bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1713: Delineation of cell subpopulations and cell cell interactions to determine correlations between drug response and tumor microenvironment in early-stage lung cancer
Authors: Ramos Corinne , Sophie Journe Anne , Gerstenberg Amandine , Pamelard Fabien , Stauber Jonathan
Published: June 2022

Purpose The tumor microenvironment (TME) is an integral player in cancer initiation, tumor progression, response and resistance to anti-cancer therapy. Understanding the complex interactions of tumor immune architecture has therefore become increasingly desirable to guide patient selection. Conventional studies that underestimate the potential value of the spatial architecture of the TME are unable to completely elucidate its complexity. To overcome these limitations, we used quantitative image analysis based on multiplexed immunohistochemistry and deep learning technologies to interrogate complex information from the tumor microenvironment and find predictive insights into treatment response for non-small cell lung cancer (NSCLC).

Method We used tumor tissues samples from resected NSCLC (adenocarcinoma and squamous cell carcinoma) tumors treated with adjuvant pembrolizumab therapy. Patient frozen tissue sections were prepared and stained using a panel of 20 immune or tumor-related antibodies. Different metal tags were assigned to each antibody. The stained cells were then detected and analyzed by the Hyperion™ Imaging System. Further data analysis was performed using proprietary workflow to identify which cells are influencing the treatment response and how they are spatially distributed relative to each other.

Results Tumor samples of NSCLC with similar clinical characteristics at the T1N0M0 had different cells types, including CD4+ T cells, CD8+ T cells, CD20+ B cells, CD14+ macrophages, CD56+ natural killer cells, granulocytes, etc. More immunosuppressive cells were found in the sample of patient that did not respond to immunotherapy illustrating that differences in the tumor microenvironment of samples may contribute to different patients’ resistance to immunotherapy. Moreover, unsupervised clustering analysis reveal cell type signatures that are relevant to the prognoses of NSCLC.

Conclusion High-multiplexed proteomics analyses using the Hyperion imaging system help identify and analyze the tumor microenvironment heterogeneity of NSCLC and explain the factors leading to different prognoses for NSCLC patients despite the same TNM stage. Furthermore, additional information for screening drugs for targeted therapy was found, that could greatly improve NSCLC treatments’ individualization.

Citation Format: Corinne Ramos, Journe Anne Sophie, Amandine Gerstenberg, Fabien Pamelard, Jonathan Stauber. Delineation of cell subpopulations and cell cell interactions to determine correlations between drug response and tumor microenvironment in early-stage lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1713.
Abstract 172: Limiting medulloblastoma progression by hindering the Hedgehog signaling pathway with novel atypical protein kinase C inhibitors
Authors: Lajmi Luke , Breedy Sloan , Ratnayake Wishrawana S. , Acevedo-Duncan Mildred
Published: June 2022

Medulloblastoma is the most common pediatric brain tumor, constituting 20% of all childhood brain cancers (Clin. Cancer Res. 2008, 14 (4), 971-976., Oncogenesis 2019, 8 (11), 1-11). It is a highly invasive tumor that originates in the cerebellum and has the ability to metastasize throughout the central nervous system early in its course. The median age of diagnosis is 5 years, with 80% of cases being diagnosed in the first 15 years (Asian J. Neurosurg. 2015, 10 (1), 1-4). Current treatment options include maximal safe resection, chemotherapy and craniospinal radiation (J. Neurosurg. Pediatr. 2019, 24(4), 353-363). Despite such aggressive therapeutic treatments, nearly 30% of afflicted patients succumb to this disease, and survivors cope with the long-term side effects of these treatments that have significant impacts on their quality of life (J. Neurosurg. Pediatr. 2019, 24(4), 353-363). This underscores the need for less invasive and more effective treatment methods to improve overall patient outcomes. We believe that oncogene expression resulting from the Hedgehog (Hh) signaling pathway is responsible for upregulating malignant cancer cell growth, cell survival, and migration. Preliminary dose curve studies, in which D341 and D283 medulloblastoma cell lines were treated with novel atypical protein kinase C (aPKC) inhibitors 2-acetyl-1,3-cyclopentanedione (ACPD) and 3,4-diaminonapthalene-2,7-disulfonic acid (DNDA) (Int. J. Oncol. 2017, 51(5), 1370-1382), have been shown to significantly decrease cell proliferation with increasing inhibitor concentration. Cell line D341 showed 44.2% inhibition when treated with 10 µM of ACPD and 75.2% inhibition when treated with 1 µM of DNDA while cell line D283 showed a 63.2% inhibition at 5 µM of ACPD and 73.6% inhibition at 10 µM. Future studies will be conducted to determine the inhibition mechanism of the Hh pathway in medulloblastoma by probing for different cell cycle and oncoprotein markers using Western blotting, employing the WST-1 assay to evaluate cell viability and cytotoxicity, and implementing migration and invasion assays.

