell 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 6029.
Abstract 6043: Manipulation of mitochondrial metabolism, through SIRT3 agonists, in a novel immunoreactive melanoma organoids model
Authors: El-derby Azza M. , Wajih Nadeem , Arbiser Jack , Votanopoulos Konstantinous , Soker Shay
Published: April 2023

The interplay between tumor cell metabolism and immune cells in the tumor microenvironment has emerged as a determinant factor in cancer progression and treatment. Sirtuin 3 (SIRT3) is a mitochondrial deacetylase that modulates ROS levels and governs many enzymes that regulate mitochondrial metabolism and homeostasis. SIRT3 has a dual role in cancer; it is regarded as an oncogene and tumor suppressor gene simultaneously. It has tumor-promoting action via keeping ROS levels low and favoring cell proliferation; on the contrary, it can trigger cell death under stress conditions by modulating tumor cell glycolysis. Recent data suggest a role for SIRT3 in cancer immunology with PD-L1 levels are found to be inversely associated with SIRT3 level in cervical cancer, and CD8+ T cells from SIRT3 knockout donor mice exhibited low GVHD in recipients. In this study, we explored the potential role of SIRT3 agonists in sensitizing the immunoreactivity of melanoma cells in a novel immunoreactive melanoma organoids model. We used the B16-F10 melanoma cell line spheroids to determine the effects of SIRT3 agonists on cellular viability, using an ATP assay. Validation of SIRT3 expression was determined via Western blot. Non-toxic amounts of SIRT3 agonists were also applied to melanoma organoids prepared from patient-derived metastatic melanoma from a lymph node. Preliminary results revealed a significant decrease in the viability of the organoids treated with R-propranolol (20µM) and Imipramine blue (0.25µM) agonists. We further investigated the effect of SIRT3 agonists R-propranolol and Imipramine blue on immunotherapy targeting PD-1, Pembrolizumab (100 nM) and Nivolumab (100 nM), and CTLA-4 inhibitor Ipilimumab (100 nM). We found a limited effect of Pembrolizumab and Nivolumab. Anti-tumor cytotoxicity was markedly increased in combination of Imipramine blue with Pembrolizumab and R-propranolol with Nivolumab. We propose that these effects are mediated through the activation of the endogenous immune cells and/or via the regulation of the PD-L1 and NKD2 ligand expression levels in the melanoma cells to become more sensitized to immune cells. Further studies will aim to validate these results in immunoreactive organoids from both mice and human patients. Our studies will shed light on the immune and metabolic axes and the use of specific agonists to develop new immune therapy treatment strategies for melanoma.

Citation Format: Azza M. El-derby, Nadeem Wajih, Jack Arbiser, Konstantinous Votanopoulos, Shay Soker. Manipulation of mitochondrial metabolism, through SIRT3 agonists, in a novel immunoreactive melanoma organoids model. [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 6043.
Abstract 6051: Dissecting unique metabolic vulnerabilities in triple-negative breast cancer metastasis
Authors: Ramchandani Divya , Yoffe Liron , Singh Arshdeep , Vahdat Linda , Mittal Vivek
Published: April 2023

Introduction. Triple-negative breast cancer (TNBC) exhibits the worst outcome due to an aggressive clinical course, high rates of metastasis, and therapeutic resistance. Lack of effective FDA approved anti-metastatic targeted therapies has generated an unmet medical need for developing effective novel treatments. Targeting metastatic seeding and colonization remains a therapeutic challenge. Only recently, attention has been drawn to the idea that cancer cells selectively and dynamically adapt their metabolism along the changing microenvironments of the metastatic cascade. However, the precise nature of the metabolic reprograming in vivo is poorly understood and constitutes a major barrier in formulating a rationally designed therapeutic approach. Using a genetic lineage tracing approach, we identified a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit enhanced invasion and metastasis, increased self-renewal, higher chromosomal instability, and marked resistance to chemotherapy. We posited that this lineage tracing system may allow assessment of precise metabolic alterations as a function of tumor progression directly in vivo.

Methods and data. Transcriptomic analysis of sorted cellular fractions from progressing primary TNBC tumor identified metabolic heterogeneities between metastatic SOX2/OCT4+ and the SOX2/OCT4- counterparts. SOX2/OCT4+ metastatic cells exhibited elevated levels of mitochondrial respiration (OXPHOS) and fatty acid (FA) metabolism; a finding supported by proteomic analysis. Using a combination of CRISPR knockout of key pathway genes, seahorse metabolic flux analysis, metabolomics, and stable isotope tracing, we provide evidence for increased dependence of the metastatic SOX2/OCT4+ population on these unique metabolic adaptions. Importantly, we have identified upstream regulators of these pathways and demonstrate impact of both genetic and pharmacological inhibition of the metabolic regulatory networks on metastasis in vivo.

