bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3809: Differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner
Authors: Bareja Chanchal , Uboveja Apoorva , Saluja Daman
Published: June 2023

The p53 tumor suppressor family is classically activated after DNA damage and plays a central role in cell fate decisions. Although, the p53 family activates many of the same genes in response to DNA damage, p73 plays distinct biological functions in development and metastasis. It is likely that p73 activates a unique transcriptional network which is critical for its anti-metastatic and anti-invasive action. Long non-coding RNAs (lncRNAs) are a class of mRNA- like transcripts longer than 200 nucleotides. They lack protein-coding ability and are believed to be involved in various kinds of biological processes. Increasing evidence suggests that lncRNA are frequently aberrantly expressed in cancers. Therefore, the roles of dysregulated functional lncRNA in human malignant tumors have attracted considerable scientific interest. The objective of our study is to find out novel long non-coding RNAs that can act as transcriptional targets of p73 and to delineate their role in p73-mediated anti-metastatic response. For this purpose, we performed transcriptome sequencing in HCT116p73wt and HCT116p73KD cells and screened the data for modulation of expression of lncRNAs in differential manner. Quantitative Real Time PCR was further carried out to validate the data obtained after screening RNA seq Data. Promoter analysis was carried out for the identification of p73 binding sites in the selected upregulated or downregulated lncRNAs which was further confirmed by Luciferase reporter, ChIP and site directed mutagenesis assays. About six lncRNAs were observed to be significantly upregulated while four were down-regulated upon knockdown of p73. The promoters of selected lncRNAs were analysed in silico using TF Bind and JASPAR software for p73 binding sites and luciferase reporter assays suggested regulation of lncRNAs by p73. Chromatin immunoprecipitation showed promoter enrichment of the selected lncRNAs. Together, our study provides insights into the differential regulation of long non- coding RNAs in p73 dependent manner which further will provide the mechanism of their action at the genome level.

Citation Format: Chanchal Bareja, Apoorva Uboveja, Daman Saluja. Differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner. [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 3809.
Abstract 381: Inhibition of CCR6-CCL20 axis enhances cytotoxic effect of chemotherapeutics in colon cancer
Authors: Mir Hina , Singh Shailesh
Published: June 2023

Response of chemotherapeutic offered to treat colon cancer are often short-lived due to chemoresistance and severe toxicity associated with the therapy. Hence, development of less toxic treatment options is imperative to improve efficacy and therapeutic outcome. We have shown that chemokine CCR6 and its natural ligand is expressed in colon cancer and correlates with advanced disease. Aim of this study was to establish the association of CCCR6/CCL26 signaling axis on development of chemoresistance and chemotherapeutic response. Our data shows higher expression of receptor, poor cytotoxic effect of 5-Fluorouracil (5-FU) when cells are treated with CCL20 while better response to 5-FU when CCR6 signaling is blocked using anti-CCR6 monoclonal antibody. The observed differences in the cytotoxic effects of 5-FU could be primarily due to CCR6-mediated alteration of cell cycle, activation of cell survival and anti-apoptotic signaling. Further investigations in a preclinical model will rationalize developing CCR6 directed therapies to improve response to conventional chemotherapeutics and offer less toxic treatment options to colon cancer patients.

Citation Format: Hina Mir, Shailesh Singh. Inhibition of CCR6-CCL20 axis enhances cytotoxic effect of chemotherapeutics in colon 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 381.
Abstract 3856: Evaluation of replication protein A inhibitor, NERx329 in combination with EGFR mutant targeted therapy
Authors: Bhowmick Reshma , Turchi John J. , Jalal Shadia I.
Published: June 2023

Lung cancer is the leading cause of cancer related deaths worldwide with the majority (~80%) of patients diagnosed with non-small lung cancer (NSCLC). Targeted therapies directed against tumor initiating growth factor receptors are effective treatment options for driver mutation positive NSCLC. Osimertinib is a third-generation EGFR-Tyrosine Kinase inhibitor (TKI) that has activity against EGFR exon 19, exon 21, and T790M mutations. Unfortunately, Osimertinib treated patients acquire resistance and median progression free survival is 18 months. Moreover, Osimertinib does not cure stage IV EGFR mutant NSCLC. The acquisition of secondary mutations leading to activation of bypass pathways, MET amplification, EMT, compensatory pathway activation and histological transformation to small cell phenotype are major causes of acquiring resistance to EGFR TKIs. Proliferating cancer cells regularly experience a low level of replication stress. Additionally, receptor tyrosine kinases such as EGFR are known to interact with DNA repair proteins and impact DNA damage repair following chemotherapy, radiation therapy, and EGFR TKI treatment. However, the role of DNA repair pathways in EGFR-TKI resistance is unknown and the interaction between EGFR pathway, DNA damage, and repair pathways has not been fully elucidated. At low-level replicative stress promotes genomic instability but at a high level through mitotic catastrophe it causes cell death. Replication protein A is a critical sensor of the DNA damage response detecting replication stress. The small molecule inhibitor of RPA, NERx 329, sequesters active RPA and induces replication catastrophe and cell death. Here we demonstrate that NERx329 in combination with Osimertinib enhanced Osimertinib mediated cell death. CCk-8 assays performed after 48h of NERx329 and Osimertinib combination treatment result in a robust decrease in IC50 compared to single agent therapy. Dissection of signaling pathways leading to this death-promoting effect of NERx329 showed a combination of bypass pathway marker (AXL) and compensatory pathway marker (STAT) inhibition in AXL-high expressing EGFR mutant lung cancer cells. Whereas inhibition of STAT pathway was responsible for the death-promoting effect of NERx329 in AXL-low expressing EGFR mutant lung cancer cells. AXL and STAT inhibition are associated with induced DNA damage and impaired DNA repair. From these data, we infer that DNA damage repair pathways could be involved in TKI resistance and NERx329 could be a promising drug candidate for combination targeted therapy.

