bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3663: Allostatic load, tumor genomics, and outcomes in NSCLC
Authors: Nguyen Ryan H. , Weinfeld Michael , Pasquinelli Mary , Weinberg Frank , Reizine Natalie , Feldman Lawrence , Gadi Vijayakrishna K.
Published: June 2022

Introduction: Allostatic load (AL) is a multi-system measure of chronic stress shown to be an emerging framework for understanding disparities in cancer outcomes, but there have been no AL studies in lung cancer.

Methods: We conducted a retrospective review of patients diagnosed with stage IV lung adenocarcinoma from 2016 - 2020 at the University of Illinois Hospital and Health System. An AL index included 9 biomarkers (systolic blood pressure, diastolic blood pressure, resting heart rate, BMI, albumin, eGFR, creatinine, glucose, and use of medications for hypertension, diabetes, or hypocholesteremia) with 1 point assigned for each biomarker meeting a pre-determined cutoff. AL was dichotomized between AL-high (5-9) and AL-low (0-4) with median progression-free survival of first-line therapy (mPFS1) and median overall survival (mOS) estimated for both groups with Kaplan-Meier curves. Rates of clinically actionable driver mutations (driver-POS), including EGFR, ALK, ROS1, BRAF V600e, RET, MET, NTRK, and KRAS G12C, were compared for both groups using chi-squared test. Mean AL was compared between those who were driver-POS and those who did not have a clinically actionable driver mutation (driver-NEG) using independent samples t-test.

Results: A total of 61 patients were identified with 56% female and 74% former or current tobacco users. The cohort was 49% Black, 21% White, and 13% Hispanic or Latino. Mean AL at diagnosis was 3.3 (+/- 1.4). AL-high patients comprised 20% (12) of the cohort compared with 80% (49) AL-low. Between AL-high and AL-low cohorts, there was no statistically significant difference in mPFS1 (15 vs. 13 months, P = 0.98) and mOS (34 vs. 18 months, P = 0.33). Rates of driver-POS were lower in AL-high compared with AL-low (25% vs 69%, P <0.01). Mean AL was higher in the driver-NEG population compared with driver-POS population (3.79 vs 3.03, P = 0.04).

Conclusions: Patients whose tumors did not have a clinically actionable driver mutation were associated with a higher AL, suggestive of higher chronic stress playing a role in more aggressive tumor biology. No statistically significant survival difference was observed in AL-high versus AL-low populations for mPFS1 and mOS. Larger, prospective trials are planned to correlate AL at diagnosis with clinical and biomarker data. AL, which serves as a measure of physiological dysfunction due to chronic stress associated with social disadvantage, may have implications for tumor biology and clinical outcomes in lung cancer.

Citation Format: Ryan H. Nguyen, Michael Weinfeld, Mary Pasquinelli, Frank Weinberg, Natalie Reizine, Lawrence Feldman, Vijayakrishna K. Gadi. Allostatic load, tumor genomics, and outcomes in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3663.
Abstract 3694: The role of the p40 family in pancreatic cancer
Authors: Sheinin Monica
Published: June 2022

Pancreatic cancer is the fourth leading cause of cancer in the US, and mortality is rising rapidly with little improvement in survival rates. Treatment remains a major challenge of this disease and these poor outcomes have researchers targeting the tumor microenvironment for therapy. The diagnosis and therapeutic options remain an obstacle due to the immune system response and associated non- specific symptoms. Immunotherapy is an important treatment for pancreatic cancer, and its applications are currently being studied. Targeting T cells to recognize and kill tumor cells could be a potential strategy. Due to the behavior of pancreatic cancer and the tumor microenvironment, immunosuppressive cells such as myeloid - derived suppressor cells and regulatory T cells limit the efficacy of T cell infiltration. The cytokine interleukin-12 (IL-12) plays an essential role to regulate both innate (natural killer cells) and adaptive (cytokine T-lymphocytes) immunities in cancer therapies. IL-12 is composed of two subunits, p40 and p35. We found that various human pancreatic cancer cells produce greater levels of p40 monomer and p40 homodimer (p402), than IL-12, and IL-23. We further demonstrate that selective neutralization of p40 by p40 mAb (a3-3a), by p402 mAb (d7-12c) stimulated death in pancreatic cancer cells. Our studies aim to identify the roles of p40 and p402 in pancreatic cancer cells and underlie the importance of p40 monomer and p40 homodimer in helping cancer cells escape cell death.

Citation Format: Monica Sheinin. The role of the p40 family in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3694.
Abstract 3720: TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase for the treatment of diffuse large B-cell lymphoma
Authors: Perabo Frank , Chandhasin Chandtip , Yoo Sanghee , Rosario Joselyn Del , Chen Young K. , Filvaroff Ellen , Stafford Jeffrey A. , Quake Stephen , Clarke Michael F.
Published: June 2022

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL) in the US accounting for about 22% of newly diagnosed cases of B-cell NHL. It is considered to be more aggressive and to have worse prognosis due to low response to existing treatments. KDM4 is a family of histone demethylases that can drive tumor growth by regulating transcription, cell cycle, and DNA replication/repair. Overexpression of KDM4 alters post-translational histone modification and is associated with many types of cancer, including DLBCL. TACH101 is a novel, potent small molecule inhibitor of KDM4 that is being developed for treatment of advanced cancers, including DLBCL.

