bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 58: Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies
Authors: Keane J T. , Dagher Oula K. , Liu Fang , Posey Avery D.
Published: March 2024

The effector functions of CAR T cells can be enhanced through delivery of immunostimulatory cytokines, such as IL-12, IL-18, or IL-23. However, systemic delivery of recombinant cytokines or paracrine secretion by CAR T cells can elicit toxicities due to non-specific targeting of the cytokines. Here, we designed immunocytokines with antibodies specific for the invariant linker peptides that join variable domains in the CAR scFv and immunostimulatory cytokines. The immunocytokines were capable of targeting CAR molecules agnostic of target antigen, demonstrating broad applicability without the need for further genetic engineering of CAR therapies, such as co-expression of a surrogate molecule (tEGFR, etc.). The addition of immunocytokines enhanced CAR T cell cytolytic activity and secretion of type I cytokines, even at low concentrations of immunocytokine. In a stringent Nalm6 xenograft model, the addition of immunocytokines delivering IL-12 enhanced the anti-tumor activity of anti-CD19 CAR T cells and overall survival compared to controls of antibody alone or PBS. This technological development presents the opportunity to specifically enhance the activity of multiple CAR T cell therapies in the absence of non-specific toxicities.

Citation Format: J T. Keane, Oula K. Dagher, Fang Liu, Avery D. Posey. Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 58.
Abstract 5800: Ultrasound-mediated nano-drug delivery system to enhance cancer treatment efficacy: An in vitro study
Authors: Kashkooli Farshad Moradi , Jakhmola Anshuman , Hornsby Tyler K. , Ferrier Graham A. , Tavakkoli Jahangir (Jahan) , Kolios Michael C.
Published: March 2024

Conventional non-targeted nano-sized drug delivery systems face limitations such as restricted drug penetration depth and treatment efficacy in solid tumors. External stimulus such as ultrasound waves can trigger targeted, on-demand drug release from the nanocarriers. We demonstrate a first-of-its-kind use of low-intensity pulsed ultrasound (LIPUS) to induce Doxorubicin release from the surface of gold nanoparticle drug carriers. Our 1-MHz LIPUS beam is capable of penetrating tissue to sufficient depths with the required power/intensity for drug release applications with a nominal intensity of around 3 W/cm2 in the region of interest (ROI). This level of intensity is necessary to elevate the tissue temperature to the mild hyperthermia range of 41 to 45 °C. Our in vitro experiments demonstrate a remarkable 22% enhancement in drug release with the addition of LIPUS. Measurements of drug release kinetics, with and without LIPUS, suggest that LIPUS shifts the drug release mechanisms from static (Fickian) to dynamic (non-Fickian) release in vitro. Thus, revealing the intricate interactions between ultrasound and drug release from gold nanoparticle drug carriers. To assess the efficacy of the delivery system, we developed an in vitro cell culture model with a customized ultrasound exposure setup. The LIPUS device was situated at the bottom base of a water tank, directing ultrasound beam upward toward a region of interest where the MDA-MB-231 breast cancer cells were located 35 mm away from the LIPUS element. We found that the LIPUS device operating at an acoustic power of around 4 W increased cell killing efficacy by approximately 15% ± 2% when compared to conventional nano-drug delivery, highlighting a significant improvement attributed to ultrasound-induced mechanical effects. At the cellular level, these results underscore the potential advantages of ultrasound-mediated nano-drug delivery over conventional chemotherapy and non-targeted nano-drug delivery.

Citation Format: Farshad Moradi Kashkooli, Anshuman Jakhmola, Tyler K. Hornsby, Graham A. Ferrier, Jahangir (Jahan) Tavakkoli, Michael C. Kolios. Ultrasound-mediated nano-drug delivery system to enhance cancer treatment efficacy: An in vitro study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5800.
Abstract 5819: An anti-TROP2-CPT113 ADC, exhibits potent targeted antitumor activity and favorable pharmacokinetic properties
Authors: Guo Huihui , Kong Xiangfei , Du Yong , Ye Zhicang , Chen Yongxiang , Guo Zhixiang , Zhang Lingli , Bai Lu , Jia Junxiang , Zheng Yunxia , Zheng Wei , Zheng Jun , Li Wenjun , Huang Yuanyuan , Fan Zhongliang , Liu Mengmeng , Chen Binbin , Dai Meng , Wang Juan , Chen Miaomiao , Yang Qingliang , Zhao Robert Y.
Published: March 2024

