bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA-Methylome–Based Tumor Hypoxia Classifier Identifies HPV-Negative Head and Neck Cancer Patients at Risk for Locoregional Recurrence after Primary Radiochemotherapy
Authors: Tawk Bouchra , Rein Katrin , Schwager Christian , Knoll Maximilian , Wirkner Ute , Hörner-Rieber Juliane , Liermann Jakob , Kurth Ina , Balermpas Panagiotis , Rödel Claus , Linge Annett , Löck Steffen , Lohaus Fabian , Tinhofer Ingeborg , Krause Mechtild , Stuschke Martin , Grosu Anca Ligia , Zips Daniel , Combs Stephanie E. , Belka Claus , Stenzinger Albrecht , Herold-Mende Christel , Baumann Michael , Schirmacher Peter , Debus Jürgen , Abdollahi Amir
Published: April 2023

Purpose:

Tumor hypoxia is a paradigmatic negative prognosticator of treatment resistance in head and neck squamous cell carcinoma (HNSCC). The lack of robust and reliable hypoxia classifiers limits the adaptation of stratified therapies. We hypothesized that the tumor DNA methylation landscape might indicate epigenetic reprogramming induced by chronic intratumoral hypoxia.

Experimental Design:

A DNA-methylome–based tumor hypoxia classifier (Hypoxia-M) was trained in the TCGA (The Cancer Genome Atlas)-HNSCC cohort based on matched assignments using gene expression–based signatures of hypoxia (Hypoxia-GES). Hypoxia-M was validated in a multicenter DKTK-ROG trial consisting of human papillomavirus (HPV)–negative patients with HNSCC treated with primary radiochemotherapy (RCHT).

Results:

Although hypoxia-GES failed to stratify patients in the DKTK-ROG, Hypoxia-M was independently prognostic for local recurrence (HR, 4.3; P = 0.001) and overall survival (HR, 2.34; P = 0.03) but not distant metastasis after RCHT in both cohorts. Hypoxia-M status was inversely associated with CD8 T-cell infiltration in both cohorts. Hypoxia-M was further prognostic in the TCGA-PanCancer cohort (HR, 1.83; P = 0.04), underscoring the breadth of this classifier for predicting tumor hypoxia status.

Conclusions:

Our findings highlight an unexplored avenue for DNA methylation–based classifiers as biomarkers of tumoral hypoxia for identifying high-risk features in patients with HNSCC tumors.

See related commentary by Heft Neal and Brenner, p. 2954
Abstract CT264: Taking sotorasib with an acidic beverage improves sotorasib exposure for subjects on omeprazole
Authors: Strydom Natasha , Cardona Panli , Spring Marintan , Purkis Jessica , Houk Brett
Published: April 2023

The solubility of KRASG12C inhibitor sotorasib is pH dependent and coadministration with a proton pump inhibitor (omeprazole) decreased sotorasib maximum concentration (Cmax) by 56.9% and the area under the concentration curve from time zero to the time of the last quantifiable concentration (AUClast) by 42.4% under fasted conditions. This study in healthy volunteers evaluated if taking sotorasib with an acidic beverage such as Coca Cola would alter sotorasib absorption when coadministered with omeprazole and improve sotorasib exposure. Two separate phase 1, open-label, fixed sequence, 2-period studies enrolled at least 14 healthy subjects each. In the first study, each subject received a single 960 mg dose of sotorasib under fasted conditions on day 1, followed by omeprazole 40 mg once daily for 5 days on days 4 to 8, and then received omeprazole 40 mg in combination with sotorasib 960 mg on day 9 with water. For the second phase 1 study all conditions were kept the same except subjects received omeprazole in combination with sotorasib on day 9 with an 8-ounce (240 mL) glass of acidic beverage (Coca-Cola) instead of water. In both studies, intensive pharmacokinetic plasma sampling occurred predose and at 13 timepoints up to 48 hours post-dose. Sotorasib plasma concentrations were measured using a validated high-performance liquid chromatography tandem mass spectrometry method. Non-compartmental analysis was used to calculate pharmacokinetic parameters. Safety and tolerability were monitored throughout the study. Increased sotorasib exposure was observed when sotorasib and omeprazole were taken with an acidic beverage compared to when sotorasib and omeprazole were taken with water for both AUClast (23200 vs 16700 h∙ng/mL respectively) and Cmax (4850 ng/mL vs 3110 ng/mL respectively). Compared to the geometric mean ratio of sotorasib and omeprazole taken with water, taking sotorasib and omeprazole with an acidic beverage resulted in an increase of 19.0 percentage-point in AUClast and a 24.6 percentage-point increase in Cmax. Coadministration of sotorasib and omeprazole with Coca Cola was safe and well tolerated. For patients on acid-reducing drugs, sotorasib taken with an acidic beverage is an effective strategy to increase sotorasib exposure and counteract the reduced exposures resulting from the drug-drug interaction effect of acid-reducing drugs.