Citation Format: Reshma Bhowmick, John J. Turchi, Shadia I. Jalal. Evaluation of replication protein A inhibitor, NERx329 in combination with EGFR mutant targeted 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 3856.
Abstract 3857: Exploring mechanisms of resistance to elraglusib in pancreatic cancer PDX models
Authors: Weiskittel Taylor , Li Ding , Han Lingling , McDermott Joseph , Caneiro Benedito , Li Hu , Billadeau Daniel , Mazar Andrew
Published: June 2023

Metastatic pancreatic cancer patients who previously progressed on nab-paclitaxel/gemcitabine (average of 3 previous lines if treatment) were treated with elraglusib in combination with a rechallenge of this combination. The majority of patients demonstrated a prolonged progression free survival. While this early clinical evidence is highly encouraging, the biological determinants of elraglusib response are unknown. In vitro screens of elraglusib on pancreatic PDX cell lines showed that some tumor cells possess modest intrinsic resistance to elraglusib as measured by LD50 which ranged from 316.9 nM to 1080 nM in a screen of 10 cell lines. To explore this phenomena further, we induced resistance in three cell line models by growing cells in the presence of increasing concentrations of elraglusib. We then sequenced the transcriptome of the parental and resistant lines to identify potential mechanisms of elraglusib resistance. Comparison of RNA sequencing profiles identified upregulation of the Aldo-keto reductase (AKR) family of genes. This upregulation was confirmed by qRT-PCR and by immunoblot demonstrating that AKRs were upregulated at both the transcriptomic and protein level. We further quantified the AKR levels in the remainder of the 10 cell lines and found that many of the more resistant lines had high expression of AKR1C2. However, not all of the resistant lines had high expression which could be because they have a separate mechanism of resistance to elraglusib. A large body of literature has found that AKR genes provide resistance to many other small molecule inhibitors either by direct metabolism or by indirectly relieving oxidative stress. Given these results, we identify aldo-keto reductases as potentially associated with elraglusib resistance, but further investigation is warranted to establish a causal link between AKR and the resistant phenotype.

Citation Format: Taylor Weiskittel, Ding Li, Lingling Han, Joseph McDermott, Benedito Caneiro, Hu Li, Daniel Billadeau, Andrew Mazar. Exploring mechanisms of resistance to elraglusib in pancreatic cancer PDX models. [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 3857.
Abstract 4129: Preclinical assessment of GlycoConnect™ ADCs with potency-modulated derivatives of PNU-159,682
Authors: van Delft Floris L. , van Geel Remon
Published: June 2023

PNU-159,682 is an oxidized secondary metabolite of nemorubicin (MMDX), and substantially more potent (2100-6400-fold) than the commonly used chemotherapeutic anthracycline doxorubicin/adriamycin, but without the dose-limiting cardiotoxicity. Based on these beneficial features, PNU-159,682 is currently being clinically evaluated (phase 1) for solid tumor indications as a payload in various antibody-drug conjugates (NBE-002, SO-N102). It must be noted, however, that the HNSTD of current PNU-based ADCs in non-human primates (~1 mg/kg) may end up in a clinical dose that is sub-optimal to achieve high tumor uptake, due to putative target-mediated drug disposition in healthy tissue.

We embarked on a program to develop a set of 2nd-generation PNU-159,682 analogues with attenuated potency, to enable the generation of ADCs with a potentially improved pharmacokinetic profile. Moreover, we reasoned that the adaptation of the new PNU analogues to our proprietary best-in-class ADC technology (GlycoConnect™)1, in combination with our polar spacer technology (HydraSpace™)2 might enable PNU-based ADCs with significantly expanded therapeutic index (TI).

We here exhibit the chemical synthesis of a panel of PNU-analogues with attenuated potency and enhanced tolerability, based on specific tailoring of the aminosugar morpholino group. Homogeneous and stable GlycoConnect™/HydraSpace™ ADCs were subsequently generated and evaluated in vitro and in vivo to assess efficacy and tolerability. These studies demonstrate the potential of attenuated PNU derivatives for application in stable and site-specific ADCs with anticipated higher clinic dose and potentially improved therapeutic index.

1 Wijdeven et al., Enzymatic glycan remodeling-metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering. mAbs 2022, 14.

2 Verkade et al. A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates. Antibodies 2018, 7, 12, doi:10.3390/antib7010012.