Trophoblast cell surface antigen 2 (TROP2) is highly expressed in a number of epithelial cancer types and is considered to be a poor prognostic marker. We have developed an anti-TROP2 antibody-drug conjugate, DXC1002, that contains a potent inhibitor of DNA topoisomerase I payload (CPT113) attached to the antibody mainly in the Fab region via a unique hydrophilic CrossConju™ linker. The anti-tumor activities of DXC1002 were studied in various human cancer cell lines and xenograft mouse models and pharmacokinetic profile of DXC1002 were evaluated in cynomolgus monkeys and rats. DXC1002 demonstrated potent anti-tumor activity in TROP2 tumor cells of various expression levels in vitro, and at low dose of 1 mg/kg for one injection, in both high and moderate Trop2 expression xenograft models. In plasma stability studies, wherein DXC1002 was incubated in the plasma of five different species for 168 hours (7 days) at 37 oC, over 90% ADC was intact, exhibiting good stability of the CrossConju™ linker. Pharmacokinetic studies of DXC1002 in monkeys (TROP2 cross-species) and rats (non-TROP2 cross-species) showed half-lives of about 61.27 and 236.77 hours, respectively. Systemic clearance in monkey and rat was 0.41-0.67 and 0.279-0.309 mL/h/kg, respectively. The longer half-lives and slower clearance observed in rats the results offered a more direct assessment of the linker stability as target-mediated clearance played no role in rats. In summary, with the conjugation of a DNA topoisomerase I inhibitor payload to Trop2 antibody via the CrossConju™ linker, DXC1002 demonstrates remarkable anti-tumor properties and pharmacokinetic profile, with a potential to be further validated in clinical applications.

Citation Format: Huihui Guo, Xiangfei Kong, Yong Du, Zhicang Ye, Yongxiang Chen, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Mengmeng Liu, Binbin Chen, Meng Dai, Juan Wang, Miaomiao Chen, Qingliang Yang, Robert Y. Zhao. An anti-TROP2-CPT113 ADC, exhibits potent targeted antitumor activity and favorable pharmacokinetic properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5819.
Abstract 5843: Exploring the implications of LPAR-1 expression in TKI-resistant NSCLC
Authors: Srinivasan Varsha , Li Zhaohui , Kannan Raghuraman , Upendran Anandhi
Published: March 2024

Introduction: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer in the United States with a 5-year survival rate hovering between 20-30%. Mutations in EGFR can initially cause the tumors to respond to tyrosine kinase inhibitors (TKI). Osimertinib, a third generation TKI, is the preferred first or second line of treatment and despite being effective, recent reports indicate that patients develop resistance within 10-18 months of treatment. TKI resistance limits therapeutic options for patients and therefore, alternative strategies are being explored. In this context, Lysophosphatidic acid receptors (LPARs), a family of G-protein coupled receptors consisting of LPARs 1-6 expressed differentially in prostate, breast, ovarian, pancreatic, and brain cancers are being explored as potential targets for drug resistant cancers. Specifically, LPAR-1 has been shown to play a role in tumor cell clonogenicity, migration, and response to therapy in EGFR mutant NSCLC cells. Pharmacological inhibition or CRIPSR based deletion of LPAR-1 in EGFR mutant NSCLC cell lines HCC827 and NCI-H3255, increased TKI sensitivity by reducing clonogenic potential. Further, detailed pathway analysis in TKI sensitive and resistant EGFR mutant cell lines suggests that LPAR-1 expression influences drug sensitivity. Surprisingly, the role of LPAR-1 in TKI sensitivity is not well understood. In the present study, we have performed detailed investigation on the implications of LPAR-1 expression in the development of Osimertinib resistance.

Methods: Osimertinib resistant EGFR mutant cell lines (OR) were generated using dose-escalation method and drug-tolerant persisters (DTPs) were generated using a short-term high exposure method. Gene and protein expressions were examined using qPCR and western blotting. Subcutaneous tumor cell derived xenografts were generated for in vivo studies.

Results: LPAR-1 is downregulated in Osimertinib resistant NSCLC cells. Further, we also observe that LPAR-1 levels are lower in DTP cells, confirming a progressive loss of LPAR-1 with Osimertinib treatment. In vivo studies in cell derived xenografts correlated with the in vitro studies.

Conclusion: Our studies indicate LPAR-1 downregulation correlates to Osimertinib resistance in EGFR mutant NSCLC cell lines. LPAR-1 downregulation has a key role in development of Osimertinib resistance. Future studies will focus on delineating LPAR-1 signaling pathways related to TKI resistance and investigating the effect of LPAR-1 overexpression using CRISPR technology.

Citation Format: Varsha Srinivasan, Zhaohui Li, Raghuraman Kannan, Anandhi Upendran. Exploring the implications of LPAR-1 expression in TKI-resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5843.
Abstract 5860: Understanding the biological role of MMP16 in cisplatin resistance in ovarian cancer
Authors: Bonilla-Liriano Jesennia , Rivera-Serrano Mariela , Reyes-Burgos Victor , Rolon-Perez Jose , Valiyeva Fatima , Vivas-Mejia Pablo
Published: March 2024

High-Grade Serous Ovarian Cancer (HGSOC) is the most common and lethal of Ovarian Cancer (OC) types, which accounts for eighty percent of deaths. Treatment for OC includes surgery and platinum-based therapy. However, around 70% of women with HGSOC become resistant to chemotherapy. Therefore, new therapeutic agents with higher specificity and efficacy are needed for women with advanced and drug-resistant OC. Matrix metalloproteases (MMPs) are a group of enzymes involved in the degradation of the extracellular matrix (ECM). Deregulation of MMPs contributes to tumor growth and metastasis in several cancer types. Preliminary studies of our research team found that MMP16 is higher in cisplatin-resistant as compared with cisplatin-sensitive OC cells. The aim of this project is to understand the role of MMP16 in the cisplatin resistance of OC cells. We hypothesize that MMP16 increased expression may contribute to cancer progression and drug-resistance. We performed experiments in A2780/A2780CP20 and OVCAR3/OVCAR3CIS human OC cells. To assess the MMP16 protein levels we used western blot analysis. To measure the mRNA levels, we used qRT-PCR. Kaplan-Meier survival curves were generated by interrogation of the KM Plotter patient database. The MMP16 protein and qRT-PCR experiments confirmed that protein and mRNA levels are more abundant in cisplatin-resistant as compared with cisplatin-sensitive cells. Interrogation of the KM plotter database showed that OC patients with higher MMP16 mRNA levels live less as compared with OC patients with lower MMP16 levels. Ongoing experiments are testing the biological effect of small interference RNA (siRNA) targeting MMP16 in cisplatin-resistant OC cells.