Conclusion. By identifying metabolic adaptations unique to the metastatic compartment of the primary tumor, we provide evidence for using clinical grade pharmacological inhibitors including metformin, tetrathiomolybdate and FA pathway inhibitors like TVB-2640 and BMS-303141, as an anti-metastatic approach in TNBC. This knowledge of metabolic remodeling could therefore be employed as a next-generation therapeutic approach for high-risk TNBC patients.

Citation Format: Divya Ramchandani, Liron Yoffe, Arshdeep Singh, Linda Vahdat, Vivek Mittal. Dissecting unique metabolic vulnerabilities in triple-negative breast cancer metastasis [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 6051.
Abstract 6053: Sulfation is required for prostate cancer xenograft tumor formation but is dispensable for cell viabilityin vitro
Authors: Chen Ko-Chien , Liu Yonhong , Lin Chenchu , Yen Er-Yen (Nick) , Citron Francesca , Ma Xingdi , Lin Tan , Lorenzi Philip , Muller Florian , DePinho Ronald
Published: April 2023

Sulfation of proteins, carbohydrates, lipids, and xenobiotics is an essential post-translational modification (PTM) process thought to play critical roles in diverse biological processes ranging from detoxification, cell signaling, and extracellular matrix architecture, to immune modulation. Sulfation is accomplished by the universal sulfate donor, PAPS (3'-Phosphoadenosine-5'-phosphosulfate), which is synthesized by bifunctional enzymes PAPSS1 and PAPSS2 (PAPS synthases). The PAPSS2 gene situates near PTEN and is frequently deleted with PTEN across cancer types. Approximately 20% of prostate cancer patients exhibit loss of PTEN, and ~50% of these cases also sustain a loss of PAPSS2. However, the loss of PAPSS2 appears to be tolerated and possibly compensated by its functionally redundant paralogue, PAPSS1, located on chromosome 4q24. The functional redundancy between PAPSS1 and PAPSS2 suggests that these two genes may be collateral lethality pair provided that sulfation is essential for cancer cell viability. Thus, we hypothesize that targeting PAPSS1 in PTEN/PAPSS2-null prostate cancer can generate cancer-specific vulnerabilities while leaving normal cells undisturbed. To assess this possibility, knockdown and knockout of PAPSS1 in cell lines of PAPSS2-null and PAPSS2-wildtype background were generated to characterize cell viability in vitro and tumor formation in vivo. PAPS and APS, an intermediary product of the sulfation pathway, are measured to verify that no alternative pathways for sulfate donors exist and that the co-extinction of PAPSS1/2 eliminates all avenues of generating sulfate donors. Combined extinction of PAPSS1/2 across multiple cancer cell lines was shown to be tolerated in vitro, and recurrent changes in morphology were observed. Loss of sulfation verified by the disappearance of sulfotyrosine and mass spectrometry measurements of PAPS and APS are pending. Our In vitro results surprisingly indicate that a major PTM, like sulfation is entirely dispensable for cancer cell viability under normal culture conditions. However, PAPSS1/2-null cell lines demonstrated a profound delay in tumor formation and prolonged survival, suggesting that sulfation may be required for stromal and innate immune modulation.

Citation Format: Ko-Chien Chen, Yonhong Liu, Chenchu Lin, Er-Yen (Nick) Yen, Francesca Citron, Xingdi Ma, Tan Lin, Philip Lorenzi, Florian Muller, Ronald DePinho. Sulfation is required for prostate cancer xenograft tumor formation but is dispensable for cell viabilityin vitro [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 6053.
Abstract 606: Cis-B7:CD28 interactions at invaginated synaptic membranes activate CD28 and promote T cell function
Authors: Zhao Yunlong , Caron Christine , Chan Ya-Yuan , Lee Calvin K. , Xu Xiaozheng , Bui Jack D. , Hui Enfu
Published: April 2023

Absence of adequate CD28 costimulation is a common feature of dysfunctional T cells in cancer. However, although augmentation of CD28 signaling is well known to be essential for restoring dysfunctional T cell immune responses upon immune checkpoint blockade treatment, it is unclear how CD28 in tumor-infiltrated T cells is activated in the absence of corresponding ligands on cancer. In current dogma, the T cell CD28 can be activated in trans by CD80 (B7-1) or CD86 (B7-2) expressed on professional antigen presenting cells (APCs) enriched in secondary lymphoid organs. Notably, besides professional APCs, B7 ligands are also displayed by T cells, but their functions are unclear. Here we report that B7 ligands expressed on T cells interact with CD28 in cis at membrane invaginations of the immunological synapse, as a result of phosphoinositide-3-kinase (PI3K) mediated membrane remodeling. Cis-B7:CD28 interactions triggered CD28 signaling through protein-kinase-C-theta (PKCθ) and promoted T cell survival, migration and cytokine production. In a B7 deficient tumor model grafted with primed CD8+ T cells, blockade of T cell intrinsic B7:CD28 interactions accelerated tumor growth and decreased intratumoral T cells. Thus, B7 ligands on T cells can evoke cell autonomous CD28 costimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.