Citation Format: Luke Lajmi, Sloan Breedy, Wishrawana S. Ratnayake, Mildred Acevedo-Duncan. Limiting medulloblastoma progression by hindering the Hedgehog signaling pathway with novel atypical protein kinase C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 172.
Abstract 1738: DNA-launched nanoparticle vaccines induce cytolytic T cell responses for in vivo tumor control
Authors: Liaw Kevin , Xu Ziyang , Chokkalingam Neethu , Kulp Daniel , Weiner David
Published: June 2022

Nanotechnology-based approaches have achieved promising results for the development of novel immunotherapeutic cancer vaccines through the decoration of antigens on nanoparticle scaffolds for multivalent display. However, their translation have been limited due to challenges with purification, scalability, and robust induction of cytolytic T cell responses critical to anti-tumor activity. We designed a nanovaccine construct by fusing the HPV E7 immunodominant epitope onto lumazine synthase scaffold. We demonstrate that through electroporation-facilitated DNA delivery encoding the antigen-scaffold construct, this nanovaccine is expressed and self-assembles in vivo into 60-mer nanoparticles. Compared to CpG-adjuvanted peptide and monomeric DNA vaccination, this DNA-launched nanovaccine induced greater CD8+ cellular responses against the E7 epitope and target tumor cells. In both flank and lung models of TC-1 HPV-associated cancers, the nanovaccine achieved significant tumor regression and prolonged survival compared to CpG-adjuvanted peptide. Anti-tumor immunity was retained upon rechallenge >150 days after initial challenge, supporting the induction of long-term immune memory. In addition, we demonstrate that this DNA-launched nanovaccine platform can be used to form hybrid nanoparticles displaying multiple antigens simultaneously. These hybrid nanoparticles exhibit synergistic efficacy, with improved tumor control over administration of separate single-antigen nanovaccines. These promising results, along with facile characterization, scalability, and stability, support that DNA-launched nanovaccines can induce potent anti-tumor cellular immunity and are ideally positioned for further translational development for clinical evaluation.

Citation Format: Kevin Liaw, Ziyang Xu, Neethu Chokkalingam, Daniel Kulp, David Weiner. DNA-launched nanoparticle vaccines induce cytolytic T cell responses for in vivo tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1738.
Abstract 1760: The antibody-drug conjugate GENA-111 conjugated to auristatin F shows therapeutic potency in BCAM positive epithelial cancer
Authors: Yu Hyunkyung , Bellocq Nathalie , Ha Youngeun , Kim Hyunuk , Kim Yunyeon , Jeon Bu-Nam , Marx Léo , Pantin Mathilde , Yoo Hyunjin , Byun Seungmin , Chung Joo-Yeon , Cha Mi Young , Garrouste Patrick , Lévy Frédéric
Published: June 2022

Antibody-drug conjugates (ADCs) are considered as promising cancer treatment modalities that combine the selectivity of antibodies and the cytotoxic properties of payloads using chemical linkers. However, despite their success, ADCs still suffer from drawbacks, e.g., systemic toxicity, which limit their potential clinical applications. The systemic toxicity of an ADC is mainly related to the stability of its linker, and to the selectivity of its antibody towards the targeted antigen expressed on cancer cells. Lu/BCAM (Lutheran/basal cell adhesion molecule) is a member of the immunoglobulin superfamily and is a receptor for laminin, a protein that facilitates cell adhesion, migration, and invasion. A growing number of studies show that BCAM plays an essential role in tumor progression and is overexpressed on epithelial cancers e.g., skin cancer. Here we describe GENA-111, a human monoclonal anti-BCAM IgG4 (S228P) antibody that binds to human BCAM with high affinity, and that is significantly internalized by BCAM-positive tumor cells. We also describe the GENA-111-auristatin F ADC, wherein the GENA-111 antibody has been armed with an auristatin F derivative using a new linker technology and a stabilized thiol maleimide conjugation. This new linker technology comprises a cleavable peptidic sequence that facilitates multidrug attachment and the production of ADCs with tailored drug-to-antibody ratios (DARs). Cytotoxic drugs are rapidly and selectively released from the linker by the carboxypeptidase activity of Cathepsin B. In vitro cytotoxicity examination showed the potent cytotoxic effects of this GENA-111-auristatin F ADC on BCAM-expressing tumor cells, with a positive correlation between cytotoxicity and BCAM expression. Moreover, this ADC was also shown to significantly reduce the growth of tumor cells, including A431, T47D, and Huh7. The GENA-111-auristatin F ADC was evaluated in a xenograft mouse model established by subcutaneous injection of A431 cells, a BCAM positive human skin cancer cell line. The results of this study will be presented and discussed. Taken together, our data suggest that an ADC targeting BCAM e.g., GENA-111-auristatin F, might be a promising treatment strategy for BCAM positive epithelial cancer patients.