Citation Format: Jesennia Bonilla-Liriano, Mariela Rivera-Serrano, Victor Reyes-Burgos, Jose Rolon-Perez, Fatima Valiyeva, Pablo Vivas-Mejia. Understanding the biological role of MMP16 in cisplatin resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5860.
Abstract 5888: Novel immune-therapeutic target for lung cancer uncovered by SpliceIO™, an innovative platform that identifies disease-specific alternative splicing
Authors: Manzanares Miguel A. , Anderson Kendall , Zumrut Hasan , Gera Sakshi , Habib Nayab , Williams Nicole , Stanton Shaleigh , Floyd Taylor , Shah Sanjana , Trahan Devon , Akerman Martin , Arun Gayatri
Published: March 2024

Alternative splicing (AS) plays a critical role in generating tumor-specific neoantigens targetable through an arsenal of immunotherapeutic modalities. Previous studies have demonstrated tumor-promoting activity of deregulated AS isoforms and mutated splicing factors, both of which increase the yield of neoantigen-encoding AS isoforms. Therefore, the identification of AS isoforms has the potential to produce novel therapeutic targets, especially in lung tumors where splicing deregulation is frequent. Envisagenics has developed SpliceIO, a proprietary software platform for AS-derived neoantigen discovery. SpliceIO uses RNA-seq data to uncover extracellular neoantigens encoded by transmembrane proteins. This predictive ensemble maximizes the value of RNA-seq data to predict fundamental aspects of neoantigen activity, such as protein localization, epitope accessibility, and cancer pathway relevance. Here, we summarize the discovery and validation of novel AS-derived neoantigens identified using SpliceIO in non-small cell lung cancer (NSCLC). Our novel SpliceIO-identified neoantigens in NSCLC were validated following our well-established experimental pipeline. We have confirmed RNA expression of the neoantigen-expressing transcripts in lung cancer cell lines and patient samples using a combination of RT-PCR as well as RNA-ISH. Further, we have validated the membrane-bound extracellular localization of neoantigen-containing isoforms at the protein level using Western blotting, GFP and c-Myc tagged isoform generation, eukaryotic cells transfection, and microscopy. Our top target showed high tumor specificity, heterogeneous distribution in tumor samples, high prevalence, confirmed novel extracellular N-terminal domain, and strong biological relevance in lung cancer. This demonstrates the potential of utilizing RNA splicing for identifying many more membrane associated proteins for tumor specific immunotherapeutic targeting. The combination of computational and experimental approaches to neoantigen identification using SpliceIO allows for novel target discovery amenable for targeted immunotherapeutic development for lung cancer treatment and other indications driven by deregulated AS.

Citation Format: Miguel A. Manzanares, Kendall Anderson, Hasan Zumrut, Sakshi Gera, Nayab Habib, Nicole Williams, Shaleigh Stanton, Taylor Floyd, Sanjana Shah, Devon Trahan, Martin Akerman, Gayatri Arun. Novel immune-therapeutic target for lung cancer uncovered by SpliceIO™, an innovative platform that identifies disease-specific alternative splicing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5888.
Abstract 5890: ZNF451 and doxorubicin resistance
Authors: Rees Amy , Blackman Alexandra , Evans Della , Delaney Joe
Published: March 2024

Precision medicine most often utilizes mutations in patient selection, however targetable mutations are exceedingly rare in high-grade serous ovarian cancer (OV). Aneuploidy and chromosome instability are major contributors to such genomic changes in OV. Genes are pervasively disrupted by copy-number alterations: 39% of all genes in the median solid tumor of all cancer types, and 67% in OV. A common second-line OV chemotherapeutic is peggylated-liposomal doxorubicin, a topoisomerase-II poison. To address which genes in OV contribute to doxorubicin sensitivity and resistance, we performed a CRISPR-Cas9 screen on 19,114 human genes in the presence or absence of doxorubicin. Positive control genes involved in drug-efflux and DNA repair were significant hits. The gene with both the highest significance and greatest magnitude of doxorubicin sensitization upon knockout was the E3 SUMO2/3 ligase ZNF451. ZNF451 exhibits a copy-number loss in 14% of patients and corresponds to ZNF451 protein reduction. ZNF451 is an attractive target for future combination therapies with doxorubicin to induce conditional lethality. ZNF451 may be valuable to study as a prognostic biomarker for topoisomerase-II inhibitor chemotherapy.