Citation Format: Yunlong Zhao, Christine Caron, Ya-Yuan Chan, Calvin K. Lee, Xiaozheng Xu, Jack D. Bui, Enfu Hui. Cis-B7:CD28 interactions at invaginated synaptic membranes activate CD28 and promote T cell function [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 606.
Abstract 61: PRDM16 is key for promoting bone metastatic prostate cancer cell survival during entry into dormancy
Authors: Nasr Mostafa , Li Tao , Bishop Ryan T. , Lynch Conor C.
Published: April 2023

Recurrent metastatic prostate cancer (PC) typically manifests in the skeleton. Despite our knowledge that DTCs give rise to incurable bone metastatic PC, the molecular mechanisms underpinning their entry into dormancy and exit, remain under-investigated. To address this, we developed a novel in vitro model of PC dormancy as determined by increased entry into G0/G1 state (flowcytometry) and increased p38/ERK activity ratio & p27 (immunoblotting). Interestingly, we also observed dormancy entry resulted in the formation of clusters that remained dormant for long periods (21 days) and could be reawakened via serum addition. RNASeq and bioinformatic analysis of control vs. dormant cells revealed PRDM16 to be significantly elevated during entry into dormancy across mouse (RM1) and human cell lines (LAPC4, 22RV1). In vitro, we observed that genetic silencing of PRDM16 leads to a significant reduction in the ability of PC cells to enter dormancy. Analyses show that PRDM16 increases the expression of anti-apoptotic proteins including BCL-2 while inhibiting pro-apoptotic proteins such as BIM and NOXA. Our preliminary data shows that PRDM16 expression in CRPC cells can be induced by BMP7 and conversely decreased by the BMP signaling antagonist, noggin, factors with known critical roles in regulating bone homeostasis. In vivo, we used intrailiac-artery technique to deliver actively growing RM1 cells or dormant RM1 clusters (D-RM1) to the hind limbs (n = 8) of syngeneic mice (C57BL/6). Bioluminescence shows that D-RM1 remains dormant for more than 30 days. Subsequent histological analyses show that D-RM1 home preferentially to the endosteal niche where, compared to RM1 controls, they remain largely negative for ki-67 and cleaved caspase 3 and exhibit high expression of PRDM16. In conclusion, our data points to PRDM16 as a key regulator of survival pathways during PC entry into dormancy.

Citation Format: Mostafa Nasr, Tao Li, Ryan T. Bishop, Conor C. Lynch. PRDM16 is key for promoting bone metastatic prostate cancer cell survival during entry into dormancy [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 61.
Abstract 6101: PUM3 is a novel R-loop regulator and is essential for efficient DNA damage repair
Authors: Lin Chenyu , Ouyang Jian , Wine Robert , Qiu Chen , Williams Jason , Hall Traci M. , Zou Lee , Miles Wayne
Published: April 2023

R-loops have important roles in regulating transcription and genome integrity. To identify novel proteins that function to modulate DNA damage repair, we screened a library of CRISPR-engineered RNA binding protein knock-out cells. From this analysis, we found that a poorly characterized protein, PUM3, is important DNA damage repair and that PUM3-deficient cells were very sensitive the chemotoxic agents. We show that under normal conditions, PUM3 is localized to the nucleolus and nucleus, where it binds to R-loops and modulates transcription. In this setting, PUM3 functions to suppress/resolve R-loops and that PUM3-/- cells have elevated R-loop levels. Following DNA damage, PUM3 is rapidly re-localized from the nucleolus to sites of DNA damage in the nucleus by members of the DDX/DHX family of RNA-helicases. Using small molecule inhibitors and quantitative assays, we find that PUM3 functions in homologous recombination (HR) mediated DNA repair and that PUM3-/- cells have significantly reduced HR-efficiency. PUM3 function at sites of DNA breaks are not affected by transcription or R-loops, implicating PUM3 as functioning after DNA resection during DNA repair. Analysis of TCGA patient data shows that PUM3 levels correlate with Cisplatin resistance and patient outcome. Collectively, our data shows that PUM3 is a previously unknown regulator of R-loops and DNA damage repair that has an important role in chemotherapy sensitivity in cancer patients.