Citation Format: Hyunkyung Yu, Nathalie Bellocq, Youngeun Ha, Hyunuk Kim, Yunyeon Kim, Bu-Nam Jeon, Léo Marx, Mathilde Pantin, Hyunjin Yoo, Seungmin Byun, Joo-Yeon Chung, Mi Young Cha, Patrick Garrouste, Frédéric Lévy. The antibody-drug conjugate GENA-111 conjugated to auristatin F shows therapeutic potency in BCAM positive epithelial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1760.
Abstract 1763: Gastric cancer antibody fragment drug-conjugates (FDCs): Succeeding in solid tumors where ADCs fail
Authors: Deonarain Mahendra P. , Yahioglu Gokhan , Stamati Ioanna , Edwards Bryan , Diez-Posada Soraya , Perez-Castro Isabel , Pomowski Anja , Bouche Laura , Stewart Ashleigh , Maciuszek Monika , Ness Sam , Ngiam Malcolm , Xue Quinn
Published: June 2022

Antibody Drug Conjugates (ADCs) are failing due to 3 critical limitations: Low potency, ineffective solid-tumor penetration and poor tolerability. The industry is full of approaches where full-length Immunoglobulins have been engineered to carry defined numbers of payloads with higher-loadings of less potent payloads appearing to be well-tolerated. However, antibody fragments (e.g. single-chain Fcs-scFvs), which have many advantages including rapid tumor penetration, faster clearance, inexpensive manufacture, have been technologically challenging to apply in oncology. Our novel approach enables scFvs to have a high Drug:Antibody loading ratio (DAR) whilst retaining effective binding and other favorable biophysical properties, leading to a new product class tailored for solid tumors. Antikor has two FDC products in development for solid tumors, notably gastric: anti-HER2 FDC (ANT-043) and a second target (ANT-045) which will be disclosed during this presentation. ANT-043 has pM potencies in a range of HER2-expressing cell-lines, including trastuzumab-resistant models, excellent tumor ablation effects in breast, ovarian and gastric cancer xenograft models and superior tolerability compared to an ADC in rat toxicology studies at a dose of 1mg/kg/weekly. Quantitative payload tumor uptake and fluorescent immuno-histological studies demonstrate superior solid tumor penetration across the entire tumor and diffusion from blood vessels. In collaboration with our partners, Essex Biotechnology, Antikor is taking ANT-043 into IND-enabling studies for clinical development. ANT-045, which emerged from Antikor’s proprietary FDC ‘discovery engine’, is progressing towards IND-filing and updated data will illustrate how ANT-045 could have a broader patient benefit in gastro-intestinal cancers. ANT-045 has excellent in vitro cell-kill potency (pM IC50s) and excellent stability and superior in vivo tumor cure efficacy, compared to a leading clinical stage benchmark ADC. This presentation will focus on Antikor’s FDC discovery platform (stable high-DAR scFv-display libraries, tailored linker-payloads and design features) that has the potential to generate first-in-class products for difficult to treat solid tumors for patient benefit and promising to succeed where ADCs have failed to deliver.

Citation Format: Mahendra P. Deonarain, Gokhan Yahioglu, Ioanna Stamati, Bryan Edwards, Soraya Diez-Posada, Isabel Perez-Castro, Anja Pomowski, Laura Bouche, Ashleigh Stewart, Monika Maciuszek, Sam Ness, Malcolm Ngiam, Quinn Xue. Gastric cancer antibody fragment drug-conjugates (FDCs): Succeeding in solid tumors where ADCs fail [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1763.
Abstract 1764: Development of an alphalex™-auristatin low pH targeting conjugate for the treatment of solid tumors
Authors: Gayle Sophia , Aiello Robert , Bechtold Jane , Bourassa Patricia , Csengery Johanna , Hagen Connor , Howard Katia , Jones Kelli , Lopresti-Morrow Lori , Maguire Robert , Paradis Timothy , Pasqualini Theresa , Tweed Joseph , Tylaska Laurie , Zhang Qing , Paralkar Vishwas
Published: June 2022

Auristatins such as monomethyl auristatin E (MMAE) are a class of high potency microtubule targeting compounds that have an extremely narrow therapeutic window. Targeting potent auristatins to the tumor is the only feasible method of unlocking the clinical potential of such toxic molecules. While there are currently four marketed antibody-drug conjugates (ADCs) featuring auristatins, these ADCs face the same fundamental issues - tumor restriction by target antigen and the potential for off target release of payload.

Alphalex࣪ is a tumor targeting technology consisting of a unique variant of a family of pH-Low Insertion Peptides (pHLIP®) that target acidic cell surfaces (references 1-2), a cleavable small molecule linker, and an anti-cancer agent warhead. Alphalex࣪ thereby allows for antigen independent targeting of the tumor and enables intracellular delivery of the warhead by leveraging the low pH microenvironment of the tumor, a universal feature common to all tumors due to the Warburg effect.

Here we report the preclinical efficacy, safety, and antigen-independent tumor-targeting properties of alphalex࣪ conjugated to MMAE. We demonstrate the ability of alphalex࣪-MMAE to display potent in vitro and in vivo efficacy in colorectal, non-small cell lung, and prostate carcinoma cell lines. We further show that alphalex࣪-MMAE efficiently and safely delivers efficacious levels of MMAE selectively to tumor and demonstrates extreme plasma stability, with 0.02% warhead release over 24h in the rat. Based on the excellent preclinical safety and efficacy profile of alphalex࣪-MMAE, Cybrexa will move forward with the goal of initiating IND-enabling studies in 2022.

References:

1. Wyatt LC, Lewis JS, Andreev OA, Reshetnyak YK, Engelman DM. Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotechnol. 2017 Jul;35(7):653-664.

2. Wyatt LC, Moshnikova A, Crawford T, Engelman DM, Andreev OA, Reshetnyak YK. Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors. Proc Natl Acad Sci USA. 2018 Mar 20;115(12):E2811-E2818.