Citation Format: Amy Rees, Alexandra Blackman, Della Evans, Joe Delaney. ZNF451 and doxorubicin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5890.
Abstract 5961: NRF2-responsive kinases in esophageal squamous cell carcinoma
Authors: Subramaniyan Boopathi , Xiong Zhaohui , Paiboonrungruang Chorlada , Li Yahui , Major M Ben , Chen Xiaoxin
Published: March 2024

Esophageal squamous cell carcinoma (ESCC) ranks as the 7th most frequently diagnosed cancer and the 6th leading cause of cancer-related death worldwide. NRF2 is identified as a therapeutic target for human ESCC, with a mutation rate of approximately 10-22%. Hyperactive NRF2 promotes proliferation, metastasis, and resistance to radiation and chemotherapy in ESCC. Although some evidence suggest NRF2 regulates kinases which contribute to these phenotypes, it remains unclear which kinases are responsive to hyperactive NRF2. Using NRF2 ChIP-seq analysis, we have enrichment of 42 kinases including Akt2 and Pkm and 31 phosphatases including Pten and Dusp16 enriched in the Keap1-/- mouse esophagus in comparison with the wild-type esophagus. We further confirmed the binding of NRF2 to the promoter regions of mouse Akt2 and Pten. mRNA expression profiling revealed enrichement of the PI3K/AKT, RAS, ERK, and GSK3 pathways in the Keap1-/- esophagus as compared to the Nrf2-/-;Keap1-/- esophagus. Immunohistochemical staining showed that pAKT, p-mTOR, pS6, PKM2 were overexpressed in the Keap1-/- esophagus, as well as in the adult NRF2-hyperactive esophagus (Sox2CreER;LSL-Nrf2E79Q/+). Using PhosphoExplorer array and PamChip array to analyze NRF2W24C-KYSE70 cells (in comparison with NRF2null-KYSE70 cells) and Sox2CreER;LSL-Nrf2E79Q/+ mouse esophageal epithelium in comparison with wild-type mouse tissue, we further identified multiple kinases including PI3K as potential NRF2-responsive kinases. These findings suggest a potential role for kinases such as PI3K in NRF2-driven ESCC, and support the idea of targeting both NRF2 and NRF2-responsive kinases as a therapeutic strategy for ESCC.

Citation Format: Boopathi Subramaniyan, Zhaohui Xiong, Chorlada Paiboonrungruang, Yahui Li, M Ben Major, Xiaoxin Chen. NRF2-responsive kinases in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5961.
Abstract 5986: Significance of LIF/LIFR axis in the progression of inflammatory breast cancer
Authors: Romo Bianca , Fuentes Zenaida , Randolph Lois , Santhamma Bindu , Nair Hareesh , Thomas Christoforos , Vadlamudi Ratna , Viswanadhapalli Suryavathi
Published: March 2024

Background: Inflammatory breast cancer (IBC) is a rare form of locally progressed breast cancer. Even though it is uncommon, IBC accounts for 7% of breast cancer deaths. There is a critical need for novel therapeutic targets to develop new therapeutics. IBC is more common in young women. Altered levels of growth factors, and cytokine signaling are suspected to play a role in the progression of IBC. Leukemia inhibitory factor (LIF) is the most pleiotropic member of the interleukin-6 family of cytokines. LIF and its receptor LIFR have been linked to the progression of many cancers including triple negative breast cancer. We recently developed a first-in-class inhibitor targeting LIFR named EC359. However, the role of the LIF/LIFR axis in IBC progression remains unknown. In this study, we examined the significance of LIFR and the effectiveness of LIFR inhibitor EC359 in treating IBC.

Methods: The effect of LIFR inhibitor EC359 on cell survival, colony formation, and apoptosis was evaluated using three well-established IBC model cells (SUM149, SUM190PT, and KPL4). Mechanistic studies were conducted using Western blotting, CRISPR KO, reporter assays, and RT-qPCR. KPL4 cell-based xenografts were used to test the efficacy of EC359 in vivo.

Results: Our results using Western blotting confirmed increased expression of LIF and LIFR in IBC cells compared to normal and ER+ breast cancer cells. Treatment with EC359 significantly decreased IBC cell viability, long term colony formation ability with an IC50 of ~10-20 nM. Similarly, knock out of LIFR significantly reduced the progression of IBC model cells in cell viability and colony formation assays. Further EC359 treatment promoted apoptosis of IBC cells. Mechanistic studies confirmed that EC359 treatment substantially reduced LIF/LIFR downstream signaling including STAT3, AKT, and mTOR signaling. We also tested the utility of EC359 in treating IBC in vivo using KPL4 xenografts. EC359 (5mg/kg/day) is highly efficacious in reducing IBC xenograft tumor growth in vivo. IHC analyses of tumors confirmed decreased cell proliferation as measured by Ki67 staining.

Conclusion: Collectively, these studies demonstrated the therapeutic benefit of using EC359 to target the LIF/LIFR axis and opened the door for the development of new treatment approaches.