Methods: TACH101 was evaluated in vitro and in vivo using cancer cell lines and patient-derived organoid (PDO) and xenograft (PDX) models of DLBCL.

Results: TACH101 is a reversible, α-ketoglutarate competitive, selective and potent inhibitor of KDM4 isoforms A-D with IC50 values < 0.100 μM for all four isoforms. An initial screen using a 301-cell line panel showed that DLBCL cell lines are sensitive to TACH101 (IC50 < 10 nM). In an additional panel of DLBCL cell lines, TACH101 inhibited proliferation in a dose-dependent manner in all DLBCL cell lines, independent of molecular subtype, with mean IC50’s of 0.03 ± 0.01 μM in ABC DLBCL cell lines (n=6); 0.02 ± 0.01 μM in GCB DLBCL cell lines (n=7) and 0.02 ± 0.01 μM in PMBL DLBCL cell lines (n=2). In OCI-LY19 DLBCL xenografts in vivo, TACH101 was well tolerated and inhibited tumor growth by 55% to 100%, depending on dosing regimen (either QD or BID following a 3 days on/4 days off schedule). In PK studies, TACH101 exhibited low clearance, moderate volume of distribution, and good oral bioavailability in mouse, rat, and dog. Moreover, treatment with TACH101 resulted in little or no inhibitory effects on CYP enzymes. Toxicity studies in rats and dogs identified potential target tissues and provided safety guidance for the use of TACH101 in human studies.

Conclusions: The KDM4 inhibitor, TACH101, had compelling activity in preclinical DLBCL models, suggesting that TACH101 could be an effective therapy for DLBCL. Preparations to advance the drug into clinical trials are underway.

Citation Format: Frank Perabo, Chandtip Chandhasin, Sanghee Yoo, Joselyn Del Rosario, Young K. Chen, Ellen Filvaroff, Jeffrey A. Stafford, Stephen Quake, Michael F. Clarke. TACH101, a first-in-class inhibitor of KDM4 histone lysine demethylase for the treatment of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3720.
Abstract 3723: Role of KAT5 in non-small cell lung cancer
Authors: Shibahara Daisuke Jefferson , Akanuma Naoki , Heo Eunyoung , Ando Mariko , Kobayashi Ikei S. , Fujii Masanori , Paul Prin , Pan Gilbert , Tenen Danniel G. , Watanabe Hideo , Kobayashi Susumu S.
Published: June 2022

Histone modifications play crucial roles in transcriptional activation, and aberrant epigenetic changes are associated with oncogenesis. Lysine (K) acetyltransferases 5 (KAT5) is reportedly implicated in cancer development and maintenance, although the role of KAT5 in lung cancer largely remain unclear. Here we demonstrate that KAT5 expression was lower in lung adenocarcinoma than normal lung cells. Overexpression of KAT5 had no effect on tumor proliferation and migration, whereas knockdown of KAT5 paradoxically decreased those activities. To assess KAT5 function in lung tumorigenesis in adults, we generated a lung cancer mouse model in which Kat5 is conditionally deleted in lung. We achieved this by crossing lung-specific EGFR-L858R-T790M transgenic mice with conditional Kat5 knockout mice, and found that complete deletion of Kat5 resulted in suppression of tumor formation. To investigate downstream genes of KAT5, we performed RNA sequencing and chromatin immunoprecipitation sequencing analysis using H1975 KAT5 inducible knockdown cells and control cells. We identified a transglutaminase enzyme, TGM5, as one of downstream of KAT5. We furthermore identified artemisinin, an anti-malarial drug, as a potent KAT5 inhibitor by using the connectivity map database, and treatment with artemisinin suppressed tumor growth and induced apoptosis in vitro an in vivo. Taken together, suppression of KAT5 activity shows a tumor-specific efficacy against lung cancer in which KAT5 expression is lower, with no definite influence on normal tissues. Thereby, targeting KAT5 is a potent candidate for the treatment of lung cancer.

Citation Format: Daisuke Jefferson Shibahara, Naoki Akanuma, Eunyoung Heo, Mariko Ando, Ikei S. Kobayashi, Masanori Fujii, Prin Paul, Gilbert Pan, Danniel G. Tenen, Hideo Watanabe, Susumu S. Kobayashi. Role of KAT5 in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3723.
Abstract 3736: SETD2 aberrancy enhanced the synergetic anti-tumor effects of DNA hypomethylating agents and PARP inhibitors in aggressive clear cell renal cell carcinoma
Authors: Zhou Xinyi , Fu Guanghou , Li Hong-Tao , Gujar Hemant , Weisenberger Daniel J. , Quinn David I. , Han Bo , Liang Gangning
Published: June 2022

Purpose: SETD2 aberrancy, as the main reason of depleted levels of H3K36me3, plays an important role in diverse forms of cancer and is most notably in clear cell renal cell carcinoma (ccRCC). Altered chromatin modelers related to impaired H3K36me3 occupancy are linked to renal cancer aggressiveness and metastasis, where the latter dramatically drops the relative 5-year survival rate of ccRCC from 91% to 11%. Developing novel treatment targeting H3K36me3-compromised cancer cell is urgently needed for diagnostic and treatment purposes.