Citation Format: Floris L. van Delft, Remon van Geel. Preclinical assessment of GlycoConnect™ ADCs with potency-modulated derivatives of PNU-159,682. [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 4129.
Abstract 416: Phosphorylation of CDK9 transduces ALK signaling to promote resistance to PARP inhibitors via RNA Pol II activated homologous recombination repair
Authors: Chu Yu-Yi , Gao Jianjun , Yu Dihua , Hung Mien-Chie
Published: June 2023

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are currently the most promising drugs to induce synthetic lethality in tumor with homologous recombination (HR) deficiency. However, development of resistance to PARPi is an almost universal occurrence in tumor despite initial sensitivity. Additionally, how to identify the right patients for PARPi monotherapy or in combination with other agents remains a substantial clinical challenge. Therefore, understanding the molecular underpinnings of PARPi resistance is critical to the development of rational combinatorial treatment strategies and identification of novel biomarkers which will in turn facilitate optimization of outcomes for patients receiving PARPi. By employing a screening procedure using phospho-RTK antibody array, our study identifies phosphorylated ALK (p-ALK) expression is higher in PARPi/platinum resistant cells relative to sensitive cells. We further demonstrate that p-ALK expression in human ovarian cancer tissue is associated with resistance to PARPi and platinum-based chemotherapy. Notably, FDA approved ALK inhibitors induce an HR-deficient phenotype and sensitize PARPi/platinum-resistant tumors to PARPi in vitro and in vivo. Mechanistically, ALK contributes to PARPi resistance by tyrosine phosphorylating CDK9 at Y19, thereby increasing the protein stability and kinase activity of CDK9 which in turn stabilizes positive transcriptional elongation factor b (P-TEFb) to activate RNA pol II and turn on HR repair genes transcription. Importantly, the phosphorylated counterparts of ALK/CDK9 complex (p-ALK/p-Y19 CDK9) can serve as a biomarker to identify patients with PARPi/platinum-resistant tumors which most likely to respond to dual PARP and ALK inhibition. Together, our findings identify p-ALK/p-Y19-CDK9 mediated stabilization of P-TEFb as a critical mechanism of PARPi resistance and supports a biomarker-guided therapeutic strategy combining ALKi and PARPi to induce synthetic lethality.

Citation Format: Yu-Yi Chu, Jianjun Gao, Dihua Yu, Mien-Chie Hung. Phosphorylation of CDK9 transduces ALK signaling to promote resistance to PARP inhibitors via RNA Pol II activated homologous recombination repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 416.
Abstract 4166: Effects of cyproheptadine in eliciting natural killer cell-mediated immune response in bladder cancer
Authors: Kumari Himani , Lin Guan-Ling , Yeh Chih-Chieh , Chen Ciao-Ni , Chuang Yu-Ming , Huang Wan-Hong , Huang Wen-Long , Lin Steven , Chan Michael W.
Published: June 2023

Bladder cancer is one of the most common occurring cancers in urothelial carcinoma. The previous study suggested that cancer cells undergo immunoevasion due to epigenetic silencing of NKG2DL, in which NKG2D-NKG2DL interaction induced natural killer (NK) cell-mediated cytotoxicity. We hypothesized that NK-mediated cytotoxicity can be reactivated by restoring the expression of NKG2DL on tumor cells. Our previous studies demonstrated that cyproheptadine (CPH) is a novel HDAC inhibitor exhibiting antitumor activity by cell cycle arrest and apoptosis. To understand the mechanism behind, RNA-seq showed an upregulation of ULBP2, a NKG2DL, in bladder cancer cells treated with CPH. Interestingly, upregulation of ULBP2, was accompanied with the enrichment of H3K27Ac and H3K4me3 at ULBP2 promoter, was observed in cells treated with CPH. Increased NK-specific lysis was observed in CPH-treated cells co-cultured with NK-92 cells. More importantly, treatment of CPH enhanced the efficacy of immune checkpoint inhibitor in a syngeneic mice bladder cancer model. In conclusion, our results suggested that CPH may be a novel HDAC inhibitor which may elicit immune response in NK-cell dependent manner.

Citation Format: Himani Kumari, Guan-Ling Lin, Chih-Chieh Yeh, Ciao-Ni Chen, Yu-Ming Chuang, Wan-Hong Huang, Wen-Long Huang, Steven Lin, Michael W. Chan. Effects of cyproheptadine in eliciting natural killer cell-mediated immune response in bladder 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 4166.
Abstract 433: Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in non-small cell lung cancer
Authors: Uzunbas Gizem Karsli , McVeigh Quinn , Gupta Akansha , Hoyt Stephanie , Cherniack Andrew , Siegel Franziska , Siegel Stephan , Meyerson Matthew , Greulich Heidi
Published: June 2023

Molecular therapies targeting EGFR-mutant non-small cell lung cancer (NSCLC) have dramatically improved prognosis for patients with the classical activating mutations, L858R and exon 19 deletion. Tumors harboring EGFR exon 20 insertions do not respond to inhibitors of classical activating mutations. Agents such as amivantamab and mobocertinib have been approved for treatment of lung cancer patients with exon 20 insertions, but agents with an improved selectivity profile versus wild-type EGFR are still needed. BAY 2927088, currently in phase I clinical trials, is a novel reversible inhibitor that targets EGFR exon 20 insertion mutations with decreased activity towards wild-type EGFR. Although most patients with advanced EGFR-mutant NSCLC eventually progress due to acquired resistance to EGFR inhibitors, the mechanisms of resistance to BAY 2927088 are currently unknown. Using two different methods of in vitro resistance generation, we identified mechanisms of intrinsic and acquired resistance to BAY 2927088 and other EGFR inhibitors. With the first method, involving initial incubation with a high concentration of inhibitor, we isolated drug-tolerant persister cells that underwent a transcriptional state transition resembling EMT. With the second method, involving gradual escalation of inhibitor concentrations, we identified NRAS hotspot mutations as a mechanism of resistance to BAY 2927088 and other EGFR inhibitors. As a parallel approach, we are developing a deep-scanning mutagenesis assay of the EGFR kinase domain to identify mutations that could cause resistance. Understanding these resistance mechanisms will help to identify second-line treatments for exon 20 insertion patients who develop resistance to EGFR inhibition.