Citation Format: Natasha Strydom, Panli Cardona, Marintan Spring, Jessica Purkis, Brett Houk. Taking sotorasib with an acidic beverage improves sotorasib exposure for subjects on omeprazole [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT264.
Abstract LB034: EWSR1, Ewing sarcoma breakpoint region 1, maintains centromere identity by binding to centromeric R-loops
Authors: Kitagawa Risa , Becker Argentina , Houghton Peter , Kitagawa Katsumi
Published: April 2023

The centromere DNA-kinetochore complex is the specialized chromatin structure that mediates chromosome attachment to microtubules and is required for high-fidelity chromosome transmission. CENP-A is the centromere-specific histone H3 variant that epigenetically determines centromere position on the chromosome to ensure kinetochore assembly. Despite its importance, the precise mechanism of maintenance of CENP-A, i.e., the maintenance of centromere identity, remains obscure. In this study, we found that EWSR1 (Ewing Sarcoma Breakpoint Region 1) is required for CENP-A deposition at centromeres by binding to centromeric RNA. We determined that the CENP-A binding domain of EWSR1 is the SYGQ2 region within the prion-like domain, which plays an important role in phase separation for chromatin remodeling. Our data suggest that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA. This is a novel mechanism to maintain the position of the centromere on the chromosome i.e., centromere identify. As EWSR1-FLI1 is the oncogenic fusion protein in Ewing sarcoma and as it inhibits EWSR1 function as a dominant negative mutant, it contributes to chromosome instability in Ewing sarcoma, which may lead to therapy resistance and metastasis.

Citation Format: Risa Kitagawa, Argentina Becker, Peter Houghton, Katsumi Kitagawa. EWSR1, Ewing sarcoma breakpoint region 1, maintains centromere identity by binding to centromeric R-loops [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB034.
Abstract LB048: AR-targeted linked-read sequencing reveals complex AR structural variants in CRPC tumors
Authors: Zivanovic Andrej , Munro Sarah A. , Miller Jeffrey T. , Li Yingming , Passow Courtney N. , Dehm Scott M.
Published: April 2023

Inhibition of the androgen receptor (AR) transcription factor is the mainstay therapeutic strategy for systemic prostate cancer. However, development of a resistance phenotype termed castration resistant prostate cancer (CRPC) enables disease progression that underlies virtually all prostate cancer mortality. In the majority of CRPC, AR is transcriptionally re-activated in part due to accumulation of numerous alterations in the AR gene. These genomic alterations include missense mutations in exons that encode the ligand binding domain of AR, amplification of the AR gene body and/or an upstream transcriptional enhancer, and AR structural variants (SVs) resulting from inaccurate repair of DNA double strand breaks. In CRPC patient samples, these AR genomic alterations co-occur within tumors, which makes elucidating the fitness contribution of each alteration difficult. While short-read DNA sequencing has allowed for characterization of these events, they are underpowered to preserve linkage information. To address this challenge, we performed AR-targeted linked-read DNA-sequencing of CRPC cell line and patient derived xenograft (PDX) models with the goal of resolving sub-clonal and complex AR SVs. This approach confirmed the heterogeneity of AR SVs in these PDXs, including phased AR SVs that can be traced back to a single high molecular weight DNA molecule. These phased AR SVs were consistently found to co-occur in CRPC specimens with amplification of AR. Our results define a new class of AR SVs where multiple rearrangements co-occur on amplified DNA molecules to yield a complex array of AR gene structures in CRPC.

Citation Format: Andrej Zivanovic, Sarah A. Munro, Jeffrey T. Miller, Yingming Li, Courtney N. Passow, Scott M. Dehm. AR-targeted linked-read sequencing reveals complex AR structural variants in CRPC tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB048.
Abstract LB049: ALKBH5 emerges as a novel regulator of the BCR pathway
Authors: Kapadia Bandish B. , Roychowdhury Anirban , Kayastha Forum , Nanaji Nahid , Windle Jolene , Gartenhaus Ronald B.
Published: April 2023

Global epitranscriptomic methylation of mRNA at the N6 position (m6A) is reported to be altered in B-cell and tumor development. However, the mechanistic underpinnings of its role in the pathogenesis of Diffuse large B cell lymphoma (DLBCL) are unknown. It is well established that the B-cell receptor (BCR) pathway is a primary oncogenic driver in aggressive B-cell lymphomas. We previously reported that the Ataxia Telangiectasia Mutated (ATM)/Hu Antigen R (HuR)axis, as well as Eukaryotic Translation Initiation Factor 4A (eIF4A), modulate BCR signaling. However, the impact of RNA modifications on BCR induction is still poorly understood. Addressing this burgeoning question, we noted enhanced expression of AlkB Homolog 5 (ALKBH5), an m6A demethylase, in naïve B-cells treated with BCR mimetics. Similarly, mRNA levels of ALKBH5 were reported to be modestly enhanced in IgM-treated human B-cells (GSE156195). Consistently, immunization of C57BL/6 mice with sheep red blood cells (SRBC) significantly enhanced ALKBH5 in splenic B-cells. Next, evaluating the impact of BCR induction in lymphomagenesis, we observed a robust increase in the demethylase expression in BCR mimetic-treated DLBCL cells. Notably, increased expression of ALKBH5 was observed in a murine EµMyc model and primary DLBCL tumors. In coherence, the enhanced transcript levels of ALKBH5 in TCGA-DLBCL have prognostic implications. To gain functional insight, we depleted ALKBH5, which resulted in a robust reduction of BCR pathway proteins. Unexpectedly, the mRNA levels of CD79a and BLK were minimally modified, while the SYK, BTK, and CARD11 messages were reduced. Further studies are ongoing to evaluate the potential mechanism of mRNA transport and/or stability.

