bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 786: Single molecule methylation sequencing of circulating DNA identifies individuals with breast cancer
Authors: Lau Billy T. , Bai Xiangqi , Notestine Alison , Parkinson Brett , Ji Hanlee P. , Nadauld Lincoln
Published: April 2023

Liquid biopsies are being investigated for the detection of cancer from blood. Epigenetic characterization of cell free DNA (cfDNA), specifically DNA methylation, is an emerging approach for sensitive detection and quantification of tumor burden. For breast cancer detection from blood, existing approaches for testing cfDNA have demonstrated a low performance in identifying affected patients. For this study, we developed a new approach that overcame this issue. Addressing the challenge of detecting breast cancer from cfDNA, this new approach relied on single-molecule methylation profiling of cfDNA. We used nanopore single molecule sequencing to provide quantitative characterization of cfDNA methylomes. This study determined if this approach provided the methylation features required for detecting the presence of breast cancer. The study population consisted of an extended cohort (~200 with untreated breast cancer and ~400 healthy controls). We identified 5mC-specific profiles from the nanopore sequencing signals of cfDNA. Importantly, this approach did not require any biosulfite or biochemical processing steps for determining methylation. Thus, we determined methylation profiles that were directly extrapolated from native cfDNA at single molecule resolution. Our results from this study demonstrated higher sensitivity of detecting breast cancer compared to other cfDNA methylation tests. We developed a new analytical framework for characterizing the methylation profiles of individuals with breast cancer compared to healthy individuals. Using a supervised bootstrapped algorithm, we identified CpG sites that were differentially methylated between the case and control groups. Afterwards, we calculated the similarity of any sample’s methylation profile to a training data. Our method also accommodates sparse and missing data. Based on separate validation datasets, we accurately identified those individuals with breast cancer. We validated our study with a different, independent cohort of healthy controls and individuals with breast cancer. Overall, this study demonstrates that a nanopore-based cfDNA sequencing and methylation profiling detects breast cancer from blood with higher performance then has been previously reported.

Citation Format: Billy T. Lau, Xiangqi Bai, Alison Notestine, Brett Parkinson, Hanlee P. Ji, Lincoln Nadauld. Single molecule methylation sequencing of circulating DNA identifies individuals with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 786.
Abstract 793: Mechanism of interferon induction by cannabidiol (CBD) and analogues in lung cancer cells
Authors: Stock Leticia , Robinson-Mailman Lydia , Nguyen Long C. , Chen Shao-Nong , Pauli Guido F. , Rosner Marsha R.
Published: April 2023

CBD, an FDA approved drug for epilepsy, may have therapeutic potential for other diseases and is currently being tested for efficacy in cancer-related clinical trials. As the literature about CBD, especially in vitro reports, is often contradictory, increasing our understanding of its specific action on a molecular level will allow to determine whether CBD can become a useful therapy or exacerbates specific cancers in a context-dependent manner. Due to its relative lipophilicity, CBD is challenging to dispense at therapeutic concentrations; therefore, one goal is to identify cannabinoid congeners with greater efficacy and reduced drug delivery challenges. We recently showed that CBD activates interferons as a mechanism of inhibiting SARS-CoV-2 replication in lung carcinoma cells. As factors produced by the innate immune system, interferons have been implicated in both pro-survival and growth arrest and apoptosis signaling in cancer. Here we show that CBD induces interferon production and interferon stimulated genes (ISGs) through a mechanism involving NRF2 and MAVS in lung carcinoma cells. We also show that CBDV, which differs from CBD by 2 fewer aliphatic tail carbons, has limited potency, suggesting that CBD specifically interacts with one or more cellular proteins rather than having a non-specific effect. We also identified other CBD-related cannabinoids that are more effective at inducing ISGs. Taken together, these results characterize a novel mechanism by which CBD activates the innate immune system in lung cancer cells and identify related cannabinoids that have possible therapeutic potential in cancer treatment.

Citation Format: Leticia Stock, Lydia Robinson-Mailman, Long C. Nguyen, Shao-Nong Chen, Guido F. Pauli, Marsha R. Rosner. Mechanism of interferon induction by cannabidiol (CBD) and analogues in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 793.
Abstract 799: Measuring the impact of the COVID-19 pandemic on breast cancer screening in Puerto Rico
Authors: Colón-López Vivian , Ortiz Hermilis Berrios , Contreras-Mora Hector , Disdier Orville M. , Pérez Cynthia M. , Ríos Nilda , Díaz Olga L. , Mejías Juan , Suárez-Pérez Erick L.
Published: April 2023

Introduction: The COVID-19 pandemic has caused major changes to healthcare services, especially those related to early detection and screening practices like breast cancer. In Puerto Rico, breast cancer is the main cause of death, representing 18.9% of cancer deaths in women, making early detection even more important to prevent morbidity and mortality. This study aims to describe the impact of COVID-19 on breast cancer screening and assess differences in health utilization by age group and health regions in Puerto Rico.