Citation Format: Gizem Karsli Uzunbas, Quinn McVeigh, Akansha Gupta, Stephanie Hoyt, Andrew Cherniack, Franziska Siegel, Stephan Siegel, Matthew Meyerson, Heidi Greulich. Mechanisms of resistance to BAY 2927088, the first reversible inhibitor targeting EGFR exon 20 insertion mutations in 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 433.
Abstract 4398: Validation of an updated algorithm to identify non-small cell lung cancer (NSCLC) patients in administrative claims databases
Authors: Patel Sandip Pravin , Wang Rongrong , Zhou Summera Qiheng , Sheinson Daniel , Johnson Ann , Lee Janet
Published: June 2023

Purpose: Administrative claims data using the ICD-9-CM or ICD-10-CM coding systems are not detailed enough to distinguish between lung cancer subtypes, which presents challenges for real-world research. This study updated and validated a previous treatment-based algorithm that uses claims data to identify NSCLC vs small-cell lung cancer (SCLC) (Turner RM, et al. Front Pharmacol 2017;8:883).

Methods: This study used Optum’s de-identified Market Clarity Data (2007-2021) which deterministically links medical and pharmacy claims with electronic health record (EHR) data from providers across the continuum of care. Included patients had lung cancer diagnosis and histology information available in the EHR, which is the gold standard for validation. Patients were required to have ≥3 months of continuous health plan enrollment before and after the diagnosis date. First, to replicate the Turner algorithm, patients were identified between Jun 2014 and Oct 2015 with any first-line NSCLC treatment and no SCLC treatment (Step 1). Next, the Turner algorithm was applied from Nov 2015 to Dec 2020 to evaluate if performance decreased given the approval of immunotherapies since 2015 (Step 2). Finally, the Turner algorithm was updated with NSCLC treatments approved since 2015 in concordance with the latest US treatment guidelines. Algorithm performance at each step was measured using sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).

Results: Sample sizes and performance statistics for each step are presented in the Table. From Step 1 to Step 2, specificity and PPV decreased while sensitivity and NPV increased. Algorithm performance improved slightly for all measures from Step 2 to Step 3.

Conclusions: This updated treatment-based algorithm improves validity and accuracy in identifying NSCLC patients in claims databases and supports its use in real-world research using claims databases when histology data is unavailable.

Step 1: Turner replication Step 2: Turner 2015-2020 Step 3: Updated Turner 2015-2020 Date range Jun 2014-Oct 2015 Nov 2015-Dec 2020 Nov 2015-Dec 2020 Sample size 406 2573 2744 Sensitivity 0.873 0.920 0.932 Specificity 0.933 0.865 0.923 PPV 0.987 0.976 0.988 NPV 0.560 0.640 0.673

Citation Format: Sandip Pravin Patel, Rongrong Wang, Summera Qiheng Zhou, Daniel Sheinson, Ann Johnson, Janet Lee. Validation of an updated algorithm to identify non-small cell lung cancer (NSCLC) patients in administrative claims databases. [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 4398.
Abstract 4408: Provisional prognostic score for monomorphic epitheliotropic T-Cell lymphoma
Authors: Haddad Philip A. , Keerty Dinesh , Dadi Neelakanta
Published: June 2023

Background: Monomorphic Epitheliotropic Intestinal T-cell Lymphoma (MEITL) is a rare extranodal intestinal PTCL. It is an aggressive disease with an overall poor prognosis. However, the disease presents a diverse spectrum of survival outcomes. We conducted this study to develop a provisional MEITL prognostic score (MPS) for this rare disease.

Methods: We used our updated MEITL database, which contains retrospective data on 218 cases. Such data included demographics such as sex, age, and race. It also included disease presentation symptoms, duration of symptoms before diagnosis, site(s) of the disease, stage, blood counts, disease immune and molecular phenotype, types of treatment, quality of response, and survival outcomes. Out of the 218 cases, only 140 cases had complete survival and outcomes data, the sample chosen for this study. Cox proportional-hazards model and Log-rank tests were used to assess the influence of clinicopathologic factors on overall survival (OS). Due to the relatively small training cohort, we included factors that statistically impacted OS as well as factors that numerically trended that way.