Methods: Phenotypic and animal experiments were conducted to test the sensitivity of kidney cancer cells with impaired H3K36me3 to hypomethylating agent (HMA), PARP inhibitor (PARPi), and HMA + PARPi combination treatments. Comet assay, flow cytometry, western blot and bioinformatic analysis were performed to explore the mechanism beneath.

Results: Combination treatment of 5-Aza-CdR (DAC) and Talazoparib (PARPi) generate more cytotoxicity in SETD2-altered ccRCC cell lines than in SETD2-intact cell lines, including inhibited cell growth, incomplete colony formation, more apoptosis, increased DNA damage and impaired DNA damage repair. The up-regulation of STING and transposable element (TE), as well as the loss of chromatin stability, were more remarkable in SETD2-altered ccRCC cell lines when treated with DAC + PARPi.

Conclusion: Combination treatment of DAC and Talazoparib showed greater synergistic anti-tumor effect in SETD2 aberrant than in SETD2 wild-type ccRCC cells, making it a potential treatment strategy for H3K36me3-compromised cancers. Further pre-clinical studies should be conducted to make a precise medicine-based approach.

Citation Format: Xinyi Zhou, Guanghou Fu, Hong-Tao Li, Hemant Gujar, Daniel J. Weisenberger, David I. Quinn, Bo Han, Gangning Liang. SETD2 aberrancy enhanced the synergetic anti-tumor effects of DNA hypomethylating agents and PARP inhibitors in aggressive clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3736.
Abstract 3754: 5-Hydroxymethylcytosine (5hmC) at or near Pathogenic Variant Hot Spots as Potential Targets for Early Cancer Detection
Authors: Lu Yabin , Lu Michael
Published: June 2022

Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome. In the current study, we demonstrate that discrete 5hmc sites within 80bp hotspot regions exist in a greater proportion of cancer versus normal cells. 5hmc was detected in 16 of 17 known hotspots having C to T or G to A mutations. The results show the presence of two characteristically distinct 5hmc groups: Tier 1 Group with 3 to 8-fold more 5hmcs detected in tumor-cells than in normal-cell derived DNA (as observed in 6 of 11 CpG sites). Tier 2 group with equal allele frequency of 5hmc among normal and tumor-cell derived DNA at 5 CpG hotspot sites as well as 5 non-CpG hotspots. Thus, detection and quantification of the Tier 1 group of 5hmc sites or its prevalence at or near cancer mutation hot spots in cells may enable early detection, screening and potentially prediction of the likelihood of cancer occurrence or the severity of the cancer.

Citation Format: Yabin Lu, Michael Lu. 5-Hydroxymethylcytosine (5hmC) at or near Pathogenic Variant Hot Spots as Potential Targets for Early Cancer Detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3754.
Abstract 3762: Applications of innovative whole-genome bisulfite sequencing (WGBS) for cancer biomarker discovery in plasma liquid biopsy
Authors: Ruiz Caila , Kim Hanjun , Zhang Zhenguo , Bhasin Jeffrey , Jin Mingda , Xu Yi , Yang Xiaojing
Published: June 2022

Circulating cell-free DNA (cfDNA) has been reported to contain valuable genetic and epigenetic information for the diagnosis and prognosis of cancer. Studies have shown that the blood from cancer patients contained more tumor-derived cfDNA compared to the healthy controls. DNA methylation, the most well-studied epigenetic marker, has been validated as one of the key drivers in the development of many diseases including cancer. cfDNA methylation thus holds great potential to become a biomarker that will enable early detection of cancer. However, cfDNA is also a challenging input for library preparation as cfDNA is mostly highly fragmented, and the amount of cfDNA from blood plasma is comparatively low. Therefore, multiple library preparation protocols were evaluated for profiling DNA methylation patterns at single nucleotide resolution from ultra-low amount of blood plasma cfDNA. These methods included two bisulfite conversion-based protocols and an enzymatic based protocol. The bisulfite conversion reaction was optimized allowing a milder treatment for less nucleic acid damage. One of the bisulfite conversion based protocols also contained a further optimized adapter ligation step, allowing a simpler procedure. Circulating cfDNA were extracted from blood plasma of a healthy control, a non-small cell lung cancer patient, and an adenocarcinoma lung cancer patient. 5-ng cfDNA was used as input and the libraries were successfully prepared using all three methods. Each library was sequenced to 300 - 600 million read pairs at a read length of 150 bp, enabling a mean coverage of about 10X per detected CpG, and over 90% of the CpG sites in human genome were detected. Unique alignment rate among all libraries were about or above 80%. The comprehensive coverage of the CpG sites across the entire human genome illustrated that the optimized bisulfite conversion reaction was compatible with fragmented cfDNA, proving the satisfactory efficacy in this simple and classic method for DNA methylation profiling of such a challenging sample type. This opens the door to apply the streamlined and simple Whole-Genome Bisulfite Sequencing (WGBS) protocol for cfDNA methylation profiling, which in turn shall facilitate further understanding in cfDNA epigenetic variations and advance the development of a cancer biomarker from the non-invasive, liquid biopsies.