Methods: This study used data on breast cancer screening medical claims from Puerto Rico Track, a project in collaboration with the Puerto Rico Public Health System and the Puerto Rico Institute of Statistics that aims to assess health access and utilization patterns in Puerto Rico. Claims including unilateral and bilateral mammography, sonommamography, and MRI were analyzed. Descriptive statistics and percentual changes between the COVID-19 baseline year (2016) compared with 2020 and 2021 were performed (overall, by age-group and health region).

Results: A total of 193,793 screening tests were performed in 2016, compared to 66,463 in 2020, and 89,322 in 2021. Overall, a third of the medical claims for breast cancer screening (33.2%) were in the age group of 51-60 years. An overall decreasing percentual change was observed comparing 2016 vs. 2020 (65.7%), where the age group with the broadest gap reduction were among women 41-50 years old (68.2%). When comparing medical claims of 2016 (193,750) versus 2021 (89,320) (pre and post pandemic), an important decreasing change (53.9%) was observed. The age group with the highest decrease comparing 2016 to 2021 was the 41-50 years one (68.2%). The Western region of Mayagüez/Aguadilla had the highest decreasing percentual change, with a reduction of 73.6% in 2020 compared to 2016, and 62.6% when compared 2021 with 2016.

Conclusion: Breast cancer screening was notably affected by the COVID-19 pandemic in Puerto Rico. A pattern of decreasing breast cancer screening was observed by health regions and by age. These efforts provide evidence of the need of tailored evidence-based interventions to increase breast cancer screening in the island.

Citation Format: Vivian Colón-López, Hermilis Berrios Ortiz, Hector Contreras-Mora, Orville M. Disdier, Cynthia M. Pérez, Nilda Ríos, Olga L. Díaz, Juan Mejías, Erick L. Suárez-Pérez. Measuring the impact of the COVID-19 pandemic on breast cancer screening in Puerto Rico [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 799.
Abstract 832: Exosome-based hybrid nanosystem for targeted TNBC therapy
Authors: Joshi Rohan , Lampe Jana , Vishwanatha Jamboor K. , Ranjan Amalendu P.
Published: April 2023

Treatment of metastatic Triple-Negative Breast Cancer (TNBC) remains a challenge despite an increasing number of newer drugs being approved in recent years. The major challenge for improved treatment outcomes for metastatic cancer originates from the fact that the drugs are unable to reach the targeted site and hence are not able to elicit the desired response. Recently, natural mesenchymal stem cells (MSC) cells and their exosomes have been shown to have tumor-homing properties. By exploiting these homing properties, we propose the development of an exosome-polymeric hybrid nanosystem (EPHN) by using exosomes for their targeting ability to enhance the targetability and thus the therapeutic efficacy of the drug against TNBC. In this study, we bioengineered MSC exosome-coated drug-loaded polymeric nanoparticles to deliver our chemotherapeutic drug in a targeted fashion. To isolate the MSC exosomes, we grew the cells in exosome-free media and used the ultracentrifugation standard method. The MSC exosomes had a size of approximately 70 ± 5 nm and a PDI of 0.3. Our exosome sample was positive for exosomal proteins and negative for all other extracellular vesicles. The optimal doxorubicin-loaded PLGA nanoparticles were prepared using a microfluidics method resulting in a higher entrapment efficiency of 42 ± 3% and a smaller size of 70 ± 3 nm and a PDI of 0.2. To make the exosome-coated NPs, we used the extrusion method. After extrusion, the zeta potential of our EPHN was closer to -11mV compared to PLGA NPs (-50mV), which demonstrates that our NPs were coated with an exosomal membrane. We were able to make MSC exosome-coated drug-loaded polymeric nanoparticles and show that they were stable and uniform in size. Microfluidics-assisted doxorubicin-loaded PLGA nanoparticles show uniform size and higher entrapment efficiency. Release kinetics showed a strong burst phase release over the first 8 hours and subsequent sustained release up to 72 hours. In vivo biodistribution of EPHN shows better tumor accumulation compared to PLGA NP. The next stage in this project is in vivo tumor efficacy testing in breast cancer tumor models. This targeted NP system can serve as a novel therapeutic platform for developing improved treatments for metastatic TNBC.

Citation Format: Rohan Joshi, Jana Lampe, Jamboor K. Vishwanatha, Amalendu P. Ranjan. Exosome-based hybrid nanosystem for targeted TNBC therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 832.
Abstract 834: Synergistic effect of bacterial ghosts loaded with belinostat and betaine nano-emulsion: A potential treatment strategy for breast cancer
Authors: Deghedy Sara H. , Mahmoud Hoda E. , Hussein Ahmed A. , El-Kholy Mohammed A. , Gaafar Passent M. , Ali Mai M. , Sultan Ahmed S.
Published: April 2023