Citation Format: Bianca Romo, Zenaida Fuentes, Lois Randolph, Bindu Santhamma, Hareesh Nair, Christoforos Thomas, Ratna Vadlamudi, Suryavathi Viswanadhapalli. Significance of LIF/LIFR axis in the progression of inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5986.
Abstract 6004: HOXC8 epigenetically controls caspse-1 expression and pyroptosis in lung cancer cells
Authors: Sun Lei , Padia Ravi , Calbay Ozlem , Chi Cheng , Akter Mahmuda , Jin Lingtao , Huang Shuang
Published: March 2024

Homeobox C8 (HOXC8) is a transcription factor preferentially overexpressed in a large percentage of lung adenocarcinoma (LUAD). To investigate the function of HOXC8 in LUAD, we showed that knockdown of HOXC8 led to massive LUAD cell death in a mechanism of pyroptosis because both YVAD, a caspase-1 (CASP1) inhibitor, and disulfiram, which prevents gasdemin D (GSDMD) pore formation, blocked cell death caused by HOXC8 depletion. Intriguingly, ASC and NLRP3, components of canonic inflammasome, were not involved in pyroptosis occurring in HOXC8-depleted cells. Instead, we found that silencing HOXC8 led to a marked increase in CASP1 abundance. As forced expression of CASP1 is sufficient to induce CASP1 activation and pyroptosis, we reason that HOXC8 deters pyroptosis by suppressing the level of CASP1. Moreover, we revealed that knockdown of HOXC8 augmented CASP1 transcription involving in HDAC1/2. In fact, HOXC8 and HDAC1 were in the same immunocomplex and the presence of HOXC8 is required for the recruitment of HDAC1 to CASP1 promoter. Since HOXC8 also binds CASP1 promoter, we conclude that HOXC8 negatively regulates CASP1 expression by drafting HDAC1/2 to CASP1. This study suggests that HOXC8 participates LUAD development by controlling CASP1 expression and pyroptosis.

Citation Format: Lei Sun, Ravi Padia, Ozlem Calbay, Cheng Chi, Mahmuda Akter, Lingtao Jin, Shuang Huang. HOXC8 epigenetically controls caspse-1 expression and pyroptosis in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6004.
Abstract 6010: BRCA2 reversion alleles confer PARPi resistance via homology-directed repair, not fork protection
Authors: Jensen Ryan , Moore Gemma , Lahiri Sudipta
Published: March 2024

BRCA2 reversion alleles have been identified in relapsed, treatment resistant cancer patients. The reversions restore the open reading frame of the BRCA2 protein often resulting in significant sequence deletions with unknown functional consequences. No reports to date have introduced patient reversion mutations into a BRCA2 null cell model to analyze which BRCA2 functions are re-established and whether all or a subset of activities are required for PARPi resistance. Our study utilized BRCA2 reversion alleles identified through sequencing of circulating tumor DNA (ctDNA) from PARPi resistant ovarian cancer patients. We rigorously evaluated whether altered BRCA2 reversion proteins restore homology-directed repair (HDR), fork protection, or gap suppression to determine the specific activities required for PARPi resistance. We found that reverted BRCA2 proteins are functional for HDR by assessing survival response to PARPi, analyzing RAD51 foci at DSBs, DR-GFP reporter assays, RAD51 binding, and CRISPR/Cas9 gene targeting assays. Surprisingly, using the DNA fibercombing approach to assess replication fork dynamics, we identify two separation-of-function mutants, T1974 and C1654_M1890, which are deficient in fork protection, yet confer PARPi resistance. The mutations are located within the BRC 5-8 domain of BRCA2 proposed to play an important role in RAD51 nucleoprotein filament stability. Biochemical analyses of the purified proteins using single molecule FRET (smFRET) indicate reduced filament stability potentially explaining the deficit in fork protection. In conclusion, these separation-of-function BRCA2 mutations provide new mechanistic insight into the underlying biology of BRCA2 and reveal the specific functions needed to overcome PARPi-mediated cell death.

Citation Format: Ryan Jensen, Gemma Moore, Sudipta Lahiri. BRCA2 reversion alleles confer PARPi resistance via homology-directed repair, not fork protection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6010.
Abstract 6025: Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display
Authors: Jaekel Anika , Sherry Kathryn , Desai Prachi , Fox David , Guo-Qing Tang , Sturzbecher-Hoehne Manuel , Mewis Dennis , Noridomi Kaori , Rayman Liam , Ye Ping
Published: March 2024

Radioligand therapeutics (RLT) aim to selectively deliver radioisotopes to cancer tissues to eradicate tumor cells while limiting the damage to surrounding tissues. They are composed of a tumor-targeting ligand and a radioactive payload. The therapeutic potential of RLT strongly depends on their biostability and residence time in tumor and healthy tissue after injection. Macrocyclic peptides are effective tumor-targeting ligands because their size and molecular characteristics confer optimal pharmacologic properties that can be fine-tuned during the development process. mRNA display is a powerful platform for macrocyclic peptide drug discovery that identifies high affinity peptide ligands through in vitro selection. Here, we describe the selection and initial characterization of macrocyclic peptides discovered by mRNA display that bind to the extracellular domain of a tumor target. Enrichment of the library for peptides that bound to the recombinant target protein was observed after four rounds of selection with no further enrichment in round five. The library of round 5 was sequenced by Illumina sequencing and the top 1,000 sequences (highest counts) were used to perform cluster analysis. We tested the top sequences of 43 different clusters in in vitro translation binding assays to validate the selection output and identify sequences for hit confirmation. Most of the sequences tested showed clear binding to the target protein and only a few peptides showed binding to a closely related protein from the same protein family. No non-specific binding of the discovered peptides to an irrelevant protein was observed showing the high specificity of the generated library. From this confirmatory selection data, we chose a subset of peptides for chemical synthesis. Binding affinity measurements were determined by surface plasmon resonance (SPR) and cellular binding of selected candidates to target-positive and -negative tumor cells was analyzed by flow cytometry. From this hit confirmation data, in vitro internalization and in vitro anti-tumor efficacy of radiolabeled peptide candidates for RLT are assessed. Our data highlights the potential of mRNA display platforms for fast and efficient discovery of highly specific macrocyclic peptides with optimal binding properties suitable for RLT.