Results: The median OS of the cohort was 11 months. The following dichotomous variables were identified as impactful prognostic factors in this cohort: Bone marrow involvement (9 vs. 14 months), Non-biclonal TCR status (10 vs. 17.5 months), CD56+ (10 vs. 26 months), Ki67>70% (4 vs. 8 months), cMyc expression >15% (7 vs. 12 months). A prognostic model was devised using these variables to identify different levels of risk. Each variable was assigned a score of 1 when present, except for cMyc, which was assigned 2 points for having the highest hazard ratio among all the variables. In this exploratory cohort, low risk was assigned a score of 0, intermediate risk a score of 1-2, and high risk a score of 3-6. This prognostic score system led to our cohort's most optimal risk discriminatory model, where low, intermediate, and high risk had a median OS of 26, 12, and 4 months, respectively (p=0.002).

Conclusion: This MPS is a promising new tool for risk-stratifying patients with MEITL. However, it still needs prospective validation.

Citation Format: Philip A. Haddad, Dinesh Keerty, Neelakanta Dadi. Provisional prognostic score for monomorphic epitheliotropic T-Cell lymphoma. [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 4408.
Abstract 4454: Loss of CX3CL1 expression mediates immune evasion in STK11 mutated lung adenocarcinomas
Authors: Eksioglu Eria , Wright Gabriela M. , Percy Trent R. , Wright Kenneth L. , Cress W. Douglas
Published: June 2023

Immunotherapy has improved survival for many cancer patients, especially for immunogenic malignancies such as lung cancer. However, even in lung cancer, the response rate for anti-PD1 therapy (for example) does not exceed 20%. Thus, it is critical to identify mechanisms that block the response to immune checkpoint therapies. STK11 (also known as LKB1) is encoded by the serine threonine kinase 11 gene (STK11). Patients harboring tumors with STK11 mutations have reduced infiltrates of cytotoxic T-cells and clinical studies have shown that they respond poorly to anti-PD1 or anti-PDL-1 therapies regardless of PDL-1 status, which otherwise predicts benefit. Herein, we have used gene expression data from a cohort of 442 lung adenocarcinoma patients to identify CX3CL1 (fractalkine) as a gene silenced in STK11 mutant tumors with potential to be a key direct modulator of the immune system relevant to STK11 loss. To further explore this hypothesis, we have edited the STK11 gene in A549 cells back to wild type (STK11 corrected). As predicted, restoration of STK11 function resulted in modest expression of CX3CL1 as measured by Western blotting. Unexpectedly, exposure of STK11 corrected cells to human immune cells isolated from the blood of healthy donors resulted in a 5 to 10-fold increase in CX3CL1 suggesting interactive signaling between tumor and immune cells. Using transwell assays and STK11 corrected A549 cells we find that restoration of functional STK11 increases immune cell migration, adhesion and invasion in vitro. Finally, cytotoxicity assays demonstrate that STK11 corrected A549 cells are 3 to 5-fold more sensitive to immune cell killing. Taken together these results suggest the hypothesis that CX3CL1 loss mediates immune evasion in STK11 mutant lung adenocarcinomas. Future work will further explore the mechanisms underlying this pathway in preclinical models.

Citation Format: Eria Eksioglu, Gabriela M. Wright, Trent R. Percy, Kenneth L. Wright, W. Douglas Cress. Loss of CX3CL1 expression mediates immune evasion in STK11 mutated lung adenocarcinomas. [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 4454.
Abstract 4591: Mechanisms of tumor-mediated endothelial cell proliferation independent of VEGF
Authors: Sheng Kevin L. , Raj Jude , Wood Kris C.
Published: June 2023

A hallmark of cancer is dysregulated angiogenesis and blood vessel co-option, which are often driven by vascular endothelial growth factor (VEGF). Despite the key role of VEGF signaling in promoting endothelial cell (EC) proliferation, survival, and migration, the clinical use of VEGF inhibitors has modest success and faces many challenges. In particular, patients demonstrate variable initial responses to anti-VEGF therapy, and many of those who respond to treatment subsequently develop resistance. These challenges suggest that there may be VEGF-independent mechanisms driving EC proliferation in the tumor microenvironment. To study these mechanisms, we established an in vitro model using a dual-reporter system in a co-culture model of ECs and tumor cells. Specifically, we used an immortalized endothelial cell line (SVEC4-10) expressing GFP and Renilla luciferase and a mouse renal carcinoma cell line (RENCA) expressing Tomato and Firefly luciferase. This model allowed us to independently evaluate the growth rates and cell viabilities of both ECs and tumor cells in direct co-culture. Our preliminary data revealed that ECs exhibit increased growth rates when directly co-cultured with tumor cells. Interestingly, this EC proliferation is VEGF-independent and not impacted by VEGF-A knockout in tumor cells or with treatment of a pan-VEGFR small molecule inhibitor (axitinib). To further characterize if our phenotype was due to other tumor secreted factors, we used a transwell setup to eliminate direct contact between ECs and tumor cells and found that EC growth rates were substantially mitigated. These results revealed that tumor cells induced EC proliferation through a physical cell-to-cell interaction that is independent of VEGF. Further characterizing this unknown mechanism may inform therapeutic interventions to overcome the clinical challenges of anti-VEGF therapies for cancer. To expand our understanding of this VEGF-independent EC proliferation, we aim to characterize the cell surface proteome from tumor-conditioned ECs using cell surface mass spectrometry. We will validate hits from this study using our in vitro model and translate these findings to in vivo mouse models. We expect that our current and future research will reveal new insights into tumor co-option of the vascular system and identify novel targets for developing therapies to effectively treat cancer.