Citation Format: Bandish B. Kapadia, Anirban Roychowdhury, Forum Kayastha, Nahid Nanaji, Jolene Windle, Ronald B. Gartenhaus. ALKBH5 emerges as a novel regulator of the BCR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB049.
Abstract LB065: Interface-guided phenotyping of coding variants
Authors: Ozturk Kivilcim , Panwala Rebecca , Sheen Jeanna , Mali Prashant , Carter Hannah
Published: April 2023

Understanding the consequences of single amino acid substitutions in cancer driver genes remains an unmet need. High-throughput mutagenesis is emerging as a powerful tool to probe the varying consequences of different amino acid substitutions across the length of a protein or protein domain, however it is currently limited to specific functional readouts such as target protein abundance or functional assays. Studying the effects of genetic perturbations on cellular programs and fitness has been challenging using traditional pooled screens. Over the last few years, there has been a surge of interest in Perturb-seq style assays to measure the transcriptional consequences of genetic perturbations ranging from whole gene knockout to amino acid substitutions in single cells. While providing greater function insight, these sequencing-based methods are not yet scalable to exhaustive mutagenesis, necessitating selection of target mutations. In this work, we hypothesized that examining the consequences of perturbing distinct protein interactions could provide a useful abstraction of the phenotypic space reachable by individual amino acid substitutions. To explore this hypothesis, we employed a Perturb-seq style approach to generate mutations at physical interfaces of the transcription factor RUNX1, with the potential to perturb different interactions, and therefore produce transcriptional readouts implicating different aspects of the RUNX1 regulon. We analyzed these readouts to identify functionally distinct groups of RUNX1 mutations, characterize their effects on cellular programs and study the implications for cancer mutations. Our work demonstrates the potential of targeting protein interaction interfaces to better define the landscape of prospective phenotypes reachable by amino acid substitutions.

Citation Format: Kivilcim Ozturk, Rebecca Panwala, Jeanna Sheen, Prashant Mali, Hannah Carter. Interface-guided phenotyping of coding variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB065.
Abstract LB074: Towards the next generation cancer dependency map
Authors: Wienand Kirsty , Stokes Samantha , Montgomery Philip G. , Sullivan Kelly , Dempster Joshua M. , Hahn William C. , Roth Jennifer A. , Vazquez Francisca
Published: April 2023

Cancer genome alterations that cause cancer cells to grow also confer vulnerabilities, which normal cells lack. The challenge is that, for the majority of cancers, we do not understand the relationship between the genetic alterations and the dependencies they cause. To solve this problem, we are creating a Cancer Dependency Map (DepMap) by systematically identifying genetic dependencies and small molecule sensitivities as well as discovering the biomarkers that predict them. Since its inception, DepMap has compiled data for over eighteen hundred cell line models, performed more than a thousand CRISPR screens, and drug sensitivity profiles using PRISM in over 500 cell lines as well as developed new analytic methods for the identification of cancer vulnerabilities. DepMap is being used by researchers around the world and numerous potential therapeutic targets have been identified using this resource. However, we are still in the early stages of having a complete map and we will discuss our ongoing work towards making the Cancer Dependency Map more powerful. The next generation DepMap will include more complex cellular models including 3D models, representing pediatric and rare cancers, and expanding the perturbation space to include combinations. Additionally, we are performing novel types of omics characterization and building new computational and visualizations tools. These new scientific directions will help make progress towards our identification of the landscape of cancer vulnerabilities and create a roadmap for cancer therapeutics.

Citation Format: Kirsty Wienand, Samantha Stokes, Philip G. Montgomery, Kelly Sullivan, Joshua M. Dempster, William C. Hahn, Jennifer A. Roth, Francisca Vazquez. Towards the next generation cancer dependency map [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB074.
Abstract LB132: In silico mutant neoantigen prediction - a promising avenue for target identification in human adrenocortical carcinoma
Authors: Landwehr Laura-Sophie , Appenzeller Silke , Ölsner Laura , Weigand Isabel , Sbiera Silviu , Fassnacht Martin , Kroiss Matthias
Published: April 2023

Introduction: Adrenocortical carcinoma (ACC) is one of the most aggressive endocrine malignancies and confers a poor prognosis in advanced stages. Effective treatments are lacking. The results of immune checkpoint inhibition were disappointing with few responders only deriving clinical benefit. For the development of novel immunotherapies such as tumor vaccines and T cell-based treatments, target identification is essential. Tumor-specific mutant neoantigens that may be recognized by T cells in the context of major histocompatibility complex (MHC) I are promising candidates.

Methods: We performed whole exome-sequencing in 10 ACC samples with matched blood controls. Somatic mutations were identified using an in house bioinformatics pipeline. By coupling POLYSOLVER for HLA typing with netMHCpan, in silico binding affinity of tumor-specific neoantigens to MHC was calculated taking into account both peptide and HLA sequence information. Strong binding (SB) was defined as <0.5% rank, weak binding (WB) as 0.5-1.9% rank and no binding (NB) as >2.0% rank of all peptides to MHC.

Results: Across 10 ACC patients, we identified 1067 unique somatic mutations (median 49.5, ranging from 15-590), affecting 989 different genes. Binding affinity changed for 414 predicted neoantigens from NB to WB, 80 from NB to SB and 82 from WB to SB. The mutant neoantigen load per patient ranged from 10 to 235 (mean 66.2) and was positively correlated to the total number of non-synonymous single nucleotide variants (R2 0.8977, 95% CI 0.79-0.99; p<0.0001).