Breast cancer resembles the second-highest incidence rate of cancers worldwide with highly remarkable incidence and mortality rates. Recently, the main target behind the current study is to reduce the toxicity along with targeted delivery of different drugs separately and simultaneously to enhance the treatment options for breast cancer. A promising strategy for overcoming poor drug delivery is to recruit the evacuated bacterial cells which are known as Bacterial Ghosts (BGs) for drug delivery. The present study focused on formulating a new complex delivery system using a highly purified and safety-confirmed BGs of Salmonella Typhimurium ATCC 1403 strain loaded with class II HDACI (Belinostat) associated with nano-emulsion droplets of constant and non-toxic natural compound (Betaine), a glycine with three additional methyl groups. The formulated new complex delivery system of BGs was tested on two breast cancer cell lines, ER-positive ZR-75-1 and TNBC, MDA-MB-231 respectively. Using WST-1 proliferation assay, our data reports a combination index (CI) <1 indicating synergistic effect between Betaine nano-emulsion at low concentrations with Poly dispersity index less than 0.5 and BGs loaded with Belinostat per constant count of BGs CFU/ml. In comparison with treatment of either Betaine or Belinostat alone, the viability inhibition and apoptosis induction of both tested MDA-MB-231 and ZR-75-1 cells were amplified in the combined treatment of 72 hours. Furthermore, the achieved delivery showed a significant downregulated expression of angiogenesis-related markers such as VEGF and TSP-1 at concentrations of 1-mM Betaine nano-emulsion and 0.2-µM Belinostat after 24 hours of treatment. In conclusion, the proposed study introduced a new formulated delivery system of BGs with synergistic mechanistic effect of two different anti-cancer compounds as a promising strategy to inhibit breast cancer cell lines proliferation and angiogenesis.

Citation Format: Sara H. Deghedy, Hoda E. Mahmoud, Ahmed A. Hussein, Mohammed A. El-Kholy, Passent M. Gaafar, Mai M. Ali, Ahmed S. Sultan. Synergistic effect of bacterial ghosts loaded with belinostat and betaine nano-emulsion: A potential treatment strategy for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 834.
Abstract 84: Single-cell transcriptional analysis of multiple myeloma bone marrow aspirates using particle-templated instant partition sequencing (PIPseq)
Authors: Katzenelenbogen Yonatan , Zada Mor , Meltzer Robert , Fontanez Kristina , Amit Ido
Published: April 2023

Single-cell RNA-seq technologies have dramatically evolved in recent years. Despite these advances, the field faces some significant limitations in the versatility of current single cell technologies. These limitations include speed, ease of use, and the ability to sample both vast and minimal numbers of cells in a single batch - particularly in human samples. The PIPseq approach is based on Particle-templated Instant Partitions (PIPs) that allow single-cell encapsulation and barcoding using uniform droplet emulsions with minimal lab hardware. PIPseq is particularly effective for routine, high-throughput analysis of multiple myeloma bone marrow biopsies. Patients with multiple myeloma undergo a series of treatments aimed at depleting their malignant plasma cells (PC). Nevertheless, a high rate of relapse is observed despite low PC levels detected in the bone marrow (BM). Deep molecular profiling of the cellular pathways of rare PC, as well as their unique microenvironment, may shed light on the mechanisms underlying resistance to conventional therapies. As part of an ongoing clinical research, a bone marrow sample from a patient with multiple myeloma was molecularly characterized using the PIPseq T2 3’ Single Cell RNA Kit. Two distinct cell populations from a frozen BM biopsy were sorted into the T2 PIPseq tube: CD45+ cells (tumor microenvironment; TME) and rare malignant PC. In spite of the limited number of cells in the biopsy, PIPseq analysis clearly resolved both the malignant PC and the TME, demonstrating its utility for extensive analyses of fresh or frozen clinical myeloma samples.

Citation Format: Yonatan Katzenelenbogen, Mor Zada, Robert Meltzer, Kristina Fontanez, Ido Amit. Single-cell transcriptional analysis of multiple myeloma bone marrow aspirates using particle-templated instant partition sequencing (PIPseq) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 84.
Abstract 853: The impact of normal tissue density on tumor growth and evolution in a 3D whole-tumor model of lung cancer
Authors: Bravo Rafael Ramon , Robertson-Tessi Mark , Anderson Alexander R. , Gatenby Robert , Antonia Scott , Gray Jhanelle , Beg Amer , Schabath Matthew
Published: April 2023

We developed a novel modeling paradigm to specifically recapitulate 3D tissue at the resolution of CT imaging and yet capture single cell information: the population-based model (PBM). This PBM method allowed us to seed a model with individual cells that operate at the CT scale of cubic millimeters and grow to the scale of 10 cubic centimeters or more.

We used our PBM to investigate how well-known tumor traits of increased resource consumption and angiogenesis maybe differentially selected by a growing tumor in different densities of normal tissue. We found that tumor phenotypes that reduced consumption and increased angiogenesis grew the fastest in low density normal tissue, and vice versa in high density normal tissue.

We ran parameter sweeps of this model using temporal patient data from the NLST dataset, which consisted of untreated tumors in the low-density lung periphery. We saw that specific movement rate and proliferation rate parameters were required to fit the growth speed of the tumors, while a low consumption and high angiogenic phenotype was required to fit the radial density distribution of the tumors.