Citation Format: Caila Ruiz, Hanjun Kim, Zhenguo Zhang, Jeffrey Bhasin, Mingda Jin, Yi Xu, Xiaojing Yang. Applications of innovative whole-genome bisulfite sequencing (WGBS) for cancer biomarker discovery in plasma liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3762.
Abstract 3766: Correlating locus-specific changes in histone trimethylation and gene expression in hypoxia
Authors: Kindrick Jessica D. , Ratcliffe Peter J. , Figg WIlliam Doug , Mole David R.
Published: June 2022

Low oxygen availability (hypoxia) is a common feature of many solid tumors and is associated with poor prognosis and resistance to therapy. The main regulator of cellular responses to hypoxia is the transcription factor HIF (hypoxia-inducible factor). In addition, several histone lysine demethylases (KDMs) are 2-OG dependent dioxygenases and require oxygen for their activity. When oxygen availability is limited, the activity of these KDMs is inhibited leading to global histone hypermethylation, potentially altering gene expression. However, some KDMs are also known to be direct transcriptional targets of HIF, increasing their expression in hypoxia and counteracting this histone hypermethylation. Furthermore, HIF can facilitate recruitment of histone methyltransferase activity to chromatin in cis, leading to local increases in methylation status. Histone methylation can be associated with both gene activation and gene repression, and it remains unclear how the balance of these widespread changes relates to locus-specific regulation of gene expression. Furthermore, previous attempts to analyze regulation of histone methylation by ChIP-seq in hypoxia have not employed normalization methods that take into account global changes in histone modification. We have performed ChIP-seq analysis of histone trimethylation (H3K4me3, H3K9me3, H3K27me3 and H3K36me3) in PC3 prostate cancer cells incubated in normoxia or (16hrs, 1%) hypoxia. We have then examined the effects of the HIF-transcriptional pathway on these marks using the same cell line following deletion of HIF-1b to block the HIF response. We employ an integrated spike-in with drosophila chromatin to provide a normalization control. We then relate our findings to changes in gene expression using RNA-seq analysis, again normalized to a spike-in control. We observe global increases in all 4 histone trimethylations across the genome in hypoxia. In particular, we observe hypoxic increases in histone trimethylation at all gene loci. However, while basal levels of histone trimethylation correlated with basal transcript levels as previously reported, hypoxic increases in promoter-associated histone H3K4me3 trimethylation bore no relation to either the direction or magnitude of gene regulation in hypoxia. Although, more subtle increases were observed across the gene body at gene loci induced by hypoxia. These findings question the currently accepted model that changes in H3K4me3 instruct changes in transcription in hypoxia and suggest that the overall effect of histone trimethylation is a more subtle balance of competing effects.

Citation Format: Jessica D. Kindrick, Peter J. Ratcliffe, WIlliam Doug Figg, David R. Mole. Correlating locus-specific changes in histone trimethylation and gene expression in hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3766.
Abstract 3768: Epigenetic dysregulation from chromosomal transit in micronuclei
Authors: Agustinus Albert S. , Raviram Ramya , Dameracharla Bhargavi , Luebeck Jens , Stransky Stephanie , Scipioni Lorenzo , Myers Robert M. , Bona Melody Di , Duran Mercedes , Weigelt Britta , Yomtoubian Shira , Toufektchan Eleonore , Mischel Paul S. , Mittal Vivek , Shah Sohrab , Maciejowski John , Gratton Enrico , Ly Peter , Bakhoum Mathieu F. , Landau Dan , Bafna Vineet , Sidoli Simone , David Yael , Bakhoum Samuel F.
Published: June 2022

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers, yet whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei, and subsequent micronuclear envelope rupture profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice as well as cancer and nontransformed cells. Some of the changes to histone PTMs occur due to micronuclear envelope rupture whereas others are inherited from mitotic abnormalities prior to micronucleus formation. Using orthogonal techniques, we show that micronuclei exhibit extensive differences in chromatin accessibility with a strong positional bias between promoters and distal or intergenic regions. Finally, we show that inducing CIN engenders widespread epigenetic dysregulation and that chromosomes which transit in micronuclei experience durable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, in addition to genomic copy number alterations, CIN can serve as a vehicle for epigenetic reprogramming and heterogeneity in cancer.