Discussion: This is the first study that demonstrates successful in silico neoantigen profiling in ACC. Mutant neoantigens were predicted to be present both in ACC with high and low tumor mutational burden. These data will pave the way for in vitro validation and hold potential to develop therapeutic cancer vaccines and T cell-based cancer immunotherapy in ACC.

Citation Format: Laura-Sophie Landwehr, Silke Appenzeller, Laura Ölsner, Isabel Weigand, Silviu Sbiera, Martin Fassnacht, Matthias Kroiss. In silico mutant neoantigen prediction - a promising avenue for target identification in human adrenocortical carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB132.
Abstract LB154: Circ0003039 interacts with calreticulin to inhibit the progression of hepatocellular carcinoma by suppressing wnt/beta-catenin signaling pathway
Authors: Wang Yue-Ning , Zhou Yu-Feng , Liu Ji , Zhang Mei-Yin , Wang Shuo-Cheng , Mai Shi-Juan , Wang Hui-Yun
Published: April 2023

Background: Circular RNAs (circRNAs) are covalently closed and single-stranded RNAs which play critical roles in various biological processes and diseases including cancers. The functions and mechanisms of circRNAs in hepatocellular carcinoma (HCC) are still need to be clarified.

Methods: Custom circRNA microarray was performed to investigate the profile of circRNAs in HCC. Expression of circ0003039 and its clinical significance in 60 pairs of HCC and matched non-cancer liver tissues were identified and confirmed by microarray and quantitative RT-PCR. The biological functions of circ0003039 were investigated by gain-and loss-of-function experiments in vitro and in vivo. RNA pull-down, mass spectrometry, RNA-immunoprecipitation, Co-immunoprecipitation, fluorescent in situ hybridization were used to identify circ0003039-protein interaction. The downstream pathway regulated by circ0003039 was explored using RNA-seq and verified using western blot and immunofluorescence.

Results: Expression of circ0003039 was reduced in HCC tissues and positively associated with the overall survival of HCC patients. Circ0003039 was located in cytoplasm of HCC cells. Ectopic overexpression of circ0003039 inhibited the proliferation, colony formation, migration and invasion of HCC cells and the growth of HCC xenograft in mice, while knockdown of circ0003039 had opposite effects in HCC cells. Mechanistically, circ0003039 bound to the N-terminal domain of CALR protein and acted as a scaffold to enhance the interaction of CALR with CAPN2, which promoted the degradation of CALR protein by the enzymatic activity of CAPN2. Rescue experiments showed that circ0003039 inhibited HCC proliferation via suppressing CALR protein. Finally, we found that circ0003039 inhibited HCC cells by suppressing CALR-mediated wnt-βcatenin signaling pathway.

Conclusions: Our study demonstrates that circ0003039 bind to N-terminal domain of CALR and facilitated CAPN2 to degrade CALR protein, which inhibits the development and progression of HCC via suppressing CALR-mediated wnt-βcatenin signaling pathway, and circ0003039 is a potential prognostic biomarker and therapeutic target for HCC.

Citation Format: Yue-Ning Wang, Yu-Feng Zhou, Ji Liu, Mei-Yin Zhang, Shuo-Cheng Wang, Shi-Juan Mai, Hui-Yun Wang. Circ0003039 interacts with calreticulin to inhibit the progression of hepatocellular carcinoma by suppressing wnt/beta-catenin signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB154.
Abstract LB157: Cereblon inhibits prostate cancer progression and metastasis by negatively regulating 6PGD
Authors: Guchhait Koushik , Yoon Hyeon Seung , Shin Seungheon , An Hyun-Su , Nam Hye Seung , Yanqui-Rivera Francisco D. , Oña Samara M. , Mendez Miguel Á. , Park Seokjae , Kim Eun-Kyoung , Hwang Jong Yeon , Han Jee-Young , Chung Doo Yong , Park Daeho , Yang Su-Geun , Park Chul-Seung , Cho Steve K.
Published: April 2023

As the third enzyme of the pentose phosphate pathway (PPP), abnormally elevated levels of 6-phosphogluconate dehydrogenase (6PGD) have been documented in various human cancers. We demonstrate that reduced cereblon (CRBN) protein expression is the underlying mechanism of elevated 6PGD expression in metastatic prostate cancer cells. We establish 6PGD as a new endogenous substrate for CRBN by demonstrating that it interacts directly with CRBN and is ubiquitinated by CRL4CRBN. In addition, CRBN negatively regulates prostate cancer cell progression and metastasis, as abnormally high 6PGD, in the absence of sufficient CRBN, enhances the metastatic potential of prostate cancer in vitro and in vivo. Our findings show convincingly that carbohydrate metabolism regulated by 6PGD is linked to prostate cancer metastasis via CRBN. Based on these data, we propose that the 6PGD-CRBN axis may be a suitable target for further research into new therapeutics for mitigating prostate cancer metastasis.