Citation Format: Rafael Ramon Bravo, Mark Robertson-Tessi, Alexander R. Anderson, Robert Gatenby, Scott Antonia, Jhanelle Gray, Amer Beg, Matthew Schabath. The impact of normal tissue density on tumor growth and evolution in a 3D whole-tumor model of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 853.
Abstract 855: Model-informed drug design (MIDD) exploration of a theoretical CD47xTAA bispecific antibody using available data from magrolimab, an anti-CD47 monoclonal antibody
Authors: Pace Emily A. , Millard Bjorn L.
Published: April 2023

Purpose: The goal of this study is to outline the process and utility of using fit-for-purpose, semi-mechanistic PK/PD modeling to inform the design of a theoretical CD47 bispecific antibody targeted to a generic tumor-associated antigen (CD47xTAA bsAb) with improved efficacy, tolerability, and PK compared to single target anti-CD47 therapies. Approach: To help guide the CD47xTAA bsAb design, a 3-compartment human model was built to include a target-mediated drug disposition (TMDD) component from CD47 expressed on red blood cells (RBC), white blood cells (WBC), and cells in the peripheral compartment. The model was parameterized using available literature data and calibrated against magrolimab human PK data that exhibited potential TMDD. The magrolimab model was used to identify key CD47 receptor occupancy (RO) thresholds associated with tolerability (the priming dose threshold) and efficacy (the recommended Phase 2 dose/RP2D threshold). Next, assumed drug properties and TAA expression levels for a CD47xTAA bsAb were implemented in a similar human model and drug PK and doses required to match key CD47 RO thresholds were explored across a range of single-arm binding affinities. Results: The calibrated magrolimab model projected target engagement levels on RBCs and WBCs and showed rapid antibody-induced CD47 pruning on RBCs that is consistent with clinical PD measurements. At TAA binding affinities that produce sufficient binding to tumor antigen, the model predicts that lowering the CD47 binding affinity allows for a higher starting dose that matches the priming threshold, while lowering the dose required to match the RP2D threshold. The model also shows that lowering the binding affinity to CD47 may improve PK exposure in the tumor compartment by reducing systemic CD47 TMDD. Conclusions: This modeling exercise demonstrates how semi-mechanistic PK/PD models can be used in model-informed drug design (MIDD) by leveraging the targeting advantage of avid bispecific antibody therapies and by using publicly available preclinical and clinical data. Furthermore, these model projections can help inform future studies with bispecific CD47 drug candidates.

Citation Format: Emily A. Pace, Bjorn L. Millard. Model-informed drug design (MIDD) exploration of a theoretical CD47xTAA bispecific antibody using available data from magrolimab, an anti-CD47 monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 855.
Abstract 86: Chemotherapy-induced microenvironmental changes promote dormant breast cancer cell outgrowth in adipose tissue
Authors: Ren Qihao , Ye Jiayu , Yuan Xiaoyu , Stewart Sheila
Published: April 2023

Despite the high 5-year survival rate for early-stage breast cancer patients, late recurrence of the disease remains a major challenge. Breast cancer can recur in approximately 20-25% of patients months to decades after an initial diagnosis, and there are few biomarkers that predict which patients will suffer from the recurrent disease. Unfortunately, this means clinicians and their patients must take a frustrating wait-and-see approach. Notably, disseminated tumor cells (DTCs) are frequently detected in early-stage breast cancer patients, and their presence predicts a higher chance of recurrence. However, why some patients with DTCs recur while others do not remains a mystery. Previous work has suggested that changes in the tumor microenvironment (TME) play an important role in the maintenance of tumor cell dormancy. When assessing the impact of doxorubicin (DOXO), a chemotherapeutic agent often used to treat breast cancer patients, on the TME, we noted that DOXO treatment led to the activation of dormant breast cancer cells in the visceral adipose tissue. Moreover, in accordance with the clinical observation that obese patients suffer a higher risk of cancer recurrence, we found that diet-induced obesity further exacerbated DOXO ability to reactivate dormant breast cancer cells in adipose tissue and this reactivation was associated with robust macrophage infiltration. These findings were interesting in light of our observation that tumor cells can metastasize to visceral fat depots in breast cancer patients. To understand why macrophages were recruited to the adipose tissue and how they led to tumor cell reactivation, we profiled the microenvironmental changes in visceral fat from Veh- and Doxo- treated mice using single-cell RNA-sequencing and found that the infiltrating macrophages exhibited unique pro-inflammatory and lipid-regulatory phenotypes. Our study provides novel insights into how TME impacts breast cancer dormancy and also highlights the importance of adipose tissue as a unique reservoir that potentiates cancer relapse.