Citation Format: Sophia Gayle, Robert Aiello, Jane Bechtold, Patricia Bourassa, Johanna Csengery, Connor Hagen, Katia Howard, Kelli Jones, Lori Lopresti-Morrow, Robert Maguire, Timothy Paradis, Theresa Pasqualini, Joseph Tweed, Laurie Tylaska, Qing Zhang, Vishwas Paralkar. Development of an alphalex™-auristatin low pH targeting conjugate for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1764.
Abstract 1767: A next generation site-specific ADC targeting breast and gastric cancer
Authors: Italia James Sebastian , Biris Nikos , Li Zhi , Hussain Myer , Boyce John , Warner Audrey
Published: June 2022

Antibody drug conjugates are entering a renaissance period as a promising treatment option for a variety of cancers. Despite some success in the past few decades, developing effective ADCs remains a challenge due to extensive inefficiencies of industry standard conjugation technologies. Industry available methods can be limited by lack of site-specificity, inflexibility on the site of conjugation, and poor overall biophysical characteristics which can alter the efficacy, safety, and bioavailability of these therapeutics. BrickBio’s unique bioconjugation methodology enables precise (site-specific), flexible (offering numerous options for conjugation site and chemistry), efficient, and scalable generation of antibody-drug conjugates which overcome these limitations and achieve the full potential of this highly promising class of therapeutics.

In this work, we incorporate an azide-containing UAAs (unnatural amino acid) into full-length antibodies targeting receptors overexpressed in breast and gastric cancer. Specifically, we have engineered variants of IgG1 antibodies to contain the aforementioned UAA, enabling site-specific attachment of cytotoxic payloads, such as auristatins, via click chemistry to generate ADCs with homogeneous DARs and at different conjugation sites. The resulting BrickADCs exhibit high therapeutic efficacy against receptor-positive cell lines, particularly lines expressing low levels of HER2, in vitro as well as in mouse xenograft models. Not only are the BrickADCs 100x more potent in demonstrated cell lines, but current research indicate that they have an improved safety profile over T-DM1. In other efforts, the BrickADC platform is leveraging a proprietary Modular Multisite Platform (MMP) to address the unmet need of eventual ADC resistance by conjugating multiple distinct payloads in a homogenous fashion. Continued preclinical work is underway for these pipeline candidates as well as investigation into other target areas.

Citation Format: James Sebastian Italia, Nikos Biris, Zhi Li, Myer Hussain, John Boyce, Audrey Warner. A next generation site-specific ADC targeting breast and gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1767.
Abstract 1778: NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer
Authors: Cai Yanyan , Zhao Peihua , Wu Fan , Shao Hong , Razavi Pedram , Xu Guotai , Scaltriti Maurizio , Chandarlapaty Sarat
Published: June 2022

Genomic loss of in Neurofibromin 1 (NF1) is a recurrent mechanism driving metastatic progression and resistance to endocrine therapy in ER-positive breast cancer, via activation of the MAPK pathway, induction of cyclin D1 expression and modulation of estrogen receptor signaling. To identify specific mediators of tumor growth in NF1 mutant cancers, we performed a genetic knockout (KO) screen in isogenic WT and NF1 KO breast cancer cells. After employing stringent cutoff criteria, we found 28 genes that are specifically essential for NF1 KO cells but not WT cells. Among these genes, NR2F2, encoding an orphan nuclear receptor, was one of the top candidates. We validated that its loss significantly impaired the growth of multiple models of NF1 null, ER+ breast cancer.

Loss of NF1 leads to the hyperactivation of Ras which sequentially activates multiple effector pathways, including the MAPK pathway and the PI3K-AKT pathway. We found that MAPK activation upon NF1 loss led to elevated NR2F2 expression levels. A negative correlation between NF1 and NR2F2 expression was further confirmed in public gene expression databases. These data suggested that induction of NR2F2 expression might be one mechanism that mediates endocrine resistance caused by NF1 loss. Consistently, we found that exogenous expression of NR2F2 could confer endocrine resistance while its knockout hypersensitized ER+ breast cancer cells to endocrine therapies and significantly attenuated NF1 loss induced resistance.

RNA-seq analysis revealed that estrogen response gene sets were among the most enriched signatures upon NF1 KO, NR2F2 over-expression (OE) or NR2F2 KO. NF1 KO or NR2F2 OE decreased the expression of early estrogen response genes and impaired their E2-induced expression, most of which could be rescued by NR2F2 KO, suggesting a regulatory effect of NR2F2 on ER transcriptional activity. To further define these mechanisms, ongoing integrative analyses of NR2F2 qPlex-RIME, ER and NR2F2 ChIP-Sequencing and ATAC-Sequencing will be presented.

Taken together, our studies identify NR2F2 as a MAPK driven transcript whose induction mediates endocrine resistance, and may serve as a novel therapeutic target to overcome anti-estrogen resistant breast cancer growth.