Citation Format: Chenyu Lin, Jian Ouyang, Robert Wine, Chen Qiu, Jason Williams, Traci M. Hall, Lee Zou, Wayne Miles. PUM3 is a novel R-loop regulator and is essential for efficient DNA damage repair [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 6101.
Abstract 6105: Unravelling the mechanistic basis of chromoplexy, a mutational process driving early cancer genome evolution
Authors: Serbyn Nataliia , Smit Myrthe M. , Gummalla Vimathi S. , Brunette Gregory J. , Pellman David S.
Published: April 2023

Genome evolution can happen gradually or via bursts of rearrangements. Chromoplexy is an example of a process driving rapid genome evolution. This mutational signature is detected in ~18% of human cancers (PCAWG Consortium, 2020) and is frequently observed in prostate adenocarcinoma, lymphoid malignancies, and thyroid adenocarcinoma. Chromoplexy is inferred to happen as one catastrophic event that generates copy-neutral chains of translocations involving multiple chromosomes (Baca et al., 2013). Existing studies of chromoplexy monitor the outcome of massive cancer genome reorganization, thus early molecular events leading to catastrophic chromosome rearrangements remain elusive. In this work, we aimed to recapitulate molecular mechanisms underlying chromoplexy. For this, we set out to establish a cell line model and use fluorescence-based reporter systems to enrich for and allow isolation of cells containing signatures of chromoplexy. We additionally address whether colocalization of multiple double-strand breaks, for example in transcription hubs or abnormal nuclear structures, might stimulate chained inter- and intra- chromosomal translocations typical for chromoplexy. If successful, this work will provide a mechanistic understanding of an important mutational process driving rapid genome evolution in cancer, congenital disease, and potentially organismal evolution.

Citation Format: Nataliia Serbyn, Myrthe M. Smit, Vimathi S. Gummalla, Gregory J. Brunette, David S. Pellman. Unravelling the mechanistic basis of chromoplexy, a mutational process driving early cancer genome evolution. [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 6105.
Abstract 6139: Clinical implication of EZH2 inhibitors in hepatocellular carcinoma
Authors: Sohn Bo Hwa , Lee Sung Hwan , Jeong Yun Seong , Lee Ju-Seog
Published: April 2023

Background & Aim: Aberrant upregulation of EZH2 is frequently observed in hepatocellular carcinoma (HCC) and significantly associated with poor prognosis of HCC patients. While EZH2 inhibitors (EZH2i) are recently approved for treatment of lymphoma and sarcoma, their antitumor effect alone is not optimally efficacious against solid tumors like HCC. Ferroptosis is a newly discovered nonapoptotic type of cell death characterized by the iron-dependent accumulation of lipid hydroperoxides in cell membrane to lethal levels. Owing to anti-cancer effects of ferroptosis, many oncogenes are known to suppress ferroptosis. However, their mechanisms in HCC are currently unknown. We aim to uncover a novel mechanism for EZH2-mediated resistance to ferroptosis in HCC.

Methods: Multi-step analyses were performed with genomic data from mouse models (Ezh1/2 KO), cancer cell lines treated with EZH2i GSK126, and primary tumors including HCC to identify genetic networks or signaling/metabolic pathways associated with EZH2 in cancer cells. Functional connection of identified networks or pathways from the prediction analyses was tested in cell line models with various molecular biology approaches including western blots, colony forming assay, overexpression and/or depletion of genes of interest.

Results: Gene network analysis predicted that ferroptosis pathway is inhibited by EZH2. Interestingly, lipid biosynthesis is also identified as an inhibited metabolic pathway by EZH2. Since polyunsaturated fatty acid (PUFA) is essential for executing ferroptosis, we hypothesize that EZH2 suppresses ferroptosis by inhibiting synthesis of PUFA. HCC cells with high EZH2 expression have higher IC50 values to erastin, a ferroptosis inducer, than those with low EZH2 expression. Moreover, inhibition or depletion of EZH2 sensitize HCC cells to erastin-induced ferroptotic cell death, strongly suggesting that EZH2 accounts for resistance to ferroptosis. Mechanistically, EZH2 increases H3K27me3 methylation near PUFA gene promoters, leading to suppression of PUFA gene expression and subsequently low cellular PUFA level. Most interestingly, treatment of EZH2i significantly sensitized HCC cells with high EZH2 expression to erastin-induced ferroptosis, suggesting potential therapeutic opportunity for treatment of HCC patients with high EZH2 expression.

Conclusion: EZH2 is a novel suppressor of ferroptosis by negatively regulating lipid metabolism. Thus, our study provides scientific evidence for developing a novel therapeutic strategy for treatment of HCC patients with co-treatment of ferroptosis inducers and EZH2 inhibitors.