Citation Format: Albert S. Agustinus, Ramya Raviram, Bhargavi Dameracharla, Jens Luebeck, Stephanie Stransky, Lorenzo Scipioni, Robert M. Myers, Melody Di Bona, Mercedes Duran, Britta Weigelt, Shira Yomtoubian, Eleonore Toufektchan, Paul S. Mischel, Vivek Mittal, Sohrab Shah, John Maciejowski, Enrico Gratton, Peter Ly, Mathieu F. Bakhoum, Dan Landau, Vineet Bafna, Simone Sidoli, Yael David, Samuel F. Bakhoum. Epigenetic dysregulation from chromosomal transit in micronuclei [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3768.
Abstract 3771: Mitochondrial metabolism and racial disparity of bladder cancer
Authors: Reddy Karthik Reddy Kami , Park Jun Hyoung , Bollag Roni J. , Bellman Allison , Terris Martha , Lerner Seth P. , Ballester Leomar Y. , Lotan Yair , Kaipparettu Benny Abraham , Putluri Nagireddy
Published: June 2022

Potential differences and mechanism in metabolism among bladder cancer (BLCA) patients of diverse race or ethnicities remain largely unexplored. Even though the incidence rate of BLCA in African Americans (AA) is nearly half as that of European Americans (EA), but AA have the worst survival. We performed the transcriptomics and metabolomics profiling in ancestry verified patients from AA and EA BLCA and observed mitochondrial complex activities were uniquely enriched in AA tumors compared to EA tumors. In addition, in vitro assay demonstrated differences in mitochondrial complex protein and activity between AA and EA BLCA. We further confirmed the reprogramming of mitochondrial metabolism using in vitro 13C labeled tracers in both AA and EA BLCA. Integration of metabolomics and transcriptomics data reveals the enrichment of mTOR pathway in AA BLCA. Our findings indicate that an elevated mitochondrial oxphos activity through mTOR activation could be a factor for AA BLCA progression and provide the rationale to examine mitochondrial specific inhibitors along with mTOR inhibitors to target BLCA on subset of patients from the AA community.

Citation Format: Karthik Reddy Kami Reddy, Jun Hyoung Park, Roni J. Bollag, Allison Bellman, Martha Terris, Seth P. Lerner, Leomar Y. Ballester, Yair Lotan, Benny Abraham Kaipparettu, Nagireddy Putluri. Mitochondrial metabolism and racial disparity of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3771.
Abstract 379: Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells
Authors: Wu Jianghong , McGinley Shawn , Wang Yuan , Gallagher Peter , Ma Haiching
Published: June 2022

NanoBRET࣪ target engagement (TE) is the first biophysical technique that broadly enables the quantitative determination of protein inhibitor occupancy in live cells, without disruption of cellular membrane integrity. This quantitative capability is achieved in live cells via BRET with an optimized set of cell-permeable tracers, allow the measurement of compound binding to a selected cellular target protein. RAS is a well-known oncogene that is frequently mutated in most lung, pancreatic and colorectal cancers and is associated with poor disease prognosis. Mutated RAS is locked in the activated GTP bound state and facilitates enhanced RAS signaling in cancer cells. While being a desirable target, the absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful. The recent discovery of a unique switch II binding pocket and successful inhibition of the KRAS (G12C) mutant by covalent inhibitors have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Here, we performed NanoBRET࣪ TE cellular assay with RAS inhibitors against transfected RAS and its mutants. Our data demonstrate that NanoBRET࣪ TE cellular assay can measure the apparent affinity of RAS inhibitors by competitive displacement of a NanoBRET࣪ RAS switch I/II pocket tracer, reversibly bound to the LgBiT- and SmBiT- KRAS, KRAS (G12C), KRAS (G12D), KRAS (G12V), KRAS (G13D), KRAS (Q61H), KRAS (Q61L), KRAS (Q61R), or HRAS fusion constructs co-transfected in live HEK293 cells. Z factor analysis indicates the assay is High Throughput Screening (HTS) compatible and reproducible. In addition, we are able to confirm the signaling inhibition of downstream phospho-ERK1/2 by KRAS (G12C)-specific inhibitors AMG 510 (Sotorasib), ARS-1620, MRTX849, and MRTX1257 in MIA PaCa-2 pancreas and SW837 colon cancer cells bearing KRAS (G12C) mutation by Western blot assay. Our results suggest NanoBRET࣪ TE cellular assay can serve as a great tool to facilitate RAS pathway drug discovery against human cancers.

Citation Format: Jianghong Wu, Shawn McGinley, Yuan Wang, Peter Gallagher, Haiching Ma. Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 379.
Abstract 3793: Single-cell transcriptional lineage tracing reveals complexity and plasticity of breast cancer pro-metastatic phenotypes
Authors: Roda Niccolò , Cossa Andrea , Ruscitto Federica , Priami Chiara , Blandano Giada , Dalmasso Alberto , Bertolini Francesco , Pelicci Pier Giuseppe
Published: June 2022