Citation Format: Koushik Guchhait, Hyeon Seung Yoon, Seungheon Shin, Hyun-Su An, Hye Seung Nam, Francisco D. Yanqui-Rivera, Samara M. Oña, Miguel Á. Mendez, Seokjae Park, Eun-Kyoung Kim, Jong Yeon Hwang, Jee-Young Han, Doo Yong Chung, Daeho Park, Su-Geun Yang, Chul-Seung Park, Steve K. Cho. Cereblon inhibits prostate cancer progression and metastasis by negatively regulating 6PGD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB157.
Abstract LB158: Rapid cancer cell perineural invasion utilizes amoeboid migration
Authors: Wang Qi , Marcadis Andrea , Kao Elizabeth , Chen Chun-Hao , Gusain Laxmi , Powers Ann , Bakst Richard , Deborde Sylvie , Wong Richard J.
Published: April 2023

The invasion of nerves by cancer cells, or perineural invasion (PNI), is potentiated by the nerve microenvironment and is associated with adverse clinical outcomes. However, the cancer cell characteristics that enable PNI are poorly defined. Here, we generated cell lines enriched for a rapid neural invasive phenotype by serially passaging pancreatic cancer cells in a murine sciatic nerve model of PNI. Cancer cells isolated from the leading edge of nerve invasion showed a progressively increasing nerve invasion velocity with higher passage number. Transcriptome analysis revealed an upregulation of proteins involving the plasma membrane, cell leading edge, and cell movement in the leading neural-invasive cells. Leading cells progressively became round and blebbed, lost focal adhesions and filipodia, and transitioned from mesenchymal to amoeboid phenotype. Leading cells acquired an increased ability to migrate through microchannel constrictions and associate with dorsal root ganglia than non-leading cells. ROCK inhibition reverted leading cells from an amoeboid to mesenchymal phenotype, reduced migration through microchannel constrictions, reduced neurite association, and reduced PNI in a murine sciatic nerve model. Cancer cells with rapid PNI exhibit an amoeboid phenotype, highlighting the plasticity of cancer migration mode in enabling rapid nerve invasion.

Citation Format: Qi Wang, Andrea Marcadis, Elizabeth Kao, Chun-Hao Chen, Laxmi Gusain, Ann Powers, Richard Bakst, Sylvie Deborde, Richard J. Wong. Rapid cancer cell perineural invasion utilizes amoeboid migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB158.
Abstract LB162: A multiplex organoid avatar drug testing platform for precision medicine
Authors: Patel Shrey , Khalili Sana , Moy Victoria , Gray Emma , Lyons Sawyer , Brents Riley , Banister Carolyn , Buckhaults Phillip J.
Published: April 2023

The promise of precision medicine is to improve patient outcomes by making better therapeutic choices based on cancer causing somatic mutations. This strategy has shown limited success in colorectal cancer, in part because the genetic rules governing resistance and sensitivity are not fully elucidated. Direct drug sensitivity testing of individual patient-derived organoids is an attractive addition to this decision process because it does not require understanding of all drug-somatic mutation interactions. Individual patients avatars can be challenged with a variety of drugs and response used to inform patient care. We have developed a panel of colon cancer organoid avatars and uniquely tagged each one with a lentiviral vector possessing identifying DNA sequence bar codes flanked by common PCR primers. Each bar-code is uniquely identifiable and quantifiable from a mixture of organoids by nanopore sequencing of a common PCR product. Treatment of the mixture of organoid avatars over time under multiple drug conditions and sampling at different time points allowed the real-time monitoring of relative Darwinian fitness of each organoid in the mixture. We demonstrated the utility of this approach at identifying both expected and novel drug responses for individual organoids. Resistance to both nutlin and irinotecan were correctly predicted by TP53 somatic mutations while sensitivity to both lapatinib and ibrutinib were best predicted by mutations in EGFR signaling pathway. Patterns of drug response revealed unexpected common mechanisms. Future work will focus on informing patient care decisions with these results and determining if improvements in patient outcomes are realized.

Citation Format: Shrey Patel, Sana Khalili, Victoria Moy, Emma Gray, Sawyer Lyons, Riley Brents, Carolyn Banister, Phillip J. Buckhaults. A multiplex organoid avatar drug testing platform for precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB162.
Abstract LB202: Obesity-trained C1q+ macrophages compromiseT cell anti-tumor immunity
Authors: Zhang Tao , Liu Shimeng , Brown Myles
Published: April 2023

Adipose tissue in the mammary gland undergoes dramatic remodeling during obesity and is involved in ﻿numerous metabolic diseases. ﻿However, how obesity-driven remodeling in adipose tissue regulates anti-tumor immunity remains unclear. Here we provide the detailed cellular atlases of mouse mammary fat pad and tumor at single-cell resolution in lean and obese mice. By leveraging scRNA-seq, we found that high-fat feeding causes a marked increase of macrophage in adipose tissue. ﻿Interestingly, a subset of macrophages expressing high levels of C1q complex genes is accumulated in obese subjects. We found that glucocorticoid may as a key regulator of C1q+ macrophage identity. Synthetic glucocorticoid, Dexamethasone, triggers C1q expression in macrophages. We found that C1q+ macrophage derived C1q complex could suppress CD8+ T cell response, thus fueling breast cancer progression. In mouse TNBC tumor, we uncovered a specific inflammatory responsive tumor subset in obese tumors with high CCR2 ligand expression. Obesity-induced TNFα exposure triggers of breast cancer cells reprogramming and CCR2 ligand production. Consequently, CCR2 ligands promote macrophage into tumor. Moreover, depletion of macrophages by anti-CSF1R suppresses tumor growth in obese mice. Our findings define a novel fundamental mechanism of C1q+ macrophage involved in anti-tumor response in obesity.