Citation Format: Bo Hwa Sohn, Sung Hwan Lee, Yun Seong Jeong, Ju-Seog Lee. Clinical implication of EZH2 inhibitors in hepatocellular 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 6139.
Abstract 6142: Inhibition of both MAPK and AKT pathways overcomes resistance of NRAS-mutant melanoma stem cells to apoptosis
Authors: Alobaidi Ryyan , Islam Nusrat , Zhang Yanjun R. , Shamo Mathew M. , Allsup Samuel , Simbulan-Rosenthal Cynthia M. , Rosenthal Dean S.
Published: April 2023

Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and characterized by elevated expression of stem cell markers, such as CD133. We have shown that siRNA knockdown of CD133 enhanced apoptosis induced by the MEK inhibitor trametinib in melanoma cells. The current study investigates the underlying mechanisms of CD133's anti-apoptotic activity in patient-derived BAKP melanoma, harboring the difficult-to-treat NRASQ61K driver mutation, after either CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. CD133 knockout in BAKP cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in trametinib-treated cells, coincident with elevated pro-survival p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. Inhibition of MEK with trametinib, in combination with pan-AKT inhibitor capivasertib (AZD5363) in BAKP cells with either CD133 overexpression or knockout in vitro reduced cell survival as measured by XTT, FACS analysis and colony formation assays. Further, in vivo studies with nude mice xenografted with Dox-inducible BAKP melanoma cells, showed significantly decreased tumor growth after xenografted mice were treated with trametinib alone or in combination with AZD5363. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the AKT and MAPK survival pathways with both trametinib and AZD5363 highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma.

Citation Format: Ryyan Alobaidi, Nusrat Islam, Yanjun R. Zhang, Mathew M. Shamo, Samuel Allsup, Cynthia M. Simbulan-Rosenthal, Dean S. Rosenthal. Inhibition of both MAPK and AKT pathways overcomes resistance of NRAS-mutant melanoma stem cells to apoptosis [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 6142.
Abstract 615: Spatially resolved expression of T cell receptors elucidates spatial relationships between T cells, immune infiltration, and cancer-associated pathways
Authors: van Raay Katrina , Kriner Michelle , Reeves Jason , Piazza Erin , Kaplan Hargita , Vivian John , Fernandez Francis , Hoang Margaret , Beechem Joseph
Published: April 2023

Spatial distribution of T cells is key in understanding the escape of tumors from immune surveillance via the adaptive immune response, including interactions between immune cells and the surrounding tumor microenvironment. T cells are critical to the adaptive immune response to pathogens and cancers, mediating an antigen-specific response through both specificity and diversity of T cell receptor (TCR) clonotypes. Many methods exist to determine specific clonotypes and overall TCR diversity present from bulk tissues or sorted cell populations; however, nearly all fail to capture spatial orientation and arrangement of T cells engaging with their microenvironment, and most require large amounts of starting material from precious samples. Here, we present a TCR expression profiling panel for the GeoMx® Digital Spatial Profiler that can be combined with the GeoMx Cancer Transcriptome Atlas (CTA) or Human Whole Transcriptome Atlas (WTA) on archival formalin-fixed paraffin embedded (FFPE) tissue specimens. This represents the first commercial spatial expression profiling assay for the simultaneous quantification of TCR constant, variable, and joining segments in situ.

We show reliable sensitivity and specificity (>90%) with respect to orthogonal sequencing and robust detection of TCR chains with evidence of clonal expansion and CD8 infiltration across tumor regions in colorectal cancer tissue. These events also corresponded to increased signatures of exhaustion from the T cells and suggest that the T cells resident in or near the tumor are tumor-specific and poised for activation via checkpoint blockade. Signaling pathways and tumor-specific signatures were also evaluated to look for mechanisms through which tumor cells respond to T cell infiltration. We further validated the performance of the TCR probe pool in cell pellet arrays with orthogonal TCR sequencing, tonsil and colorectal cancer tissues.

Together, the combination of our TCR add-on panel with the CTA or WTA illuminates T cell phenotypes, signaling pathways, population dynamics, and transcriptomic changes, yielding an unparalleled view of the T cell response in any context.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Citation Format: Katrina van Raay, Michelle Kriner, Jason Reeves, Erin Piazza, Hargita Kaplan, John Vivian, Francis Fernandez, Margaret Hoang, Joseph Beechem. Spatially resolved expression of T cell receptors elucidates spatial relationships between T cells, immune infiltration, and cancer-associated pathways [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 615.
Abstract 6152: Exploiting ferroptosis in mutant BAP1 uveal melanoma
Authors: Cunanan Camille J.
Published: April 2023