Citation Format: Anika Jaekel, Kathryn Sherry, Prachi Desai, David Fox, Tang Guo-Qing, Manuel Sturzbecher-Hoehne, Dennis Mewis, Kaori Noridomi, Liam Rayman, Ping Ye. Discovery of novel macrocyclic peptide radioligands for tumor therapy by mRNA display [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6025.
Abstract 6052: Systematic identification of novel targeted protein degradation mechanisms using SITESEEKER® technology
Authors: Dillon Christian
Published: March 2024

The rise of Targeted Protein Degradation (TPD) is rapidly changing perceptions about therapeutic target druggability and is rewriting many preconceived notions on drug design. However, the majority of monovalent molecular glue and heterobifunctional degraders in clinical and preclinical development rely predominantly on the recruitment of a single E3 ligase, Cereblon. Arising resistance, toxicities and restricted Protein-of-Interest scope may limit clinical potential, highlighting the need to uncover novel TPD mechanisms. To expand the potential of this modality, we have developed SITESEEKER®, a screening technology operating at substantially greater magnitude of complexity than extant target discovery platforms. SITESEEKER® utilizes computationally-derived encoded mini-protein fragments with huge shape diversity to systematically identify novel degrader mechanisms and define functionally-active binding sites on targets. SITSEEKER® allows for the discovery of targets that may be missed through traditional gene editing or knockdown approaches and can additionally provide valuable mechanistic insights that help to inform, unlock and truncate the path from target ID to drug discovery. We describe the identification of a cache of degrader motifs which showcase the breadth of proteome space yet to be explored within TPD. Moreover, we identify peptide motifs capable of driving degradation in a selective manner with potential to be translated into tissue- or cancer-selective degraders. The functional dependencies of prioritized degraders have been mapped to their cognate E3 ligase using combinatorial screening, giving rise to a number of E3 ligases with potential to be hijacked for TPD. PhoreMost is progressing a pipeline of monovalent and heterobifunctional oncology degrader programs arising from its platform. We demonstrate the effective hijacking of selected E3 ligases, identified by SITESEEKER®, through the discovery of high affinity small molecule binders and subsequent discovery of efficient heterobifunctional degraders against selected Proteins-of-Interest.

Citation Format: Christian Dillon. Systematic identification of novel targeted protein degradation mechanisms using SITESEEKER® technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6052.
Abstract 6103: The association of genetic polymorphisms in DNA repair genes XRCC1 and 3 with cervical cancer risk among Bangladeshi females
Authors: Rahman Md Mustafizur , Das Laboni , Rahman Sadia , Hossain Amir , Sultana Razia
Published: March 2024

Introduction and Objective: Worldwide, cervical cancer ranks the second most common cancer in women. Sequence variations in DNA repair genes X-ray repair cross complementing (XRCC) 1 and 3 genes can lead to can result cellular function aberration leading to cancers. Single nucleotide polymorphisms (SNPs) in XRCC1 (Arg399Gln) and XRCC3 (Thr241Met) genes have been shown to cause individual variation in their DNA repair capacity. The aim of this study was to investigate the association of XRCC1 Arg399Gln and XRCC3 Thr241Met SNPs with susceptibility to cervical cancer among Bangladeshi populations.

Methods: In the current case-control study 124 cervical cancer patients and 148 age matched healthy controls were recruited. Genomic DNAs were isolated from peripheral blood collected from the participants and genotyped for candidate SNPs using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

Results: For XRCC1, heterozygous Arg/Gln and combined heterozygous plus variant homozygous Gln/Gln genotypes showed 1.78-fold (95% CI 1.0037 to 2.8771, p=0.0484) and 1.8627-fold (95% CI 1.1470 to 3.0250, p = 0.0119) increased risk of cervical cancer, respectively, when compared with normal homozygous Arg/Arg genotype. The variant Gln allele was positively associated with cervical cancer by 1.68-fold increase (95% CI 1.1732 to 2.3980, p = 0.0046). Similarly, for XRCC3, Thr/Met heterozygous and combined Thr/Met + Met/Met genotypes were found to be associated with 1.6993-fold (95% CI 1.0398 to 3.0166, p=0.0354) and 1.8312-fold (95% CI 1.0890 to 3.0791, p = 0.0225) higher risk, respectively, when compared with normal homozygous Thr/Thr genotypes. The variant Met allele showed significant association with 1.71-fold increased risk.

Conclusion: XRCC1 (Arg399Gln) and XRCC3 (Thr241Met) SNPs may be positively associated with increased cervical cancer risk in Bangladeshi females.