Citation Format: Kevin L. Sheng, Jude Raj, Kris C. Wood. Mechanisms of tumor-mediated endothelial cell proliferation independent of VEGF. [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 4591.
Abstract 4627: Immune profiling of immunotherapy and adjuvant chemotherapy pretreatment NSCLC tissues by CODEX
Authors: Monkman James , Moradi Afshin , O'Leary Connor , Wu Zhenqin , Hamel Steven , Mason David , Schneider Fabian , Mansfield James Robert , Meyer Aaron , O'Byrne Ken , Kulasinghe Arutha
Published: June 2023

Non-small cell lung cancer (NSCLC), including adenocarcinoma and squamous cell carcinoma subtypes, is a leading cause of cancer deaths worldwide. Treatment of NSCLC has advanced from chemotherapy modalities to the use of immunotherapy, namely immune checkpoint inhibitors (ICIs) which enhance the adaptive immune response against tumour cells. Thus, while the immune cell composition of tumours and its influence on treatment outcomes is poorly understood, it likely holds the key to effective, personalized treatment regimens.

Here we profiled an adjuvant chemotherapy (n=61) as well as a second line immunotherapy (n=42) NSCLC cohort by the Phenocycler CODEX technology (highplex spatial proteomics) to investigate the association between immune composition and patient outcome. We applied a panel of 38 markers to delineate naïve, memory, cytotoxic and hyperactivated T cell states, as well as B cells, Tregs and myeloid lineage innate immune cell types. Our study sought to understand the heterogeneity of tumour-immune composition across patients and investigate the spatial neighbourhoods and clusters that these cells inhabit within TMA cores. We used Phenoplex™ software for tissue segmentation (into classes for tumor, stroma, artifacts, blood vessels, etc.), cellular segmentation, and cellular phenotyping based on thresholds for each marker, and then performed spatial analyses (distances, interactions, neighborhoods) using SpatialMap1 to identify cellular motifs associated with clinical phenotypes.

Our study has identified spatial features associated with immune contexture linked to therapy outcome in both chemotherapy and immunotherapy modalities. Taken together, our study demonstrates the utility of spatial proteomics to identify cellular features associated with outcome to therapy in lung cancer.

1) Trevino A, Ivison G, Hamel S, Chiou A (2022). SpatialMap: Analysis of Spatial Biology Data. R package version 0.4.57.

Citation Format: James Monkman, Afshin Moradi, Connor O'Leary, Zhenqin Wu, Steven Hamel, David Mason, Fabian Schneider, James Robert Mansfield, Aaron Meyer, Ken O'Byrne, Arutha Kulasinghe. Immune profiling of immunotherapy and adjuvant chemotherapy pretreatment NSCLC tissues by CODEX. [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 4627.
Abstract 4742: Familial adenomatous polyposis epigenetic landscape as a precancer model of colorectal cancer
Authors: Lee Hayan , Krieger Gat , Clark Tyson , Zhu Yizhou , Khan Aziz , Hanson Casey R. , Horning Aaron , Esplin Edward D. , Badu Mohan , Paul Kristina , Chiu Roxanne , Bahmani Bahareh , Nevins Stephanie , Weimer Annika K. , Jaimovich Ariel , Curtis Christina , Greenleaf William , Ford James M. , Lipson Doron , Shipony Zohar , Snyder Michael P.
Published: June 2023

Aberrant shifts in DNA methylation have long been regarded as an early biomarker for cancer onset and progression. However, it is unclear when methylation aberrance starts and how it interacts with other epigenomic modifications. To address how epigenomic changes occur and interact during the transformation from normal healthy colon tissue to malignant colorectal cancer (CRC), we collected 51 samples from 15 familial adenomatous polyposis (FAP) and non-FAP colorectal cancer patients. We generated 30-70x of whole-genome enzymatic methylation sequencing (WGEM-seq) data via the novel Ultima Genomics ultra high-throughput sequencing platform. We observed hypermethylation and hypomethylation emerge early in the malignant transformation process in gene promoters and distal regulatory elements. We performed multifaceted analysis on methylation alterations with whole-genome sequencing (WGS), transposase accessibility (ATAC-seq), high-resolution chromatin accessibility (Tri-C), and gene expression (RNA-seq) data. Our multidimensional analysis demonstrates how collectively epigenomic alterations have affected gene expression throughout normal colon mucosa, benign and dysplasia polyps to adenocarcinoma. Epigenomic changes start as early as benign polyps, followed by other epigenomic shifts, including bivalent domains. Various epigenomic aberrances are associated with concomitant gene expression level changes. Our integrative analysis of multi-epigenomics data implicates collective and cumulative epigenomic instability in the early onset of colon carcinogenesis.