Citation Format: Qihao Ren, Jiayu Ye, Xiaoyu Yuan, Sheila Stewart. Chemotherapy-induced microenvironmental changes promote dormant breast cancer cell outgrowth in adipose tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 86.
Abstract 861: Modelling the role of EGFR mutation in the initiation of never-smoker lung cancer
Authors: Coggan Helena M. , Weeden Clare E. , Dalwadi Mohit , Swanton Charles , Pearce Philip , Page Karen M.
Published: April 2023

Genetic mutations associated with never-smoker lung cancer frequently appear in normal lung tissue, in both cancer and non-cancer patients. It is unclear what circumstances are required for a mutation to fix in healthy tissue and eventually lead to a tumor. To investigate this, alveolar type-II murine cells were grown in a 3D nutrient system, forming spheroidal clusters. The volumes of these clusters were measured at 7 and 14 days and a mathematical model was devised to describe their growth. In the absence of an EGFR mutation, variations in growth between clusters can be explained by a skew-normal distribution of initial division rates. These rates decline exponentially as the clusters age, and the decline rate is the same across clusters. When cells possess an epidermal growth factor receptor (EGFR) mutation or are grown in the presence of interleukin-1 beta (il-1b) growth factor, clusters form more complex shapes, which we believe may be driven by branching processes and cell-cell interactions. Accordingly, more complex models are needed to explain their development. This work lays the groundwork for new, parameterized models of mutational spread in lung tissue, and a deeper understanding of the role of mutation in carcinogenesis.

Citation Format: Helena M. Coggan, Clare E. Weeden, Mohit Dalwadi, Charles Swanton, Philip Pearce, Karen M. Page. Modelling the role of EGFR mutation in the initiation of never-smoker lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 861.
Abstract 862: Pairwise and higher-order epistatic effects among somatic cancer mutations across oncogenesis of lung adenocarcinoma
Authors: Alfaro-Murillo Jorge A. , Dasari Krishna , Townsend Jeffrey P.
Published: April 2023

The somatic evolution of cancer depends on the rate at which new mutations are acquired, as well as the selective pressure acting on them. Further, mutual exclusivity or co-occurrence of mutations suggests that variants can exert antagonistic or synergistic epistatic effects on the selection of new mutations, contributing to a complex landscape of evolutionary trajectories. Estimation of pairwise and higher-order epistatic effects—essential to estimation of the trajectory of likely cancer genotoypes—has remained a challenge. We have developed a continuous-time Markov chain model that enables the estimation of mutation origination and fixation (flux), dependent on somatic cancer genotype. Coupling the continuous-time Markov chain model with a deconvolution approach provides estimates of underlying mutation rates and selection across the trajectory of oncogenesis. We demonstrate computation of fluxes and selection coefficients in a somatic evolutionary model of lung adenocarcinoma informed by a compilation of samples from multiple data sets including The Cancer Genome Atlas and the AACR Project GENIE. The approach enables inference of the most likely routes of site-specific variant evolution and estimation of the selection strength operating on each step along the route, a key component of what we need to know to develop and implement personalized cancer therapies. Finally, by comparing smoker and nonsmoker tumor cohorts, we reveal and describe distinct evolutionary trajectories, influenced not only by differential mutation, but also by the differential physiological effects that smoking can have on the selective regime of lung tissue. These distinctions disambiguate the physiological and mutational influences of smoking and can inform personalized treatment of the two groups.

Citation Format: Jorge A. Alfaro-Murillo, Krishna Dasari, Jeffrey P. Townsend. Pairwise and higher-order epistatic effects among somatic cancer mutations across oncogenesis of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 862.
Abstract 87: The role of miro1-mediated mitochondrial positioning in the development and metastasis of breast cancer
Authors: Gravelle Randi , McCoy Margaret , Shannon Nathaniel , Chang Martin , Cunniff Brian
Published: April 2023

Miro1 is an outer mitochondrial membrane protein that links mitochondria to motor protein complexes to support subcellular mitochondrial trafficking. Preliminary observations and published work has established that metastatic breast cancer cells have increased expression of Miro1 in comparison to breast cancer cells with low metastatic potential. Furthermore, tumors from breast cancer patients with high Miro1 expression have poorer survival rates compared to patients harboring tumors with low Miro1 expression. This data suggests that Miro1 has a functional role in aggressive breast cancers although this has not been widely studied or confirmed in advanced preclinical models. The objective of our research is to investigate the role of Miro1 in breast cancer tumorigenesis and metastasis. We hypothesize that that Miro1-mediated mitochondrial dynamics support subcellular signaling events driving cell migration, invasion, and tumorigenesis. To examine the function of Miro1 in promoting metastatic phenotypes in vitro, we generated human breast cancer cell lines (MDA-MB-231, MDA-MB-468, T-47d, and MCF7) with Miro1 knockdown using adenoviral shRNA. Knockdown of Miro1 to levels greater than 50% of that in control cells resulted in mitochondria sequestered around the nucleus and reduced cell migration. Additionally, MDA-MB-231 cells with ≥50% Miro1 knockdown resulted in a significant decrease in cell invasion through Matrigel coated membranes in transwell invasion assays. Our research has also uncovered altered phosphorylation of key cell migration and stress-response proteins including vinculin, focal adhesion kinase and ERK ½ when Miro1 is knocked down. To investigate the role of Miro1 in vivo, we generated a novel transgenic mouse model with inducible, tissue specific Miro1 deletion in mammary epithelial cells with concurrent polyomavirus middle T-antigen oncogene activation. We found that when breast cancer formation is induced utilizing the transgenic middle-T antigen system, our control mice form tumors at all mammary gland sites and have metastasis to the lungs. Conversely, mice with concurrent Miro1 deletion and middle T- antigen activation fail to develop tumors. This data suggests that Miro1 has a functional role in both mammary tumor onset and tumor cell migration and invasion, providing a possible new biomarker of tumor status and pathway for therapeutic intervention.