Citation Format: Md Mustafizur Rahman, Laboni Das, Sadia Rahman, Amir Hossain, Razia Sultana. The association of genetic polymorphisms in DNA repair genes XRCC1 and 3 with cervical cancer risk among Bangladeshi females [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6103.
Abstract 6190: Patterns of EGFR and HER3 co-expression in solid tumors
Authors: Wood Matthew , Morrison Cody , Khalili Jahan Salar , Zhu Yi
Published: March 2024

EGFR and HER3 are members of the ERBB receptor family and transmit growth and survival signals to cancer cells upon ligand binding. While both receptors are well established targets for cancer therapy, greater understanding of the relationship of their expression patterns in human cancer is needed to establish the scope of therapeutic potential. To evaluate the co-expression pattern of EGFR and HER3, cancer FFPE tissue microarrays from several cancer types were evaluated by immunofluorescence imaging and digital pathology, including cell-by-cell marker intensity and spatial analyses within the tumor and stromal compartments. Tissue cores were assigned H-scores, and individual cells were binned into 0, 1+, 2+, 3+ scoring based on established digital pathology methods using QuPath software. Spatial cytometry was used to further evaluate single-cell expression intensity. Analysis was conducted widely on multiple normal human tissues as well as cases of NSCLC adenocarcinoma n=86, NSCLC squamous cell carcinoma n=84, SCLC n=43, TNBC n=45, Biliary tract cancers n=80, Nasopharyngeal carcinomas n=60. Overall, the results of this analysis show the intermediate to high prevalence of EGFR and HER3 co-expression within each indication, as well as the frequency of EGFR and HER3 expressing cells. Furthermore, EGFR and HER3 heterodimers were confirmed by proximity ligation assay in certain index cases. Notably, of several observations, we observe NSCLC cases show a profound absence of EGFR- HER3+ tumor cells. Additionally, we observe that co-expression of EGFR and HER3 in normal human tissues is rare and of low signal intensity.

Citation Format: Matthew Wood, Cody Morrison, Jahan Salar Khalili, Yi Zhu. Patterns of EGFR and HER3 co-expression in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6190.
Abstract 62: Intra-tumor persistence of activated anti-mesothelin hYP218 CAR T cells is associated with increased efficacy in gastric and colorectal cancers
Authors: Mir Sameer A. , Venugopalan Abhilash , Zhang Jingli , Khanal Mana Kamana , Stathopoulou Chaido , Sengupta Manjistha , Jiang Qun , Hassan Raffit
Published: March 2024

Purpose: Patients with gastric and colorectal cancers that have progressed after standard therapies have poor prognosis. Given the high expression of mesothelin in these tumor types, we examined the potential utility of the anti-mesothelin hYP218 CAR T cells to treat these cancers.

Experimental Design: Cell surface mesothelin expression in a panel of gastric and colorectal cancer cell-lines was analyzed using flow cytometry. In vitro efficacy of hYP218 CAR T cells against these cell lines was evaluated using cytotoxicity and cytokine release assays. In vivo efficacy of hYP218 CAR T cells was studied using gastric (HGC27) and colorectal (SW48) tumor xenografts in NSG mice. NSG mice were injected with 1 × 106 cancer cells and the tumors were allowed to grow until they reached a volume of 80-100mm³, after which the mice were treated with either 5 × 106 hYP218 CAR T cells, 5 × 106 untransduced T cells, or saline and monitored over time. Persistence of CAR T cells in the tumor was evaluated over time and expression of activation and exhaustion markers were studied.

Results: hYP218 CAR T cells demonstrated strong cytotoxic activity against mesothelin positive gastric and colorectal cancer cell lines and exhibited increased pro-inflammatory cytokine production. In both the HGC27 and SW48 tumor models hYP218 CAR T cells demonstrated significant reduction in tumor volume and overall survival compared to mice treated with untransduced T cells or saline. CAR T cells isolated from tumors of both the xenograft models, at day 40 after treatment, displayed higher expression levels of activation markers CD39 and CD69 when compared to the infused product. Furthermore, a concomitant elevation in the levels of effector cytokines, TNF-α and IFN-γ, was detected in the blood of treated mice demonstrating the functional capacity of the CAR T cells to not only persist but also to maintain an activated state.

Conclusions: hYP218 CAR T cells show anti-tumor efficacy against gastric and colorectal tumor xenografts due to increased accumulation of activated CAR T cells in the tumor and warrants further investigation for treatment of patients with these cancers.