Breast cancer (BC) represents a major clinical hurdle, with metastasis being the most devastating events in patients’ history. Traditional lineage tracing approaches have been widely employed to study the metastatic progression of BC. However, these approaches were limited to the investigation of clonal dynamics and did not provide detailed molecular information underlying the pro-metastatic phenotype. In this work, we exploited a library of degenerated barcodes which were both integrated in the genome and expressed as synthetic transcripts. Coupling this barcoding strategy to scRNA-seq analysis, we tracked the clonal and transcriptional evolution of more than 37,000 MDA-MB-231 (a human triple-negative BC cell line) cells in vivo. Clonal distribution analysis revealed that BC clones display proliferative heterogeneity in primary tumors, with a small fraction of clones outcompeting all the others. Furthermore, primary breast tumors turned out to be composed of two phenotypically distinct groups of clones: highly proliferative, on one side, and migratory/stressed/stem-like, on the other. Then, we showed that the striking majority of metastatic cells derived from a single clone which was under-represented in its primary tumor. Importantly, these pro-metastatic clones upregulated genes and pathways associated with extracellular matrix (ECM) interaction, migration, and type-I interferon (IFN) response. In this scenario, independent pro-metastatic clones heterogeneously upregulated these pathways, thus suggesting a multi-faceted functional phenotype. Despite this variability, we detected a set of genes whose expression was statistically associated with the pro-metastatic phenotype, and we validated the relevance of these molecular features both at the biological and clinical level. First, we observed a reduced metastatic burden upon the in vivo knock-down of genes involved in ECM interaction (e.g., ANGPTL4, KCNQ1OT1, and ITGB4) and type-I IFN response (e.g., LY6E and IFI6). Second, we derived two compact gene signatures showing robust predictive power of BC patient survival, independently of other known clinical factors. In conclusion, our work revealed previously unappreciated cellular mechanisms of BC metastasization, providing new actionable therapeutic targets and new molecular predictors of BC prognosis.

Citation Format: Niccolò Roda, Andrea Cossa, Federica Ruscitto, Chiara Priami, Giada Blandano, Alberto Dalmasso, Francesco Bertolini, Pier Giuseppe Pelicci. Single-cell transcriptional lineage tracing reveals complexity and plasticity of breast cancer pro-metastatic phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3793.
Abstract 3801: Comprehensive characterization of the biology of inflammatory bowel disease-associated colorectal cancer using the GeoMx® Digital Spatial Profiler (DSP)
Authors: Juncker-Jensen Anna , Chandramohan Lakshmi
Published: June 2022

Inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) significantly increases the risk for development of colorectal cancer (CRC). However, the exact molecular mechanisms that underlie the transition from inflammation to cancer are not fully understood, and a systematic characterization of the immunopathogenesis of IBD-associated CRC is lacking. To address this issue, we will perform a comprehensive and spatial tissue analysis using the Cancer Transcriptome Atlas (CTA) on the NanoString GeoMx® Digital Spatial Profiler (DSP). The CTA panel is designed to profile the global immune response and all aspects of tumor microenvironment biology, including the 18-gene Tumor Inflammation Signature (TIS).

For DSP analysis, a total of 20 FFPE samples (5 colon cancer patients, 5 UC patients, 5 CD patients, and 5 matched normal samples will be spatially profiled for up to 1,800 genes included on the CTA. Selection of regions of interest (ROI) will be guided by H&E staining and fluorescent markers (CD45, PanCK, Syto13), and profiling of tumor and TME regions will be achieved through segmenting by PanCK+/PanCK- followed by collection of indexed oligonucleotides and sequenced on NextSeq 550 Illumina instrument. NGS reads will be processed into digital counts, normalized and mapped back to each ROI, generating a map of transcript activity within each ROI. For all samples crypt and villus regions will be selected for a detailed spatial analysis. Subsequently, using the proprietary multiplex immunofluorescence (mIF) platform MultiOmyx™ we will characterize the protein immune cell landscape in IBD-associated CRC, using a custom immune panel applied to adjacent slides.

With this study we hope to highlight genes and pathways involved in the transition from inflammation to cancer by combining large-scale spatial gene profiling by DSP CTA with a comprehensive spatial protein profiling by MultiOmyx. It is our hope that this will provide some much-needed understanding of the underlying mechanisms leading from an IBD disorder to the development of colon cancer.

Citation Format: Anna Juncker-Jensen, Lakshmi Chandramohan. Comprehensive characterization of the biology of inflammatory bowel disease-associated colorectal cancer using the GeoMx® Digital Spatial Profiler (DSP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3801.
Abstract 3809: Adaptation of prostate cancer cell lines to tumor microenvironment culture conditions induces expression of androgen receptor splice variant 7 and contributes to drug resistance
Authors: Li Yunmin , Wong Albert , Lu Ann , Lim James
Published: June 2022

The tumor microenvironment (TME) has several characteristics that distinguish it from normal tissue, including elevated interstitial fluid pressures and hypoxic environments. Both pressurized and hypoxic culture conditions can influence gene and protein expression of tumor cells which can result in tumor metastasis and drug resistance. Enzalutamide is effective in treating metastatic castration-resistant prostate cancer (mCRPC), but majority of patients eventually develop resistance to enzalutamide. One of the biomarkers of enzalutamide resistance is the expression of androgen receptor splice variant 7 (AR-V7), but the mechanism of how AR-V7 expression is upregulated in prostate cancer is unclear. To investigate the influence of TME culture conditions on prostate cancer cell lines, 22Rv1 and LNCaP cell lines were incubated under pressurized and hypoxic culture conditions (2 PSI + 1%O2) for a minimum of 7 days. We compared cell growth, morphology changes, gene expression profiles and drug response to enzalutamide treatment. The growth of 22Rv1 and LNCaP cells was markedly inhibited under TME culture conditions, accompanied by distinct changes in cell morphology. 22Rv1 and LNCaP cells cultured under TME conditions exhibited increased resistance to enzalutamide treatment. Higher levels of AR-V7 expression were detected in 22Rv1 and LNCaP cell lines maintained for 7 days in TME culture conditions. RNA-seq data identified 700+ genes that were differentially expressed in 22Rv1 and LNCaP cell lines adapted to TME culture conditions. GSEA analysis identified signaling pathways associated with hypoxia, glycolysis and epithelial-mesenchymal transition were upregulated under TME culture conditions, and the pathways of xenobiotic metabolism and oxidative phosphorylation were downregulated by TME culture conditions. In summary, adaptation to TME culture conditions can promote the expression of AR-V7 in prostate cancer cell lines and can contribute to enzalutamide drug resistance. Adaptation of cell lines to TME culture conditions can provide meaningful insights into mechanisms of drug resistance while providing a novel approach to identify targets and biomarkers of drug resistance.