Citation Format: Yanyan Cai, Peihua Zhao, Fan Wu, Hong Shao, Pedram Razavi, Guotai Xu, Maurizio Scaltriti, Sarat Chandarlapaty. NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1778.
Abstract 1783: JNK/MDR1 confers chemo-resistance in breast cancer cells carrying estrogen receptor activating mutations
Authors: Taya Marwa , Merenbakh-Lamin Keren , Rubinek Tami , Wolf Ido
Published: June 2022

Background: Endocrine therapy is the mainstay treatment for estrogen receptor-α (ER) positive breast cancer (BC) patients. However, all metastatic BC patients develop resistance, which in nearly 40% of patients is due to activating mutations, mostly Y537S and D538G, in the ligand-binding domain (LBD). Tumors harboring these mutations are more aggressive and the patients' prognosis is worse. We hypothesized that refractory response to further treatment lines, i.e, chemotherapy, partially accounts for the worse prognosis. Hence, our goal was to study whether LBD-ER BC cells are resistant to chemotherapy and reveal the mechanism of action.

Methods: We used MCF-7 cells stably expressing WT-ER, Y537S or D538G-ER mutations. Cell proliferation and viability were determined using MTT, methylene blue and colony assays. We evaluated apoptosis by monitoring cleaved-PARP and flow cytometry using Annexin/7AAD staining. MDR1 expression was studied using qRT-PCR and western-blot. JNK/c-Jun pathway was studied by pJNK and cJun expressions, cJun transcriptional activity, ChIP assay and pharmacological inhibition.

Results: LBD-ER cells exhibited higher proliferation and viability and exerted less apoptosis compared to WT-ER cells following treatment with paclitaxel, doxorubicin, and 5-FU. MDR1 expression and JNK pathway activity was higher in D538G cells compared to both WT-ER and Y537S cells. Importantly, inhibition of the JNK pathway, using SP600125 and BI-78D3, lead to decreased MDR1 expression, increased cleaved PARP, and decreased viability in the D538G cells. In addition, our results revealed that JNK increased c-Jun binding activity of the MDR1 gene in the D538G mutated cells.

Conclusions: These results indicate that LBD-ER cells confer resistance to chemotherapy. The mechanisms leading to it are diverse, and we found that in D538G increased JNK pathway activation resulted in elevated MDR1 expression and chemo-resistance. This paves the way to therapies aiming at inhibition the JNK pathway in order to restore chemo-resistance. This study also highlights the marked differences between these seemingly similar mutations, strengthening the necessity to adopt a personalized treatment approach for BC patients who developed endocrine resistance.

Citation Format: Marwa Taya, Keren Merenbakh-Lamin, Tami Rubinek, Ido Wolf. JNK/MDR1 confers chemo-resistance in breast cancer cells carrying estrogen receptor activating mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1783.
Abstract 1795: Molecular vulnerabilities of the polyaneuploid cancer cell state
Authors: Loftus Luke
Published: June 2022

Once cancer has metastasized it remains incurable owing to resistance to nearly all systemic therapies. Classical views of therapeutic resistance include tumor heterogeneity and genetic instability, but our group and others have identified an unappreciated cancer cell phenotype, the polyaneuploid cancer cell (PACC) state, that provides an alternative model. The PACC state is induced upon exposure to a variety of applied stressors, including multiple different classes of chemotherapy, and is characterized by an enlarged cell size, polyploidy of the typically aneuploid cancer genome, and the ability to repopulate tissue through depolyploidization. We have demonstrated that PACCs are present in a variety of cell lines, arise in response to a multitude of stressors in vitro, and are resistant to cisplatin, docetaxel and Ipatasertib treatment. Thus, we hypothesize that the PACC state is driving cancer’s therapeutic resistance to nearly all available treatment regimens and enabling cancer relapse. Elimination of the PACC state is essential to prevent cancer recurrence, and ultimately enable a cure for cancer.To identify candidate drug targets specific to the PACC state, we performed RNA sequencing, NanoString mRNA panels, bulk proteomics and cell surface proteomics of PACCs versus their parental cell line. PACCs were induced from prostate cancer lines PC3 and LNCaP, and breast cancer line MDAMB231 with both cisplatin and docetaxel treatment. Initial datasets with PC3 cells found that 1013 genes are enriched (greater than 1.5 Log2 Fold Change) in cisplatin induced PACCs while 88 genes are enriched in docetaxel induced PACCs. 22 genes were enriched with both chemotherapy treatments implying that there are genes common to the PACC state. NanoString mRNA and proteomic analysis further validated a subset of the overlapping genes, including ATF3, BIRC3, IL6, and NOX5.Our existing data reveals that there are molecular vulnerabilities of the PACC state that can be manipulated for therapeutic elimination. We will further validate candidate genes across numerous stressors and cell lines and then assess the PACC state’s dependence on these pathways through inhibition and engineered cell lines. Information from these studies will directly lead to development of a therapeutic strategy specifically targeting the PACC state, with the intention of eliminating cancer resistance.