Citation Format: Randi Gravelle, Margaret McCoy, Nathaniel Shannon, Martin Chang, Brian Cunniff. The role of miro1-mediated mitochondrial positioning in the development and metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 87.
Abstract 871: Accurate estimation of open chromatin and intrinsic biases in bulk and single-cell CUT&Tag data
Authors: Hu Sheng'en , Chen Qingying , Zang Chongzhi
Published: April 2023

Histone modifications (HM) and transcription factors (TF) are aberrantly regulated in the pathogenesis of humans and are a major class of cancer cell dependencies. Precise detection of HM and TF binding genome-wide is essential for a better understanding of cancer epigenetics. Cleavage Under Targets & Tagmentation (CUT&Tag) is an easy and low-cost epigenomic profiling method that can be performed on a low number of cells and on the single-cell level. A large number of CUT&Tag datasets have been generated in cancer samples, providing a valuable resource. Unbiased analysis of CUT&Tag data is important for cancer epigenetics research. CUT&Tag experiments rely on the hyperactive transposase Tn5 for tagmentation. We performed integrative analysis of CUT&Tag and other high-throughput data, and found that Tn5 is subject to intrinsic sequence insertion biases (intrinsic biases). Preference of Tn5 captured reads toward chromatin accessibility regions (open chromatin biases) also confound the distribution of CUT&Tag reads. Both sources of biases confound the analysis of CUT&Tag data, especially for factors that are not expected to associate with accessible chromatin. High sparsity of single-cell data makes the effect of both biases more substantial compared to bulk data. We present a computational model for accurate characterization and quantitative estimation of biases in CUT&Tag data. This model paved the way for further development of computational tools for improving both bulk and single-cell CUT&Tag data analysis.

Citation Format: Sheng'en Hu, Qingying Chen, Chongzhi Zang. Accurate estimation of open chromatin and intrinsic biases in bulk and single-cell CUT&Tag data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 871.
Abstract 879: Exosomal mRNAs secreted from pancreatic cancer (PC) cell lines might include good candidates of biomarkers of PC patients
Authors: Rong Yuhan , Okubo Naoki , Fukui Risa , Suzuki Akihiro , Sato Motokazu , Tokuhisa Motohiko , Takeda Yuma , Kobayashi Noritoshi , Endo Itaru , Ichikawa Yasushi
Published: April 2023

Background: Exosomal mRNAs secreted from cancer cells are regarded as important messengers in communication system between cells. In PC, some exosomal RNAs from patients’ plasma were expected as biomarkers of PC. However, exosomes in the blood of cancer patients are not derived only from cancer cells. Relationship of RNAs produced in cancer cells and those secreted into exosomes is still unclear. In this study, we evaluated relationships between mRNAs in PC cell lines and those of exosomes secreted into supernatant and determined candidates for PC biomarkers from cancer specific exosomal mRNAs.

Material and Method: Three PC cell lines (PANC-1, MIAPaCa-2 and Capan-1) were used. Three studies were performed for each cell line. After 48h serum-free culture, the supernatants were collected, and exosomes were isolated by ultracentrifugation. The remaining cells in the culture dish were also collected and mRNAs were extracted. RNA-seq was performed for mRNAs extracted from cell and exosomal RNAs extracted from supernatant. Data was analyzed by R (version 4.2.1).

Results: In MIAPaCa-2, expression of cellular mRNAs and corresponding exosomal mRNAs showed strong correlation (R was higher than 0.7). In the other 2 cell lines, they showed moderate correlation (R was higher than 0.5). Then, expression of mRNA in the cells was approximately reflected in those in the exosomes. However, some mRNAs showed high expression in exosome and low expression in the cell. We focused on these mRNAs, and then, performed a differential analysis of the whole raw RNA data by using TCC package of R and found differentially expressed mRNAs.

Conclusion: Comparing with data of GEO database showing exosomal mRNA extracted from healthy volunteers, we finally find candidates of cancer specific exosomal mRNAs of PC.

Citation Format: Yuhan Rong, Naoki Okubo, Risa Fukui, Akihiro Suzuki, Motokazu Sato, Motohiko Tokuhisa, Yuma Takeda, Noritoshi Kobayashi, Itaru Endo, Yasushi Ichikawa. Exosomal mRNAs secreted from pancreatic cancer (PC) cell lines might include good candidates of biomarkers of PC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 879.
Abstract 881: Desmocollin-3 and tumor immune microenvironment: Bioinformatics evaluation
Authors: Dheeraj Arpit , Khamar Bakulesh M. , Shukla Chandreshwar P. , Jain Nayan K. , Modi Rajiv I.
Published: April 2023