Citation Format: Yunmin Li, Albert Wong, Ann Lu, James Lim. Adaptation of prostate cancer cell lines to tumor microenvironment culture conditions induces expression of androgen receptor splice variant 7 and contributes to drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3809.
Abstract 3833: The role of chemotherapy-induced fibrosis in the maintenance of tumor initiating cells
Authors: Lujano-Olazaba Omar , Robinson Mikella , Gilbert Samuel F. , Kogan Emily , House Carrie D.
Published: June 2022

Around 80 percent of high-grade serous ovarian cancer patients will relapse with chemotherapy resistant disease. Despite extensive research and developments in immunotherapy, there are limited improvements in overall survival for these patients. Recent research has found that the tumor microenvironment (TME) plays a crucial role in tumor initiation, therapy resistance, and tumor relapse. The TME, which consists of immune cells, blood vessels, and an extracellular matrix (ECM) built primarily by fibroblasts, may enhance the survival and growth of tumor-initiating cells (TICs), chemoresistant stem-like cells thought to be responsible for tumor relapse. Cytotoxic chemotherapies have been shown to modify the TME and this modulation may favor the survival of TICs. For example, carboplatin can activate stromal cells such as fibroblasts to secrete an altered ECM profile, including increases in collagen 1a1 and fibronectin production, which can bind specific integrins upregulated in TICs. Using ex-vivo decellularized peritoneum tissues from mice exposed to chemotherapy or vehicle treatment, we observed a 2-fold increase in growth in peritoneums pre-exposed to chemotherapy relative to those pre-exposed to vehicle. We hypothesize that alterations in the ECM following chemotherapy treatment permit TIC adhesion and growth. To investigate this, mouse embryonic fibroblasts (3T3s) were exposed to chemotherapy during ECM production, decellularized, and reseeded with a panel of ovarian cancer cell lines. Our results suggest a significant increase in cell survival in matrices pre-exposed to chemotherapy compared to control matrices pre-exposed to vehicle. Using this model, we are examining the features of cells that preferentially survive on these matrices. In addition to drug resistance, we are using flow cytometry to assess TIC markers, such as CD117, and CD133, and qRT-PCR to measure stemness genes such as SOX2, OCT4, and NANOG. These studies highlight the need for further investigation into the systemic effects of chemotherapies on the TME and how these changes may be fostering cancer cell adhesion, growth, and survival. Understanding these mechanisms will allow us to develop therapies to achieve better clinical outcomes.

Citation Format: Omar Lujano-Olazaba, Mikella Robinson, Samuel F. Gilbert, Emily Kogan, Carrie D. House. The role of chemotherapy-induced fibrosis in the maintenance of tumor initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3833.
Abstract 3841: SMIM11A, a novel protein involved in the mechanism of vasculogenic mimicry formation in vitro
Authors: Mingo Gabriel , Pradenas Javiera , Babbitt Nicole , González Pamela , Bertocchi Cristina , Owen Gareth
Published: June 2022

Introduction: Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite the strong correlation with reduced patient survival, the mechanisms by which a tumor can create this self-generated irrigation system are still not fully understood. The process of VM in vitro can occur in laminin-111-containing Matrigel and requires the PI3K pathway. However, the membrane protein component and signaling pathways involved in this process are unknown.

Methods: In an established in vitro model of VM of ovarian and breast cancer cells (HEY and MDA-MB-231, respectively) on Matrigel coating, we utilized gene silencing and blocking antibodies to elucidate the signaling pathways involved in this process. RNASeq was used to identify novel transcripts and siRNA was utilized to verify the requirement of candidate RNA/protein in tubular formation.

Results: siRNA and antibody blocking of integrin β1, but not β3, prevented VM formation in vitro. Individual silencing of cortactin, TKS5, MMP-2, MMP-9 and MMP-14 affected tubular formation. RNAseq analysis suggested that VM has minimal dependence on de novo transcriptional activity yet reported a strong upregulation of small Integral Membrane Protein 11 (SMIM11A). This result was verified by qPCR and siRNA silencing of SMIM11A prevented VM formation.

Discussion: Laminin 111 may interact with integrin β1, and the consequent activation of the PI3K pathway could potentially remodel cytoskeletal proteins and promote the activity of MMPs. We report for the first time a biological role for SMIM11a. This gene is regulated at the RNA level and is required for the formation of tubular structure in vitro. As VM is strongly associated with poor patient survival, understanding the formation of this alternative irrigation system may deliver new druggable targets.