Citation Format: Tao Zhang, Shimeng Liu, Myles Brown. Obesity-trained C1q+ macrophages compromiseT cell anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB202.
Abstract LB210: RenNano® mice: A heavy-chain-only antibody platform for the generation of nanobody therapeutics
Authors: Hu Yiqing , Zhang Qi , Zhang Lijun , Zhang Yabo , Zhao Huizhen , Yao Jiawei , Yang Liu , Liu Baihong , Wang Shensen , Su Zhengfang , Hui Li , Lin Qingcong , Ma Qiangqiang , Shen Yuelei
Published: April 2023

Monoclonal antibodies have been used to successfully treat various diseases, including tumors, autoimmune diseases, and infectious diseases. Traditional antibodies are comprised of a tetramer of two heavy chains and two light chains, totaling 150 kDa in molecular weight. However, the large size of antibodies can limit the therapeutic application; in particular, the penetration of tumors and the blood brain barrier (BBB) is not always feasible. In contrast to traditional antibodies, heavy-chain-only antibodies (HCAbs) are significantly smaller (~75 kDa), as they contain only two heavy chains. Since the heavy chain variable domain of HCAbs (i.e., VHH or single domain antibody, sdAb, or nanobody) is solely responsible for antigen recognition, nanobodies can function independently as a therapeutic molecule, which may be advantageous for penetrating tumors or the BBB. Previously, we generated a fully human antibody mouse platform, RenMabTM, in which the murine heavy chain and kappa light chain variable domains were replaced by the full human heavy chain and kappa light chain V(D)J loci in situ. Here, we have further modified the RenMabTM model to generate a fully human heavy-chain-only antibody mouse model, termed RenNano®. The modified heavy chain constant regions of RenNano® mice allow them to spontaneously produce HCAbs. Flow cytometry and biolayer interferometry confirmed that RenNano®-derived HCAbs can bind antigens without light chains. Despite this reliance on the heavy chain only variable regions for antigen specificity, RenNano® mice can generate antigen-specific antibodies with high affinity (10−8 -10−9 KD) upon immunization with various antigens. In addition, many RenNano®-derived HCAbs exhibited a longer CDR3 length, which could promote the recognition of difficult-to-reach epitopes. Furthermore, RenNano®-derived HCAbs have favorable diversity, and excellent developability properties such as a higher degree of hydrophilicity. Anti-4-1BB HCAbs can also activate 4-1BB-NF-κB signaling in a dose-dependent manner, as demonstrated in reporter assays.

In summary, the full human heavy-chain-only antibody mice, RenNano®, can produce human HCAb with high affinity and good efficacy. Thus, RenNano® is a powerful platform to discover HCAb/nanobodies for various therapeutic applications.

Citation Format: Yiqing Hu, Qi Zhang, Lijun Zhang, Yabo Zhang, Huizhen Zhao, Jiawei Yao, Liu Yang, Baihong Liu, Shensen Wang, Zhengfang Su, Li Hui, Qingcong Lin, Qiangqiang Ma, Yuelei Shen. RenNano® mice: A heavy-chain-only antibody platform for the generation of nanobody therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB210.
Abstract LB216: Discovery of BCG033, a novel anti-PTK7 x TROP2 bispecific antibody-drug conjugate with promising efficacy against triple-negative breast cancer
Authors: Yao Sufei , Shang Chengzhang , An Gao , Zhang Ellen , Lin Qingcong , Yang Yi
Published: April 2023

Triple-negative breast cancer (TNBC) accounts for 15-20% of metastatic breast cancer incidence, and remains an area of unmet clinical need due to the low rates of overall survival. Recently, the TROP2-targeting ADC sacituzumab govitecan has received an accelerated approval from the FDA for adult patients with metastatic TNBC, as more than 85% of TNBC is marked by TROP2 overexpression. However, the clinical efficacy of ADC therapies targeting TROP2 alone is limited by its on-target toxicity. In an effort to offer therapeutic alternatives that limit this toxicity, we sought to identify other targets to combat metastatic TNBC in combination with TROP2. PTK7 is highly expressed in breast cancer; notably, PTK7 expression is higher in TNBC than non-TNBC, and is correlated with worse prognosis, tumor metastasis and TNBC progression. PTK7 has also been demonstrated to be enriched in tumor-initiating cells (TICs) in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). We generated fully human anti-human PTK7 x TROP2 bispecific antibodies (bsAbs) from RenLite® mice, which harbor the complete human heavy chain immunoglobulin variable domain with a common human kappa light chain for subsequent bispecific antibody assembly. These bsAbs demonstrated reactivity to human, monkey, and dog antigens, and showed enhanced internalization in vitro compared with parental PTK7 antibodies. In addition, these bsAbs showed favorable tumor cell selectivity, as there was minimal internalization of the monovalent antibodies. These bsAbs were than conjugated with Monomethyl auristatin E (MMAE) to generate anti-PTK7 x TROP2 bispecific ADC (BCG033) candidates. BCG033 candidates showed potent anti-tumor activity in several cell line-derived xenografts including TNBC xenografts, indicating that BCG033 has strong therapeutic potential in TNBC and other PTK7/TROP2 co-expressing cancers. Patient-derived TNBC xenografts with co-expression of PTK7 and TROP2 have been screened for future in vivo drug efficacy screening. In summary, BCG033 has the potential to exert anti-tumor efficacy in TNBC and other solid tumors co-expressing PTK7 and TROP2.