Citation Format: Luke Loftus. Molecular vulnerabilities of the polyaneuploid cancer cell state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1795.
Abstract 1799: Inhibition of triple negative breast cancer metastasis via Enolase-1 modulation
Authors: Tailor Dhanir , Dheeraj Arpit , Garcia-Marques Fernando Jose , Pandrala Mallesh , Bermudez Abel , Pitteri Sharon , Malhotra Sanjay V.
Published: June 2022

Triple-negative breast cancer (TNBC) is one of the aggressive forms of breast cancer and frequently relapses and metastases. Currently, very few options are available for TNBC treatment. Cancer cells exploit glycolytic machinery for the Warburg effect and aerobic glycolysis. Enolase 1 (ENO1) is the glycolytic enzyme expressed in the majority of tissues and many cancer cells have its higher expression. Apart from the glycolytic role in the cytosol, ENO1 also plays different roles in cancer cells including function as a surface receptor. We have developed a chemical small molecule (CET12) that strongly binds to ENO1 and restrain its activity and subcellular localization. CET12 treatment arrests the TNBC cells in the mitotic phase and leads the apoptotic cell death via AMPK activation. Global proteome profiling suggested that CET12 pushes the cells toward oxidative phosphorylation. It also inhibits cell migration and invasion in vitro. In vivo studies using 4T1 and EMT6 syngeneic mouse models are also in-line with in vitro results and revealed that SU0212 treatment inhibits tumor progression and metastasis. These results were further supported by tail vain lung metastasis assay and intracardiac injection mouse model. This study provides compelling preclinical data for further development of CET12 for the treatment of TNBC.

Citation Format: Dhanir Tailor, Arpit Dheeraj, Fernando Jose Garcia-Marques, Mallesh Pandrala, Abel Bermudez, Sharon Pitteri, Sanjay V. Malhotra. Inhibition of triple negative breast cancer metastasis via Enolase-1 modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1799.
Abstract 1812: Therapeutic targeting and development of IDH inhibitors for the treatment of chondrosarcoma
Authors: Sheikh Tahir N. , Lu Chao , Schwartz Gary K.
Published: June 2022

Chondrosarcomas are the second most frequently occurring type of bone malignancy, and account for approximately 25% of all bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. They are largely considered to be resistant to conventional chemotherapy and radiotherapy. Recurrent somatic mutations in the isocitrate dehydrogenase I (IDH1) genes have been identified in a spectrum of human malignancies, including chondrosarcoma. Mutations in these genes lead to impaired ability of IDH1 to catalyze the conversion of isocitrate to alpha ketoglutarate and to gain of a neomorphic enzymatic activity which results in production of oncometabolite 2-hydroxyglutarate (2HG). Our preliminary data indicated that compared to samples that were wild type, IDH1 mutant samples showed a significant increase in intra-tumoral 2HG levels. In the present study, we hypothesized that 2-HG accumulation induces DNA and histone hypermethylation and altered gene expression, ultimately resulting in a block in cellular differentiation by competitively inhibiting α-ketoglutarate-dependent dioxygenases involved in histone and DNA demethylation. Therefore, we proposed that inhibition of IDH should mediate its antitumor effects through the induction of differentiation. To test this hypothesis, we utilized human chondrosarcoma cell lines including IDH wild type (CH2879) and IDH1 mutant (JJ012) and the IDH1 inhibitor FT6535. Results from our in vitro cell viability assay and histone methylation showed that selective inhibition of IDH1 resulted in modest inhibition of cell proliferation and suppression of histone methylation in IDH1 mutant chondrosarcoma cells. Next we elected to test the IDH inhibitor in combination with differentiating agents including retinoic acid. Cell viability assays showed that when compared to either agent alone, the combination of retinoic acid and FT6535 resulted in a significant decrease in cellular proliferation. Western blot analysis showed induction of COL1A2 and SOX9 (differentiation markers) with suppression of histone methylation H3K27me3 or H3K9me3, Taken together, our data strongly suggests that combination treatment with the IDH1 inhibitor FT6535 and retinoic acid represents a novel approach to suppress cell growth by epigenetic modulation to induce differentiation and merits further evaluation as a potential treatment modality in chondrosarcoma.

Citation Format: Tahir N. Sheikh, Chao Lu, Gary K. Schwartz. Therapeutic targeting and development of IDH inhibitors for the treatment of chondrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1812.
Abstract 1829: Characterization of factors affecting sensitivity and resistance to PRMT5 inhibition in glioblastoma
Authors: Tsai Tsung-Lin (Gavin)
Published: June 2022

BACKGROUND: Glioblastoma (GBM) is a highly infiltrative and aggressive malignancy with few treatment options; all patients develop recurrence after initial therapy. Due to the dismal outcome of these patients, identification of GBM-specific targets for therapeutic development is urgently needed. PRMT5 is a protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues within histones marks, resulting in chromatin restructuring, which in turn promotes oncogenic processes. PRMT5 inhibition has shown efficacy against multiple malignancies; MTAP loss has been associated with increased sensitivity to PRMT5 inhibitors. Here, we examined the effects of pharmacological abrogation of PRMT5 function using highly specific, brain penetrant, orally bioavailable inhibitors and assessed the role of MTAP and p53 expression on sensitivity to these agents. MATERIALS AND

METHODS: Using pharmacological inhibition of PRMT5 using with the highly specific inhibitors PRT808 and PRT811, we examined their effects on apoptosis, cell proliferation, cell cycle distribution, and methylation markers in patient-derived glioma stem-like cells (GSC) with epigenetic and genetic signaling. Additional studies to identify transcriptomic (RNA-seq), and proteomic (RPPA) landscape related to PRMT5 in gliomas are ongoing.