Desmocollin-3 (DSC3) is a desmosomal adhesion molecule responsible for integrity and cell adhesion under normal physiological condition. Its expression is altered during carcinogenesis. DSC3 is a p53 responsive gene. Like other adhesion molecules, it is described to provide barrier for immune infiltration of tumor. To identify a specific immune gene signature related to DSC3, spearman correlation coefficient was determined for 207 immune related genes using Cbioportal in cancer having high and low level of DSC3 gene expression .We classified renal adenocarcinoma lung, hepatocellular carcinoma and renal cancer as DSC3low and squamous lung cancer, cervical cancer and head and Neck cancer as DSC3high based on DSC3 gene expression level. In DSC3high group, six genes were found to be correlated (Spearman correlation ≥ 0.3) with DSC3 expression (Table), while none of the 207 immune related genes were correlated with DSC3low expression. Of genes with positive correlation, CD44 is a marker for stem cell and is associated with resistance to chemotherapy while S100A12 and TOLLIP are associated with carcinogenesis and inflammation. Of immune genes with negative correlation, GATA6 is a tumor suppressive gene, IL-12A is responsible for T-cell-independent induction of IFNγ and CD63 is associated with survival as described in lung cancer. This findings suggests DSC3high expression may be associated with poor prognosis compared DSC3low . DSC3 expression is specific for squamous lung cancer and is used to differentiate it from adenocarcinoma lung. Squamous lung cancer is also known to have poor prognosis compared to adenocarcinoma of lung. We will be presenting full details about 207 selected genes and their correlation with DSC3 in our presentation.

Table: Spearman’s correlation of DSC3 with Immune genes DSC3 expression Immune genes with positive spearman’s correlation Immune genes with negative spearman’s correlation Cancer Median RSEM (N) CD44 S100A12 TOLLIP CD63 GATA6 IL12A LUSC 6208 (n=501) 0.4 0.3 0.4 -0.4 -0.3 -0.3 CESC 10086 (n=306) 0.6 0.6 0.6 -0.6 -0.4 -0.4 HNSC 15287(n=522) 0.3 0.3 0.3 -0.5 -0.3 -0.4 KIPAN 1.5 (n=720) < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 LIHC 2.0 (n=231) < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 LUAD 10 (n=496) < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3

Citation Format: Arpit Dheeraj, Bakulesh M. Khamar, Chandreshwar P. Shukla, Nayan K. Jain, Rajiv I. Modi. Desmocollin-3 and tumor immune microenvironment: Bioinformatics evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 881.
Abstract 898: Generation and validation of anti-linker monoclonal antibodies for the detection of surface expressed scFv-based CARs
Authors: Singh Amrik , L'Heureux Sarah , Sinapius Ryan , Kvorjak Michael , Wade Emma , Lohmueller Jason , Fisher Jeremy , Prioli Reginaldo
Published: April 2023

Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of cells with greater persistence, there is a need in multiple phases of the CAR-T development pipeline for highly specific detection reagents that can be leveraged to monitor the expression of CARs on the cell surface. Many commercially available CAR detection reagents, however, either lack specificity or are not versatile in their ability to detect CARs of differing antigen specificity. Here, we report on the generation and validation of rabbit monoclonal antibodies raised against two linker sequences that are commonly integrated into single-chain variable fragment (scFv)-based CARs. These antibodies serve as versatile detection reagents that can be used to interrogate the surface expression of CARs.

Methods: The monoclonal antibodies, E7O2V and E3U7Q, were generated by rabbits immunized with peptide sequences most commonly used to construct the linker region of scFv based CARs, Gly4Ser and Whitlow, respectively. E7O2V and E3U7Q were validated for specificity and versatility using flow cytometric analysis of non-transduced versus CAR-transduced cell lines and primary human T cells.

Results: Flow cytometric analysis of live Jurkat cells and primary human T cells transduced with CAR constructs revealed that E7O2V and E3U7Q could detect surface expressed CARs containing the appropriate linker sequence, independently of scFv specificity. No specific staining was observed on non-transduced cells.

Conclusions: In a flow cytometry assay, E7O2V and E3U7Q specifically detect surface expressed scFv-based CARs containing either a Gly4Ser linker or a Whitlow linker, respectively. Furthermore, these monoclonal antibodies are versatile in that they can also detect their respective linker sequence independently of scFv specificity. The potential exists to leverage these antibodies for CAR-T cell enrichment and for incorporation into multiparametric flow cytometry panels used to phenotype CAR-T cells during the discovery, manufacturing, and clinical phases of the development pipeline.