Citation Format: Sameer A. Mir, Abhilash Venugopalan, Jingli Zhang, Mana Kamana Khanal, Chaido Stathopoulou, Manjistha Sengupta, Qun Jiang, Raffit Hassan. Intra-tumor persistence of activated anti-mesothelin hYP218 CAR T cells is associated with increased efficacy in gastric and colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 62.
Abstract 6213: Determination of permissive and restraining cancer-associated fibroblast (DeCAF) subtypes
Authors: Peng Xianlu Laura , Kharitonova Elena , Kearney Joseph , Xu Yi , Chan Priscilla , Luan Changfei , Belt Brian , Panni Roheena , Hariharan Arthi , McCabe Ian , Morrison Ashley , Cliff Ashley , Linehan David C. , Iuga Alina , Rashid Naim U. , Yeh Jen Jen
Published: March 2024

Cancer-associated fibroblasts (CAFs) in pancreatic adenocarcinoma (PDAC) are known to play a significant role in regulating tumor progression, invasion, and metastasis. Multiple studies using experimental strategies, as well as single-cell RNA-sequencing (scRNAseq) technology, have shown the existence of subpopulations of PDAC CAFs, with divergent physical and biological characteristics. However, how these CAF subpopulations translate to clinical practice remains unclear. Using a combination of sc- and bulk RNAseq data, we present two CAF subtypes with permissive (permCAF) and restraining (restCAF) characteristics in patients, and developed a clinically usable single-sample classifier (DeCAF) using using 11 independent datasets with high reproducibility (AUC = 0.961). We found that the permCAF patients tend to be associated with a “myxoid” histological features, while restCAF samples were more “collagenized” (p = 0.018). Patients with permCAF tumors had a median overall survival (OS) of 18.5 m compared to 30.2 m for restCAF tumors, with a stratified HR = 1.66 (p < 0.001) for patients with permCAF tumors. Basal-like tumors were found to be associated permCAF subtype (p < 0.001). DeCAF and PurIST (basal-like/classical) subtypes were independently associated with OS (p < 0.001). Analysis of a phase Ib trial FOLFIRINOX in combination with a CCR2 inhibitor (PF-04136309; NCT01413022) , we found that in patients with classical tumors, increasing permCAF probability was associated with response (r = -0.688, p<0.001). Tumors with permCAF subtype are enriched in neutrophils using CIBERSORT and may explain the positive response to CCR2 inhibition. More intriguingly, we found similar associations with OS in kidney renal clear cell carcinoma (KIRC, p = 0.001) and mesothelioma (MESO, p = 0.019). DeCAF subtypes are associated with histological subtype in MESO (p = 0.021) and grade in KIRC (p = 0.056). Taken together, DeCAF subtypes explain the role of CAF subtypes in patients, provide a foundation for the translation of preclinical studies, and facilitate the design of future therapeutic approaches and clinical trials.

Citation Format: Xianlu Laura Peng, Elena Kharitonova, Joseph Kearney, Yi Xu, Priscilla Chan, Changfei Luan, Brian Belt, Roheena Panni, Arthi Hariharan, Ian McCabe, Ashley Morrison, Ashley Cliff, David C. Linehan, Alina Iuga, Naim U. Rashid, Jen Jen Yeh. Determination of permissive and restraining cancer-associated fibroblast (DeCAF) subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6213.
Abstract 6233: Characterizing variants of uncertain significance in the tumor suppressor gene known as phosphatase and tensin homolog (PTEN)
Authors: Singh Ajay P. , Wen Jasmine , Nguyen David , Khan Vedanta , Dhami Kiran , Meier-Ross Kaitlynn , Martin Benjamin , Chao Jesse T.
Published: March 2024

PTEN is an important tumor suppressor that plays an essential role in regulating cell proliferation. Loss of PTEN function is implicated in approximately 50% of metastatic cancers. Individuals with pathogenic PTEN variants are at significantly elevated risk of developing hereditary cancer syndromes such as PTEN hamartoma tumor syndrome. While genetic testing for PTEN is valuable for identifying individuals at risk, 47% of 3065 publicly reported PTEN variants are not conclusively classified; they either have conflicting interpretations of pathogenicity or are labeled as “Variants of Uncertain Significance (VUS)” and are not clinically actionable. The lack of a clear interpretation for these PTEN variants hinders the targeted care and surveillance of patients and places a strain on the healthcare system. To overcome this issue, our aim is to develop a clinically relevant tool for assessing the pathogenic effects of PTEN variants.We hypothesize that loss of function (LoF) PTEN variants will lead to changes in cellular phenotypic profiles that can be measured through single-cell phenotypic profiling. Thus, our goal is to investigate the functional status of exogenously expressed PTEN variants in PTEN-/- HEK 293 cells. We developed a multiplexed high content imaging assay to characterize PTEN and its key signaling partners such as phospho-Akt. Additionally, we developed a deep learning based single-cell phenotypic profiling pipeline named Paracell that uses subcellular segmentation to extract 540 phenotypic features. Cells that exhibit abnormal phenotypic profiles are indicative of loss of PTEN function. Preliminary results from testing 73 missense variants of PTEN showed that functional and LoF PTEN variants form distinctive clusters in a two-dimensional UMAP plot based on Paracell outputs, suggesting that our approach can identify potentially pathogenic variants of PTEN.Through characterizing novel VUS, we can elucidate structural domains involved in PTEN’s tumor suppressing activity. The result of these classifications will enable more optimized detection, diagnosis, and surveillance strategies for PTEN-related cancers. Ultimately, we aim to contribute to improving the genetic testing of PTEN and the development of targeted therapies.

Citation Format: Ajay P. Singh, Jasmine Wen, David Nguyen, Vedanta Khan, Kiran Dhami, Kaitlynn Meier-Ross, Benjamin Martin, Jesse T. Chao. Characterizing variants of uncertain significance in the tumor suppressor gene known as phosphatase and tensin homolog (PTEN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6233.