Citation Format: Sufei Yao, Chengzhang Shang, Gao An, Ellen Zhang, Qingcong Lin, Yi Yang. Discovery of BCG033, a novel anti-PTK7 x TROP2 bispecific antibody-drug conjugate with promising efficacy against triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB216.
Abstract LB222: An mRNA-based cancer vaccine multi-targeting KRAS mutations inhibits tumor growth by increasing immune response in KRAS mutant LL/2 mouse model
Authors: Shin Seung-Hyun , Han Youngjin , Lim Chang Gyu , Heo Yong Ho , Jeong Seongju , Kim Yu-Yon , Choi In Young
Published: April 2023

Kirsten rat sarcoma viral oncogene (KRAS) mutations are present in 26% of non-small cell lung cancer (NSCLC) patients (The Cancer Genome Atlas data). Studies have reported the presence of spatial or intra-tumoral heterogeneity of KRAS mutations. Though resistance to mutant KRAS inhibitors targeting a specific form of major KRAS mutations (e.g, G12C or G12D) is multifaceted, intra-tumoral heterogeneity of KRAS mutation is one of the crucial factors for the intrinsic resistance to the inhibitor. Multi-targeting of different KRAS mutations can prevent resistance due to the heterogeneity of KRAS mutations within a tumor. To achieve this, we incorporated an mRNA-based cancer vaccine containing transcripts for multiple KRAS mutant antigens. Our KRAS cancer vaccine (CV) candidate showed significantly attenuated tumor growth by 37% in the syngeneic KRAS G12C-expressing LL/2 tumor-bearing mice. CV treatment group showed significantly decreased tumor size by 45.4% after biopsy. Moreover, the mice treated with KRAS CV showed spleen enlarged by 42.6% indicating increased immune responses. To confirm, the expansion of T cells in the cancer vaccine-treated group, we collected tumor tissues and analyzed proportions of CD4+ and CD8+ T lymphocytes in the tumor. We found a 14.5-fold increase in infiltration of CD8+ T cells and a 0.5-fold increase in CD8+ CD44+ memory/effector T cells in the tumors from mice treated with KRAS CV. Whereas CD4+ Foxp3+ Treg cells were decreased by 3.9-fold in the tumor. HLA-A*02 is the most common MHC class I allele. To investigate the changes in MHC expression, we conducted FACS analysis using HLA-A*02 antibodies after ex vivo treatment of KRAS CV on the human peripheral blood mononuclear cells (hPBMCs). KRAS CV treatment facilitated higher proportions of HLA-A*02+ monocytes (CD14+) and B cells (CD19+). Though LL/2 tumor model has been considered an immunosuppressive model, our results suggest that the KRAS CV can significantly enhance immune responses, thereby suppressing tumor growth.

Citation Format: Seung-Hyun Shin, Youngjin Han, Chang Gyu Lim, Yong Ho Heo, Seongju Jeong, Yu-Yon Kim, In Young Choi. An mRNA-based cancer vaccine multi-targeting KRAS mutations inhibits tumor growth by increasing immune response in KRAS mutant LL/2 mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB222.
Abstract LB259: Distinct microRNA signature and suppression of ZFP36L1 define ASCL1-positive lung adenocarcinoma
Authors: Miyashita Naoya , Enokido Takayoshi , Horie Masafumi , Suzuki Hiroshi I. , Matsuki Rei , Brunnström Hans , Micke Patrick , Nagase Takahide , Saito Akira
Published: April 2023

Background: Achaete-scute family bHLH transcription factor 1 (ASCL1) is a master transcription factor involved in neuroendocrine differentiation. ASCL1 is expressed in approximately 10% of lung adenocarcinomas and exerts tumor-promoting effects. However, microRNA (miRNA) profiles regulated by ASCL1 in lung adenocarcinoma cells remain unexplored.

Method: We analyzed public database of gene expression profiling (RNA-sequencing and miRNA expression data). We also studied miRNA profiles in ASCL1-positive lung adenocarcinoma cells and identified a subset of miRNAs downregulated by ASCL1 knockdown. We examined functions of genes suppressed by miRNAs in ASCL1-positive lung adenocarcinoma cell line, VMRC-LCD.

Result: We identified miRNA profiles in ASCL1-positive lung adenocarcinomas and found several miRNAs closely associated with ASCL1 expression, including miR-375, miR-95-3p/miR-95-5p, miR-124-3p, and members of the miR-17~92 family. Similar to small cell lung cancer, Yes1 associated transcriptional regulator (YAP1), a representative miR-375 target gene, is suppressed in ASCL1-positive lung adenocarcinomas. ASCL1 knockdown followed by miRNA profiling in a cell culture model further revealed that ASCL1 positively regulates miR-124-3p and members of the miR-17~92 family. Integrative transcriptomic analyses identified the RNA-binding protein zinc finger protein 36 like 1 (ZFP36L1) as a target gene of miR-124-3p, and immunohistochemical studies have demonstrated that ASCL1-positive lung adenocarcinomas are associated with low ZFP36L1 protein levels. Cell culture studies have shown that ectopic ZFP36L1 expression inhibits cell proliferation, survival, and cell cycle progression. Mechanistically, ZFP36L1 negatively regulated tumorigenic genes, including E2F transcription factor 1 (E2F1) and snail family transcriptional repressor 1 (SNAI1), indicating a tumor-suppressing action.