Citation Format: Hayan Lee, Gat Krieger, Tyson Clark, Yizhou Zhu, Aziz Khan, Casey R. Hanson, Aaron Horning, Edward D. Esplin, Mohan Badu, Kristina Paul, Roxanne Chiu, Bahareh Bahmani, Stephanie Nevins, Annika K. Weimer, Ariel Jaimovich, Christina Curtis, William Greenleaf, James M. Ford, Doron Lipson, Zohar Shipony, Michael P. Snyder. Familial adenomatous polyposis epigenetic landscape as a precancer model of colorectal 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 4742.
Abstract 4745: Prostatic fluid-based cfDNA methylation profiling distinguish benign hyperplasia from prostate cancer
Authors: Dong Hang , Tang Haoran , Zhang Yue , Jia Shidong
Published: June 2023

Introduction: A challenge in the early diagnosis of prostate cancer (PC) is to distinguish between malignant tumor and benign prostatic hyperplasia (BPH) because of the similar symptoms. Methylation profiles provide a way for discriminating BPH from PC, which could prevent overdiagnosis and overtreatment. Applying methylation biomarkers to liquid biopsy specimens provides higher sensitivity to detect tumors.

Methods: Prostatic fluid samples were prospectively collected from 40 patients following digital rectal exam and isolated cell-free DNA to perform whole genome methylation sequencing using PredicineEPIC platform. Combing differentially methylated regions (DMR) with methylation signature identified from massive public data, a panel of methylation patterns was developed. The difference of methylation signal in profiled region were implemented by machine learning to robustly distinguish BPH from prostate cancer.

Results: We identified 324 biomarkers from prostatic fluid samples and collected 46 methylation biomarkers reported from previous research. Regions with extreme difference between benign prostatic hyperplasia and prostate cancer were identified as candidate biomarkers. A diagnostic model trained on these 55 biomarkers could distinguish BPH from PC in 10-fold cross-validation (AUC = 0.92). And this model could be applied for discriminating normal tissue from prostate tumor tissue with highly performance (AUC > 0.95). Using this set of regions, we developed an NGS-based panel for detecting methylation alteration of cfDNA from prostate liquid, which inherited the ability of differentiation.

Conclusion: These methylation patterns could be developed as the novel biomarkers to support the development of prostatic fluid-based non-invasive diagnostic test and reduce unnecessary biopsies. This panel could be applied for differential diagnosis of benign hyperplasia and prostate cancer.

Citation Format: Hang Dong, Haoran Tang, Yue Zhang, Shidong Jia. Prostatic fluid-based cfDNA methylation profiling distinguish benign hyperplasia from prostate 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 4745.
Abstract 4886: PARP-1 and γ-GCS inhibitors enhance platinum sensitivity in ovarian cancer
Authors: Devi Luxmi , Sharma Ashok , Kumar Lalit
Published: June 2023

Background: Ovarian cancer is the most important cause of gynaecological cancer-related mortality globally. Approximately 80% of patients are diagnosed with advanced stage (FIGO stage III-IV) of ovarian cancer. The standard treatment approach for advanced EOC is primary debulking surgery followed by adjuvant chemotherapy using taxanes plus platinum. Resistance to platinum based chemo remains a key reason for treatment failure in advanced epithelial ovarian cancer (EOC). Therapeutic efficacy of Poly [ADP-ribose] polymerase 1 (PARP1) and Gamma-glutamyl cysteine Synthetase (γ-GCS)inhibitors in ovarian cancer remains undefined, either as a single agent or in combination with cisplatin.

Material and Methods: We determined the combined effect of PJ34 (PARP1 inhibitor) and BSO (Buthionine-sulfoximine- (γ-GCS inhibitor) with cisplatin in ovarian cancer cell lines (OVSAHO, KURAMOCHI, and IGROV1) by MTT assay. PARP1 and GCS levels were analyzed by western blotting. We employed flow cytometry for cell cycle analysis as well as for apoptosis studies. Analysis was done using Graph Pad Prism-6. Results were considered to be statistically significant when p value was < 0.05.

Results and Conclusion: Combination of cisplatin with PJ34 and BSO augmented cisplatin toxicity in vitro by decreasing cell proliferation and enhancing cell cycle block and cell death when compared with either single agent. A significant cell death (p= <0.05) was observed with the combination of cisplatin, BSO and PJ34.

Conclusion: In conclusion, our study provide experimental evidence on the combined effect of PARP-1 and GCS inhibition on CDDP-induced cytotoxicity in epithelial ovarian cancer cells. We propose that PARP-1and BSO inhibitors can be combined with cytotoxic chemotherapeutic agents such as CDDP to enhance the cytotoxicity of chemotherapeutic agents and inhibit key signalling pathways involved in cell proliferation and apoptosis. Future comprehensive studies are needed to demonstrate further validation of above combination.

Keywords: Ovarian Cancer, cisplatin, PARP-1, PJ34, γ -GCS, β-actin, Glutathione.

Abbreviations: Epithelial Ovarian Cancer (EOC), cisplatin (CDDP), Glutathione (GSH), Gamma Glutamyl Cysteine Synthetase (γ-GCS), Poly (ADP ribosyl) polymerase-1 (PARP1), Nucleotide excision repair (NER),homologous repair (HR), Enzyme-linked immunosorbent assay (ELISA), Buthionine Sulfoximine (BSO), Quantitative Polymerase Chain Reaction (qPCR), Peripheral Blood (PB). Propidium Iodide(PI).