RESULTS: PRT808 and PRT811 induced potent reduction of symmetrical dimethylation (SDMA) protein marks in all GSC tested. However, treatment of GSC214 (mutant p53), GSC262 (mutant p53), GSC811 (truncated p53), and GSC11 (wild type p53) showed that wild type and truncated p53-expressing lines (GSC811 and GSC11) were more sensitive than GSC cell lines expressing mutant p53 (GSC214 and GSC262). There was no correlation between MTAP status and sensitivity to PRMT5 inhibition. In addition, PRMT5 inhibition resulted in reduced proliferation and alteration of several cell survival pathways; induction of apoptosis in GSC cell lines at late time points around 8-9 days after single dose treatment even though SDMA reduction occurred as early as 3 days.

CONCLUSIONS: Our results demonstrate pharmacological PRMT5 inhibition induced SDMA reduction and late apoptosis in genetically and epigenetically heterogeneous patient-derived GSC lines and that mutant p53 lines are less sensitive to PRMT5 inhibition. Additional studies related to splicing defects upon PRMT5 inhibition are ongoing and will be presented at the meeting.

Citation Format: Tsung-Lin (Gavin) Tsai. Characterization of factors affecting sensitivity and resistance to PRMT5 inhibition in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1829.
Abstract 1850: Characterizing senescence response to PARP inhibition may provide opportunities for enhanced efficacy through combinations with senolytic agents
Authors: Lombard Alan P. , Lou Wei , Evans Christopher P. , Gao Allen C.
Published: June 2022

Background: Inhibition of poly (ADP-ribose) polymerase (PARP) is an exciting treatment strategy recently approved for prostate cancer patients with homologous recombination repair defects. Despite this advance in the field, it remains unclear how PARP inhibitor (PARPi) sensitive cells respond to treatment. We previously demonstrated that treatment with the PARPi olaparib induces not only cell death, but also G2/M arrested senescence characterized by activation of the p53 signaling pathway. We hypothesize that targeting PARPi induced senescence may provide a means to enhance the efficacy of PARPi treatment. In our current work, we sought to 1) understand whether senescence induction is a generalized response to all PARPi’s and 2) characterize senescence induction to guide the development of novel treatment strategies combining a PARPi with a senolytic drug.

Methods: PARPi sensitive LNCaP and C4-2B prostate tumor cells were treated with olaparib, rucaparib, niraparib, or talazoparib for 5 days to induce senescence. Both vehicle treated and quiescent cells (LNCaP and C4-2B cultured in FBS-low (0.2%) conditions) were used as controls. Cell viability, flow cytometry, and beta-galactosidase activity assays tested response to PARPi’s. Western blot was used to detect PARP activity, apoptosis, and DNA damage response. RNA-sequencing was performed to characterize senescence induced signaling alterations.

Results: We found that exposure to rucaparib, niraparib, and talazoparib all induce a robust G2/M arrested senescence response in LNCaP and C4-2B cells, suggesting that senescence induction is a class effect of PARPi’s. PARPi induced senescence is characterized by activation of the p53 signaling pathway and significantly increased expression of the cyclin-dependent kinase inhibitor p21. Furthermore, PARPi induced senescence is distinct from quiescence, suggesting that response to PARP inhibition is phenotypically different from a more general growth arrest. RNA-sequencing revealed several signaling changes associated with senescence which may provide novel treatment opportunities.

Conclusions: Senolytics are a class of drugs thought to specifically target senescent cells. Our results demonstrate that PARPi’s induce senescence. Future work will be directed at further characterizing PARPi induced senescence, leading to rationally selected senolytic drug combinations which may enhance the efficacy of PARPi therapy.

Citation Format: Alan P. Lombard, Wei Lou, Christopher P. Evans, Allen C. Gao. Characterizing senescence response to PARP inhibition may provide opportunities for enhanced efficacy through combinations with senolytic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1850.
Abstract 1875: Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy
Authors: Grailer Jamison , Gilden Julia , Wang Jun , Garvin Denise , Stecha Pete , Hartnett Jim , Edenson Steven , Fan Frank , Cong Mei , Cheng Zhi-jie Jey
Published: June 2022

Programmed death 1 (PD-1) is an immune checkpoint (IC) receptor that negatively regulates T-cell immune function. Blocking the interaction of PD-1 with its ligand PD-L1 unleashes the immune system and enhances anti-tumor responses. Immunotherapy using PD-1 blockade has led to a paradigm shift in cancer drug discovery, due to its durable effect against a wide variety of cancers. However, despite its tremendous clinical success in cancer treatment, a significant fraction of patients and tumor types remain unresponsive to this therapy. This has led to the development of combination therapies combining PD-1 checkpoint inhibitors with a broad range of clinically active treatments including those targeting other IC receptors. A major challenge in immunotherapy drug development is the lack of quantitative and reproducible functional assays as existing methods rely heavily on primary immune cells and are highly variable. Here, we report the development of a suite of cell line-based reporter bioassays for monoclonal antibody targeting human PD-1/PD-L1, or bispecific molecules target PD-1/PD-L1 and a co-stimulatory receptor (e.g., 4-1BB, OX40, ICOS) or an immune inhibitory receptor (e.g., CTLA-4, LAG-3, TIGIT). A mouse PD-1/PD-L1 assay and a human PD-1/PD-L2 assay are also developed and can be used in parallel with human PD-1/PD-L1 assay to address preclinical study and assay specificity. Each of these assays consist of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), PD-1 and the second IC receptor, and an artificial antigen presenting cells (aAPCs). Particularly, the combination bioassays are able to measure the synergetic effect of PD-1 blockade with a second IC inhibitor receptor blockade or a costimulatory receptor activation. These reporter-based assays reflect the mechanisms of action for the therapeutic drugs of interest and are sensitive and quantitative. They are prequalified according to ICH guideline and demonstrated the performance characteristics (specificity, precision, accuracy, and linearity) to be used as potency assay for product release and stability studies during immunotherapy drug development and manufacture.