Conclusion: Our study revealed that suppression of ZFP36L1 via ASCL1-regulated miR-124-3p could induce tumor-promoting effects, providing evidence that ASCL1-mediated regulation of miRNAs shapes malignant features of ASCL1-positive lung adenocarcinomas.

Citation Format: Naoya Miyashita, Takayoshi Enokido, Masafumi Horie, Hiroshi I. Suzuki, Rei Matsuki, Hans Brunnström, Patrick Micke, Takahide Nagase, Akira Saito. Distinct microRNA signature and suppression of ZFP36L1 define ASCL1-positive lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB259.
Abstract LB261: The Akt-IWS1 signaling axis regulates microRNA activity
Authors: Christodoulou Niki , Hatziapostolou Maria , Montiel-Duarte Cristina , Edwards Elisabetta Verderio , Tsichlis Philip N. , Polytarchou Christos
Published: April 2023

MicroRNAs are small single-stranded non-coding RNAs that regulate gene expression post-transcriptionally, by targeting the 3‘-untranslated region (3’-UTR) of mRNAs. The regulation of microRNAs at the transcriptional level is well-studied, however, how their activity is regulated remains largely elusive. The family of Akt serine/threonine protein kinases, comprised of Akt1, Akt2 and Akt3, regulates important cell functions including metabolism, survival, proliferation, and migration. We have previously shown that IWS1 (Interacts with Spt6), a factor involved in mRNA splicing and nuclear export, is phosphorylated specifically by Akt1 and Akt3 at Ser720/Thr721. Here, RNA extracts from NCI-H522 lung cancer cells, transduced with shIWS1 and reconstituted with wild type (Ser720/Thr721-IWS1) or phosphorylation-deficient IWS1 (Ala720/Ala721-IWS1) were subjected to RNA and microRNA sequencing. Bioinformatic analysis suggested that microRNAs expressed at similar levels in the two cell types have a different impact on their mRNA targets. microRNA activity reporter assays and western blot analysis for microRNA targets in these cells revealed increased microRNA activity in Ala720/Ala721-IWS1-expressing cells. Our findings were verified in a second cell line, the non-transformed immortalized colonic epithelial cells NCM460. microRNA activity reporter assays, western blot analyses and cell growth assays showed that microRNA effects are enhanced in cells expressing Ala720/Ala721-IWS1. Immunoprecipitation and proximity ligation assays showed that IWS1 interacts with proteins known to be associated with RISC, and this interaction is enhanced by Akt-mediated IWS1 phosphorylation. To evaluate the dependence of IWS1/RISC interaction on IWS1 phosphorylation, we employed Akt1− / −Akt2− / −Akt3− / − immortalized mouse lung fibroblasts, transduced with myc-Akt1 or myc-Akt2 or the empty retroviral vector. Immunoprecipitation experiments in these cells, before and after treatment with IGF1, revealed that IWS1 interacts with the RISC components specifically in Akt1-expressing cells upon Akt1 activation by IGF1. Overall, our data demonstrate that IWS1 interacts with RISC in a conserved Akt1-dependent manner and regulates the activity of microRNAs with significant effects on cellular properties.

Citation Format: Niki Christodoulou, Maria Hatziapostolou, Cristina Montiel-Duarte, Elisabetta Verderio Edwards, Philip N. Tsichlis, Christos Polytarchou. The Akt-IWS1 signaling axis regulates microRNA activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB261.
Abstract LB267: Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma
Authors: Jayabal Panneerselvam , Ma Xiuye , Bondra Kathryn M. , Chen Yidong , Houghton Peter J. , Shiio Yuzuru
Published: April 2023

Retinoblastoma is a cancer of the infant retina primarily driven by the loss of the Rb tumor suppressor gene, which is undruggable. Here, we identified an autocrine signaling, mediated by secreted frizzled-related protein 2 (SFRP2), which suppresses nitric oxide and enables retinoblastoma growth. SFRP2 silencing resulted in rapid production of nitric oxide, leading to p53-dependent growth arrest of retinoblastoma cells. We show that coxsackievirus and adenovirus receptor (CXADR) is the cell surface receptor for SFRP2 in retinoblastoma cells, that CXADR functions as a "dependence receptor," transmitting a growth inhibitory signal in the absence of SFRP2, and that the balance between SFRP2 and CXADR determines nitric oxide production. Accordingly, high SFRP2 RNA expression correlates with high-risk histopathologic features in retinoblastoma. Targeting SFRP2 signaling by SFRP2-binding peptides or by a pharmacological inhibitor rapidly induced nitric oxide and profoundly inhibited retinoblastoma growth in orthotopic xenograft models. These results reveal a cytokine signaling pathway that regulates nitric oxide production and retinoblastoma cell proliferation, and is amenable to therapeutic intervention.

Citation Format: Panneerselvam Jayabal, Xiuye Ma, Kathryn M. Bondra, Yidong Chen, Peter J. Houghton, Yuzuru Shiio. Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB267.