Citation Format: Luxmi Devi, Ashok Sharma, Lalit Kumar. PARP-1 and γ-GCS inhibitors enhance platinum sensitivity in ovarian 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 4886.
Abstract 4894: The anti-ROR1 ADC STRO-003 demonstrates immune-modulating properties that may enhance checkpoint blockade
Authors: Yam Alice , Kiefel Helena , McGeehan Andrew , Yuan Robert , Zhou Sihong , Moreira Dayson , Dutta Indrani , Li Xiaofan , Tran Cuong , Yin Gang , Bedard Kristin , Hallam Trevor
Published: June 2023

There is growing evidence that tumor-targeted cytotoxins can also enhance anti-tumor immunity by inducing immunogenic cell death (ICD) in tumor cells and promoting recruitment of immune effector cells. We sought to investigate the immune stimulating potential of STRO-003, an antibody drug conjugate (ADC) composed of an anti-Receptor Orphan Receptor Kinase 1 (ROR1) antibody conjugated to a topoisomerase I targeted exatecan warhead via a cleavable linker. ROR1 is an oncofetal transmembrane receptor with restricted expression in normal tissues. Its overexpression in several cancer indications has been described, including in ovarian, non-small cell lung carcinoma (NSCLC), triple negative breast cancer (TNBC), and hematological malignancies, thus making it an ideal ADC target. We have previously demonstrated potent in vivo activity of STRO-003 in a panel of ROR1-expressing lung cancer patient-derived xenograft (PDX) models. Here we show that the exatecan warhead, SC3386, and STRO-003 ADC induced ICD in vitro as evidenced by presentation of cell-surface calreticulin and release of HMGB1. Consistent with this finding, STRO-003-treated cells elicited monocyte activation in a PBMC co-culture assay. To determine if these immunogenic properties could improve therapeutic efficacy, we evaluated STRO-003 in combination with an immune checkpoint inhibitor in a mouse syngeneic model expressing ROR1. We have demonstrated that STRO-003 in combination with checkpoint inhibition significantly enhances efficacy in a syngeneic mouse model and supports durable anti-tumor immunity. Follow-up vaccination studies were performed to further explore the significance of STRO-003-induced ICD and protective immunity in vivo. These results demonstrate that tumor cells pre-treated with STRO-003 or SC3386 undergo potent immunogenic cell damage which can, in turn, mount protective immunity in vivo.

Citation Format: Alice Yam, Helena Kiefel, Andrew McGeehan, Robert Yuan, Sihong Zhou, Dayson Moreira, Indrani Dutta, Xiaofan Li, Cuong Tran, Gang Yin, Kristin Bedard, Trevor Hallam. The anti-ROR1 ADC STRO-003 demonstrates immune-modulating properties that may enhance checkpoint blockade. [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 4894.
Abstract 5081: Anti-tumor activity of HPN217, a BCMA-targeting tri-specific T cell engager, is enhanced by γ-secretase inhibitors in preclinical models
Authors: Laurie Payton C. , Molloy Mary Ellen , Ng Patrick P. , Anand Banmeet S.
Published: June 2023

HPN217 is a Tri-specific T Cell-Activating Construct (TriTAC) currently being evaluated in a phase 1 clinical trial for relapsed or refractory multiple myeloma (MM) (NCT04184050). It consists of a single domain antibody (sdAb) specific for B-cell maturation antigen (BCMA), a serum albumin-specific sdAb for half-life extension, and a single chain Fv (scFv) specific for the CD3ε subunit of the T cell receptor (TCR) complex for T cell engagement. BCMA is a cell surface receptor highly expressed on malignant plasma cells in MM patients and plays an important role in B-cell proliferation and survival. Soluble BCMA (sBCMA) is produced when the extracellular domain of BCMA is cleaved by γ-secretase and enters circulation. sBCMA may act as a sink for BCMA targeted therapies, potentially decreasing anti-tumor efficacy. Previous studies have shown γ-secretase inhibitors (GSIs) can increase membrane bound BCMA expression on MM cells and decrease sBCMA concentrations, providing a rationale for combining GSIs with HPN217. Here we describe preclinical studies evaluating that combination in vitro and in vivo. To evaluate the effects of GSIs on BCMA expression, MM cell lines MOLP8, KMS-12-BM, and MM.1S were incubated with GSIs LY3039478, PF03084014, or RO4929097overnight. GSI treatment led to a 3-7-fold increase in BCMA expression on MM cell surface. To determine the effects of increased BCMA expression mediated by GSIs on the activity of HPN217, an in vitro T cell dependent cell cytotoxicity assay (TDCC) was conducted. GSIs enhanced the activity of HPN217, in some cases leading to a 3.5-fold increase in potency compared to HPN217 alone. Next, the impact of the combination was tested in vivo using a disseminated MOLP-8 xenograft model engrafted with T cells from a healthy human donor. Vehicle treated mice succumbed to disease burden with a median survival of 23 days as assessed by body weight loss and clinical observations. A suboptimal dose of 4 ug/kg HPN217 protected mice from disease burden for an additional 3 days. By contrast, the combination of HPN217 and LY3039478 led to a significant increase in survival compared to vehicle or either monotherapy, with a median survival of 43 days. Taken together these results demonstrate the potential utility of GSIs to enhance the anti-tumor activity of HPN217 and supports further investigation of this combinatorial approach in patients.

Citation Format: Payton C. Laurie, Mary Ellen Molloy, Patrick P. Ng, Banmeet S. Anand. Anti-tumor activity of HPN217, a BCMA-targeting tri-specific T cell engager, is enhanced by γ-secretase inhibitors in preclinical models. [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 5081.