Citation Format: Jamison Grailer, Julia Gilden, Jun Wang, Denise Garvin, Pete Stecha, Jim Hartnett, Steven Edenson, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1875.
Abstract 1885: Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality
Authors: Lye Melvin , Eberle Christoph , Wang Ann , Feld Geoffrey K. , Kim Namyong
Published: June 2022

Limited innovation in automated cell and organelle sample preparation methodology limits the effectiveness of modern analytical methods, such as single-cell ‘omics, flow and mass cytometry. These techniques traditionally rely on manual centrifugation-based protocols for cell washing and suspension preparation, hampering researchers’ access to the reproducibility and scalability benefits of automation. We have developed a suite of cell suspension preparation systems that enable semi and full automation of cell washing protocols. These Laminar Wash࣪ technologies robustly, gently, and efficiently remove debris, dead cells, and unbound reagent using laminar flow and liquid handling robotics, rather than turbulent and harsh pelleting-plus-pipetting methods. Adaptation of standard protocols to Laminar Wash automation typically improves repetitive immunostaining processes and workflows, in terms of reduced hands-on time and inter- and intra-operator variability. We demonstrate the superior live cell retention and reproducibility of Laminar Wash over centrifugation in processing murine and humanized mouse peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) for flow cytometry. Furthermore, we show how Laminar Wash improves flow cytometry data quality, in terms of debris removal and separation of immune cell subsets for both PBMCs and TILs. Overall, these results show how Laminar Wash methodology assists in standardizing sample preparation for cytometric analysis, an important and unmet need in cancer immunotherapy discovery and manufacturing workflows.

Citation Format: Melvin Lye, Christoph Eberle, Ann Wang, Geoffrey K. Feld, Namyong Kim. Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1885.
Abstract 1886: Targeting oral squamous cell carcinoma tumor microenvironment through simultaneous modulation of pattern recognition receptors and angiogenic factors using a synthetic polymer-based drug
Authors: Lee Won Yong , Ponthan Frida , Sonis Stephen T.
Published: June 2022

Squamous cell carcinomas (SCCs) are the dominant form of cancers of the upper aerodigestive tract. Main risk factors are smoking and human papilloma virus (HPV) infection. Despite the HPV vaccination efforts, the number of oropharyngeal cancers (OSCCs) has been steadily increasing especially in males. Standard of care primarily focuses on the surgical removal of cancerous tissues followed by chemoradiation therapy. However, the recurrence rate of OSCCs remains fairly high as the tumor frequently develops therapy resistance and more effective therapeutic regimens are needed. The chronic inflammatory state yet paradoxical immunosuppressive tumor microenvironment has been regarded as a cause of therapy resistance and suggested as a potential therapeutic target. Pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) contribute to pro-inflammatory tissue responses. TLR2 present in the OSCC tumor appears to play important roles in regulating immune responses and promoting pro-inflammatory tumor microenvironment. GM-1111 is a synthetic glycosaminoglycan-based anti-inflammatory molecule that potently inhibits PRRs such as TLR2 and NLRP3 inflammasome. In addition, GM-1111 inhibits multiple angiogenic molecules - CXCL12, VEGF, and FGF2. We hypothesized that GM-1111 might be able to reduce OSCC tumor growth by simultaneously disrupting pro-inflammatory as well as angiogenic signaling. To test this hypothesis, male CD1 nude mice bearing FaDu cell tumor in the back were treated with either (1) vehicle, (2) GM-1111 (30 mg/kg, q.d., s.c.), (3) vehicle with irradiation (2 Gy/day x 5 days), or (4) GM-1111 with irradiation 17 days. Established tumors grew in two phases from slow for the initial 7 days to fast growths during the remaining days. Irradiation- or drug-induced tumor regression was observable about 5 days after the last radiation therapy (day 10). Compared to the vehicle only (1) treatment group, GM-1111 treated animals (groups 1 vs. 2: p = 0.0032; groups 1 vs. 3: p < 0.0001; groups 1 vs. 4: p < 0.0001) demonstrated significantly reduced tumor growth. These results suggest that GM-1111 has a potential to reduce OSCC and further confirm our previous orthotopic human tongue SCC-25 cell xenograft study data.

Citation Format: Won Yong Lee, Frida Ponthan, Stephen T. Sonis. Targeting oral squamous cell carcinoma tumor microenvironment through simultaneous modulation of pattern recognition receptors and angiogenic factors using a synthetic polymer-based drug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1886.