Uveal melanoma (UM) arises from melanocytes of the uveal tract in the eye and is the most common intraocular malignancy in adults. Primary UM lesions often harbor mutations in G-proteins, GNAQ or GNA11, and are successfully treated by radiotherapy or enucleation. However, 50% of patients develop metastases. Of these metastatic UM (MUM) patients, 80%develop metastases to the liver, an effect strongly correlated with the loss of function of BAP1,a nuclear deubiquitinase. BAP1-mutant MUMs are less responsive to conventional treatments, such as chemotherapy and immune checkpoint inhibitors, with 50% of these patients having an overall survival of only 1 year. We propose that the unique metabolic intermediates found in liver microenvironment may dictate MUM tropism, aggressiveness, and poor therapeutic response. Here, using a combination of in vitro and ex vivo liver slice cultures, we found that the liver microenvironment induces gene expression alterations consistent with limiting the toxicity of reactive oxygen species (ROS). Importantly, we note that mutant BAP1 UM displayed improved ROS detoxification compared to its wildtype counterparts. Furthermore, publicly available datasets suggest patients with mutant BAP1 UM, the subset of patients with liver metastases, have corresponding ROS detoxification gene expression patterns. These findings suggest that the liver microenvironment and BAP1 loss directs MUM to mitigate ROS-associated toxicity. This compensatory mechanism may be therapeutically exploited and pair well as a combination treatment with the recently approved bispecific agent, Tebentafusp, to selectively target liver MUM.

Citation Format: Camille J. Cunanan. Exploiting ferroptosis in mutant BAP1 uveal 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 6152.
Abstract 6182: Tumor treating fields (TTFields) concomitant with PARP inhibitors or carboplatin for treatment of ovarian cancer cell lines
Authors: Martinez-Conde Antonia , Ene Hila , Frechtel-Gerzi Roni , Dor-On Eyal , Haber Adi , Giladi Moshe , Weinberg Uri , Palti Yoram
Published: April 2023

Purpose/Objectives: Ovarian cancer continues to be the leading cause of death among gynecological malignancies. Platinum-based chemotherapy is recommended after surgery for most patients with ovarian cancer; and PARP inhibitors (PARPi) are suggested as maintenance therapy. Both therapies induce DNA damage that require the proper activity of the Fanconi Anemia (FA)- BRCA pathway for its resolution, and hence are most beneficial in cancers with a BRCA mutation. However, 75% of patients with ovarian cancer do not harbor BRCA mutations, and thus may experience limited benefit from these treatments. TTFields are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cancer cell death. Recently, TTFields have been shown to induce a state of BRCAness in various cancer types, and to be effective in ovarian cancer pre-clinical models. The objective of the current study was to examine the effect of TTFields concomitant with carboplatin or PARPi on ovarian cancer cell lines.

Materials/Methods: A2780 (BRCA wild type) and OVCAR3 (BRCA mutated) ovarian carcinoma cells were treated with TTFields (72 h, 1 V/cm RMS, 200 kHz), alone or with concomitant application of carboplatin or the PARP inhibitors olaparib or niraparib. Efficacy was examined via measurements of cell count, colony formation, and induction of apoptosis. The overall effect was calculated by multiplying cell count with colony formation.

Results: Application of TTFields to A2780 or OVCAR3 cells resulted in reduced cell count, increased overall effect, and elevated apoptosis. Carboplatin, olaparib, and niraparib each displayed dose dependent effects in both cell lines, with higher sensitivity demonstrated in the BRCA mutated cells. Concomitant application of TTFields with any of these drugs displayed a synergistic interaction in the BRCA wild type A2780 cells and an additive effect in the BRCA mutant OVCAR-3 cells.

Conclusions: The data suggest potential benefits for TTFields concomitant with platinum-based chemotherapy and PARPi in ovarian cancer, even in the absence of background BRCA mutations, in accordance with the BRCAness state induced by TTFields. As such, TTFields may enhance the efficacy of treatment for ovarian cancer in both the adjuvant and maintenance settings.

Citation Format: Antonia Martinez-Conde, Hila Ene, Roni Frechtel-Gerzi, Eyal Dor-On, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor treating fields (TTFields) concomitant with PARP inhibitors or carboplatin for treatment of ovarian cancer cell lines. [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 6182.
Abstract 62: Untangling the connection between stress and metastatic breast cancer
Authors: He Xue-Yan , Ng David , Gao Yuan , Michalopoulou Evdokia , George Shanu , Adrover Jose M. , Sun Lijuan , Albrengues Jean , Wan Ledong , Han Xiao , Vakoc Christopher , Van Aelst Linda , Egeblad Mikala
Published: April 2023