Citation Format: Gabriel Mingo, Javiera Pradenas, Nicole Babbitt, Pamela González, Cristina Bertocchi, Gareth Owen. SMIM11A, a novel protein involved in the mechanism of vasculogenic mimicry formation in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3841.
Abstract 3844: COL11A1: A driver of tumor progression and immune evasion in head and neck squamous cell carcinoma (HNSCC)
Authors: Saunders Harmony Ivanna , Arnold Levi , Markiewicz Mary , Thomas Sufi M.
Published: June 2022

We previously reported that collagen type XI alpha 1 (COL11A1) is upregulated in head and neck squamous cell carcinoma (HNSCC) and contributes to tumor progression. Here we tested the hypothesis that COL11A1 regulates the expression of immune regulatory markers in HNSCC. Xena Browser was used to analyze 520 and 42 cancer free normal head and neck tissue samples for COL11A1 expression. The Cancer Genome Atlas (TCGA) RNASeq data from 522 HNSCC samples was downloaded and sorted by clinical parameters and RNA expression for 20,000 genes. COL11A1 expression was correlated with tumor stage (T) and clinical stage (I-IV). Data was sorted by COL11A1 high and low based on the mean COL11A1 expression level and run through the CIBERSORT analysis tool for prediction of immune cell proportions in the tumor. HNSCC lines with COL11A1 knockdown were assessed for immune regulatory markers at the RNA and protein level. COL11A1 was found to be increased in HNSCC relative to normal tissue (tumor vs. normal p<0.0001). COL11A1 expression is highest in HPV negative HNSCC. Its expression correlates with increased tumor (T1 vs T4 p=0.033) and clinical stage (stage III vs. stage IV p=0.023). CIBERSORT analysis demonstrates COL11A1hi patients have significantly decreased active natural killer (NK) cells (active vs. inactive p=0.0166), CD8+ T cells (CD8 low vs. high p<0.0001) and active dendritic cells (active vs. inactive p=0.0035) suggesting immune modulation. COL11A1 targeted lines in vitro show decreased expression of hla-a, -c and cd274 (PD-L1), suggesting COL11A1 expression modulates immune activation by regulating immune suppressive ligands on the tumor cell. COL11A1 is overexpressed in HNSCC and drives tumor proliferation, migration and invasion. Expression levels correlate with tumor and clinical stage. Bioinformatics tool CIBERSORT, predicts decreased immune cell activity in patients with high COL11A1. Targeting COL11A1 in vitro decreased expression of immune suppressive ligands. These preliminary results indicate COL11A1 may drive immune evasion in HPV negative HNSCC. Targeting COL11A1 may improve patient response to immunotherapeutic targets in HNSCC.

Citation Format: Harmony Ivanna Saunders, Levi Arnold, Mary Markiewicz, Sufi M. Thomas. COL11A1: A driver of tumor progression and immune evasion in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3844.
Abstract 3846: Parkin regulates tumor evasion by controlling antigen processing and presentation through the PTEN/AKT network
Authors: Linares Renzo Perales , Mohei Hesham , Leli Nektaria M. , Beghi Silvia , Kostopoulos Nektarios , Maity Amit , Koumenis Costantinos , Facciabene Andrea
Published: June 2022

A growing body of evidence supports the role of Parkin as a tumor suppressor involved in a number of cellular processes, including the downregulation of cell cycle and proliferation, migration, invasion, metastasis, mitophagy, and energy metabolic reprogramming. Indeed, epidemiological, genetic, and bioinformatic studies have shown multiple degrees of defects in the PARK2 gene, localized in human chromosome 6q25-27, a region frequently lost in cancers. In the present study, we show a novel role of Parkin in tumor immune surveillance through dysregulation of the PTEN/PI3k/AKT pathway, involving the modulation of antigen presentation machinery and antitumor-specific CD8+ T cell reactivity. We generated murine and human models that resemble the advanced-stage tumors where Parkin deficiencies are mostly found. In the mouse models, we observed a striking increase of tumor aggressiveness in the absence of Parkin that was associated with loss of antigen presentation and a significant decrease in the infiltration of antitumor CD8+ T cells. Importantly, loss of PARK2 also affected the efficacy of a Tumor-Associated Mitochondria Antigens (TAMAs) based vaccine showing the crucial role of Parkin in tumor development and in the context of antitumor cellular therapeutic strategies. Moreover, we also found that the lack of Parkin in the B16-OVA melanoma model also negatively impacts cytosol-derived antigen presentation and CD8+ T cell immunity. Finally, the analysis of TCGA tumor atlas validates the relevance of PARK2 loss on cancer immunotherapy effectiveness. Thus, during the progression and immunotherapy of cancer, the presence of Parkin mutations that result in loss of its functionality impact antigen presentation and facilitate tumor evasion.

Citation Format: Renzo Perales Linares, Hesham Mohei, Nektaria M. Leli, Silvia Beghi, Nektarios Kostopoulos, Amit Maity, Costantinos Koumenis, Andrea Facciabene. Parkin regulates tumor evasion by controlling antigen processing and presentation through the PTEN/AKT network [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3846.