Citation Format: Amrik Singh, Sarah L'Heureux, Ryan Sinapius, Michael Kvorjak, Emma Wade, Jason Lohmueller, Jeremy Fisher, Reginaldo Prioli. Generation and validation of anti-linker monoclonal antibodies for the detection of surface expressed scFv-based CARs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 898.
Abstract 9: Histological characterization of two genetically engineered mouse models of non-small cell lung cancer
Authors: Leon Nicole F. , Baliu-Rodriguez David , Murugesh Deepa K. , Sebastian Aimy , Loots Gabriela G. , Hum Nicholas R.
Published: April 2023

Non-small cell lung cancer (NSCLC) patients exhibit minimal symptoms until late-stage disease, resulting in approximately 80% of patients being diagnosed at advanced stages when the response to therapy is low. Effective pre-clinical models are needed to accurately reflect NSCLC disease progression and improve basic NSCLC research. This work characterizes two genetically engineered mouse models (GEMM) that reflect human NSCLC. These mouse lines utilize a cre recombinase system to induce the expression of two oncogenic mutations (KrasG12D, Trp53fl) in club cells or alveolar epithelial cells based on cell type-specific promoters (Scgb1a1, Sftpc), thereby inducing NSCLC in mice. Furthermore, these mice harbor tumor cell-specific transgenes (GFP, MetRS) for cancer tracking. The MetRS transgene allows tumor cells to incorporate azide-bearing methionine analogs (ANL) into newly synthesized proteins which can then be detected via fluorescent noncanonical amino acid tagging (FUNCAT). Cancer mutations were induced at 6 weeks of age and lung samples were collected for histological analysis at early, mid, and late time points. Histological analysis of the club cell model revealed that hyperplasias are most abundant at the early time point and develop into invasive carcinomas within the bronchioles and alveoli at later time points. Histological scoring of this model showed that tumor burden increased from 28% to 61% of the total lung area after 15 weeks. The alveolar cell model progressed rapidly and exhibited adenocarcinomas and squamous cell carcinomas throughout disease progression. Histological scoring of this model revealed that tumor burden rapidly increased from 55% to 89% of total lung area after 5 weeks. FUNCAT analysis demonstrated that the incorporation of ANL within tumor-bearing mice was cancer-cell-specific. These findings suggest that the GEMM models accurately represent human NSCLC based on their cellular profile, morphology, and localization within the lung. The results also confirm that the tumor cells, and not the normal cells/tissues, are able to incorporate ANL into their proteins via the MetRS transgene. These models have the potential to identify biomarkers of NSCLC progression to improve patient prognosis.

*This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. LLNL-ABS-839507.

Citation Format: Nicole F. Leon, David Baliu-Rodriguez, Deepa K. Murugesh, Aimy Sebastian, Gabriela G. Loots, Nicholas R. Hum. Histological characterization of two genetically engineered mouse models of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 9.
Abstract 953: Development of a predictive biomarker signature for the highly potent SIK3 inhibitor OMX-0407
Authors: Maser Ilona-Petra , Lacher Sonja , Piras Vincent , Kracher Barbara , Zanziger Kai , Yule Murray , Loferer Hannes , Bissinger Stefan
Published: April 2023

Using the iOTarg genetic screening platform, salt-inducible kinase 3 (SIK3), a serine/threonine kinase of the AMP-activated protein kinase (AMPK) family was recently identified as a novel cell signaling modulator in cancer biology. OMX-0407, an orally available, single-digit nanomolar inhibitor of SIK3 was shown to inhibit tumor growth in a distinct panel of tumor models in vitro as well as in vivo by decreasing downstream pro-survival signaling of SIK3, enhancing caspase-mediated apoptosis and repolarizing the tumor microenvironment by decreasing regulatory T cells.

In a comprehensive viability screening, more than 200 murine and human cancer cell lines and patient derived tumor models of different indications were used to identify a distinct subset of cancers highly sensitive towards OMX-0407. Representative for these tumors, OMX-0407 demonstrated dose-dependent anti-tumor efficacy in the renal cell carcinoma model RENCA, the squamous non-small cell lung cancer model (sqNSCLC) KLN205 and the murine colorectal tumor model MC38. With significantly prolonged overall survival of more than 60% tumor growth inhibition in all tumor models, OMX-0407 demonstrated equivalent or superior efficacy compared to anti-PD-1 monotherapy. In addition, OMX-0407 enhances the therapeutic potential of the standard of care chemotherapy in a broad range of human cancer cells and murine tumor models of sqNSCLC.

Using comprehensive transcriptomics and proteomics analyses the selective activity profile of OMX-0407 on individual cancer cell lines and PDX models was used to identify and validate a predictive biomarker signature. With this gene signature, 84% of the selected cancer cells lines are correctly classified and would benefit from OMX-0407 therapy. The predictive biomarker will be evaluated in the clinical Phase I trial of OMX-0407 scheduled to start enrollment shortly.

In summary, OMX-0407, a first-in-class oral SIK3 inhibitor, demonstrates potent monotherapy efficacy in tumor indications with high unmet medical need equal or superior to anti-PD-1 checkpoint blockade. By screening sensitive and non-sensitive tumor cell lines and PDX models, we identified a response-prediction biomarker signature. In upcoming clinical studies, this predictive biomarker signature will be evaluated for it’s potential to enrich for patients highly responsive to OMX-0407 therapy.

Citation Format: Ilona-Petra Maser, Sonja Lacher, Vincent Piras, Barbara Kracher, Kai Zanziger, Murray Yule, Hannes Loferer, Stefan Bissinger. Development of a predictive biomarker signature for the highly potent SIK3 inhibitor OMX-0407 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 953.