There is emerging evidence that exposure to chronic stress is associated with increased risk of metastasis and poor survival in cancer patients. However, the underlying molecular and cellular mechanisms responsible for the effects of stress on tumor progression remain unclear. To determine how stress promotes metastasis, we established new mouse models in which multiple chronic stress exposure promoted breast cancer metastasis to the lungs. We found that chronic stress generates a pro-metastatic lung microenvironment with e.g., reduced T cell infiltration and increased extracellular matrix (ECM) deposition and neutrophil infiltration. Among them, neutrophils were required for these changes as their depletion abolished the pro-metastatic effects of stress and normalized the lung tumor microenvironment. Mechanistically, we analyzed neutrophils from chronically stressed mice and identified dramatically altered transcriptomes, including increased expression of several glucocorticoid receptor regulated genes. Phenotypically, neutrophils from stress exposed mice exhibited increased ROS production, and an increased ability to inhibit T cell activation and to form spontaneous neutrophil extracellular traps (NETs). The stress-associated changes to neutrophils were dependent on glucocorticoid receptor (GR) signaling as neutrophil-specific GR knockout mice showed no increase in NET-formation, a normalized lung microenvironment and no increase in lung metastasis upon stress exposure. Among the changes in the neutrophils caused by stress exposure, we found that the formation of pro-metastatic NETs was critical as directly digesting NETs with DNase I prevented stress-induced metastasis and normalized the lung microenvironment. In sum, this work shows that chronic stress exposure causes a glucocorticoid-mediated increase in NET formation and that NETs are critical drivers in the establishment of a stress-induced, metastasis-promoting microenvironment. Targeting NETs could be an important strategy to prevent metastatic recurrence in cancer patients, many of whom will experience stress due to their cancer diagnosis and subsequent treatments.

Citation Format: Xue-Yan He, David Ng, Yuan Gao, Evdokia Michalopoulou, Shanu George, Jose M. Adrover, Lijuan Sun, Jean Albrengues, Ledong Wan, Xiao Han, Christopher Vakoc, Linda Van Aelst, Mikala Egeblad. Untangling the connection between stress and metastatic 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 62.
Abstract 620: Regulation of anti-tumor immune response by EphA2 receptor tyrosine kinase
Authors: Shi Roger Xiaojun , Lingerak Ryan , Kim So-Yeon , Wang Wei , Sossey-Alaoui Khalid , Wang Bingcheng
Published: April 2023

Eph receptors are the largest family of receptor tyrosine kinases. Overexpression of EphA2 is common across many types of human cancer, particularly in the late stage malignant diseases. We report here that EphA2 knock-out causes delayed tumor growth in several syngeneic mouse caner models, such as breast, colon, lung, and prostate cancer as well as melanoma. However, such effects are missing in immune deficient mice, which suggests the involvement of immune components. Immunophenotyping of the tumors shows elevated T cell infiltration and accompanied by reduced MDSC population in the EphA2 KO tumors. Single cell transcriptomic analysis and cytokine measurements support these results showing increased production of CCL5, CXCL9 and IFNg in the EphA2 KO tumors. Analysis on tumors at very early time points reveals elevated levels of CXCL1 and CXCL2 which recruits suppressive MDSCs. Consistently, larger population of MDSCs was found in PBMCs from the control tumor bearing mice than those bearing EphA2 KO tumors. These results suggest that EphA2 in cancer cells drive the production of CXCL1 and CXCL2 to recruit suppressive MDSCs to the tumor site, thus subvert the host anti-tumor immune surveillance. Targeting the overexpressed EphA2 in cancer can help reshape the tumor micro- environment towards type I anti-tumor immune response.

Citation Format: Roger Xiaojun Shi, Ryan Lingerak, So-Yeon Kim, Wei Wang, Khalid Sossey-Alaoui, Bingcheng Wang. Regulation of anti-tumor immune response by EphA2 receptor tyrosine kinase [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 620.
Abstract 6201: Identification of SP-002, a highly selective USP1 inhibitor effectively inhibits HRD tumor growth and displays low hematotoxicity risk
Authors: Zhou Feng , Li Zhengtao , Zhou Lei , Zhu Wei , Zhang Jiyan , Yang Wenqing , Xue Liting , Qin Xiaokang , Chen Ping , Tang Renhong
Published: April 2023

Tumors with homologous repair deficiencies (HRD) are sensitive to agents interrupting DNA repair. The deubiquitinase USP1 involves in the DNA damage response by translesion synthesis and Fanconi anemia pathway. And the synthetic lethality of USP1 and HRD is well reported. Moreover, deficiency of USP1 or its downstream pathway leads to hypersensitivity of HRD tumors to PARP inhibition. In addition, target-related hematotoxicity is widely observed in serval DNA Damage Response (DDR) pathway inhibitors, including PARP and ATR inhibitors, which limits the combination therapy. Here, we reported SP-002, a highly selective USP1 inhibitor, displayed monotherapy potential and combination activity with PARP inhibitor in HRD cancers. In the biochemical assay, SP-002 strongly inhibited USP1 enzyme with IC50 of 15.7 nM, and potently inhibited proliferation of BRCA1-mutant MDA-MB-436 breast cell (GI50, 47.4 nM). SP-002 demonstrated synergistic anti-proliferation effects in combination of olaparib, a PARP inhibitor, in MDA-MB-436 cells. And further results revealed that the combination significantly enhances the DNA damage burden and cells apoptosis. Moreover, synergistic activities of SP-002 and olaparib also were observed in a panel of HRD cell lines, such as UWB1.289, Capan-1, MDA-MB-453 and NCI-H292. In a human hematopoietic stem cells based in vitro hematotoxicity assay, SP-002 showed no significant inhibition on lineage-specific (myeloid, erythroid, and megakaryocytic) cell differentiation and survival. In comparison, positive controls, 5-fluorouracil and olaparib significantly attenuated those primary cells both on differentiation and survival, which was consistent with the hematological toxicity observed in human. Oral administration of SP-002 led to completely tumor growth inhibition in MDA-MB-436 xenograft model, and combination of SP-002 and olaparib resulted in tumor regression. In conclusion, SP-002, a selective and potent USP1 inhibitor without significant hematotoxicity, that demonstrated excellent efficacy on treatment of HRD cancers.

Citation Format: Feng Zhou, Zhengtao Li, Lei Zhou, Wei Zhu, Jiyan Zhang, Wenqing Yang, Liting Xue, Xiaokang Qin, Ping Chen, Renhong Tang. Identification of SP-002, a highly selective USP1 inhibitor effectively inhibits HRD tumor growth and displays low hematotoxicity risk [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 6201.
Abstract 6204: DNA-PK inhibition augment the cancer specific cytotoxicity of mitotic MTH1 inhibitor OXC-101
Authors: Danda Akhilesh Nagesh , Gad Helge , Scobie Martin , Martínez-López Laura , Carrillo Nuria Pastor , Orta Manuel Luis , Helleday Thomas , berglund Ulrika warpman , Sanjiv Kumar
Published: April 2023

DNA-dependent protein kinase (DNA-PK) plays an important role in the repair of DNA double-strand breaks via the non-homologous end joining (NHEJ) pathway. In addition to its role in the NHEJ pathway, DNA-PK is also known to have an emerging role in cell cycle progression and mitosis. Some studies have shown that depletion or inhibition of DNA-PK inhibitors leads to a delay in the transition from metaphase to anaphase. The mitotic MTH1 inhibitor OXC-101(TH1579, karonudib) is a dual inhibitor that both targets MTH1 and disrupts tubulin polymerization in cancer cells, causing DNA damage and mitotic catastrophe in cancer cells. Based on the involvement of DNA-PK in mitosis, we hypothesized that depletion/inhibition of DNA-PK might enhance the DNA damage and mitotic catastrophic effect of OXC-101.To test our hypothesis, we performed an in vitro drug combination experiment using the DNA-PK inhibitor nedisertib with OXC-101 in non-small cell lung carcinoma NSCLC (H460,A549), uveal melanoma (MP -38, MP -41 and MP46), osteosarcoma (U2OS) and) and neuroblastoma (IMR32, Kelly, SKNFI) cancer cell lines and non-cancerous foreskin fibroblasts(VH-10, BjhTERT). We found cancer-specific synergistic drug interactions between nedisertib and OXC-101. Combined treatment of nedisertib and OXC-101 to H460 and MP46 increased expression of s10H3 (mitotic marker) and the apoptosis marker (cPARP) compared with single treatment. However, there was no additional increase in expression of the DNA damage markerγH2AX with combined treatment. We also performed annexin V staining by FACS on MP38 andMP48 cells and observed a significant increase in the apoptotic population with combination treatment compared with single treatment. Imaging live cells, we observed a significant delay or arrest in the mitotic phase followed by death of cancer cells in mitosis in response to combination treatment, whereas cells treated individually with nedisertib or OXC-101 continued to proliferate and progress to the next phase of the cell cycle. These data suggest that the DNA-PK inhibitor may enhance the cytotoxic effect of OXC-101specifically in cancer cells. In addition, we will conduct an in-depth mechanistic study of how depletion/inhibition of DNA-PK enhances the cytotoxicity of OXC-101 and validate the drug combination in an appropriate preclinical mouse model.

Citation Format: Akhilesh Nagesh Danda, Helge Gad, Martin Scobie, Laura Martínez-López, Nuria Pastor Carrillo, Manuel Luis Orta, Thomas Helleday, Ulrika warpman berglund, Kumar Sanjiv. DNA-PK inhibition augment the cancer specific cytotoxicity of mitotic MTH1 inhibitor OXC-101 [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 6204.