bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2529: Necrosome translocates to lysosome to induce lysosome membrane permeabilization and subsequent necroptotic cell death
Authors: Mammadova Jamila , Fatirkhorani Rojin , Liu Shuzhen , Chen Ken , Wang Zhigao
Published: April 2023

Introduction: Necroptosis is a cellular process known to be downregulated by cancer cells, and its molecular steps have been described in the literature. Mixed-lineage kinase domain like (MLKL) protein is the most terminal effector of necroptosis. Necroptosis has previously been shown to be activated by the formation of necrosome complex consisting of receptor interacting serine/threonine kinase 1 (RIPK1)-RIPK3-MLKL. MLKL is activated by RIPK3 via phosphorylation, forming phospho-MLKL (P-MLKL), leading to MLKL polymerization and cell necroptosis via lysosome membrane permeabilization (LMP) and plasma membrane rupture. We propose that the necrosome locates onto the lysosome, leading to MLKL-activation induced LMP before cell membrane rupture.

Objectives: Our objective is to show that necrosome formation occurs on lysosomal membrane, which then leads to the formation of P-MLKL, and, subsequently, necroptosis via LMP and plasma membrane rupture.

Methods: HT-29 cells with hemagglutinin (HA) tagged lysosomal transmembrane protein were induced to undergo necroptosis with the treatment of TNF (T), Smac-mimetic (S) and Z-VAD-FMK (Z) (abbreviated as T/S/Z). Lysosomes were extracted first by cell rupture via homogenization and then immunoprecipitation using anti-HA magnetic beads.

Results: Our results suggest that LMP precedes plasma membrane permeability and that MLKL activation takes place on lysosomal membrane. Western blotting demonstrated that lysosomal cathepsin proteases, including CTSA, CTSB, CTSC, CTSD and CTSK, were released into cytosol during necroptosis, confirming lysosomal damage. Furthermore, P-RIPK1, P-RIPK3, P-MLKL and MLKL tetramer were found to be specifically associated with purified lysosomes upon necroptosis induction.

Conclusions: MLKL activation on lysosomal membrane and LMP constitute an important step of necroptosis. [J.M. and R.F. contributed equally to this work.]

Citation Format: Jamila Mammadova, Rojin Fatirkhorani, Shuzhen Liu, Ken Chen, Zhigao Wang. Necrosome translocates to lysosome to induce lysosome membrane permeabilization and subsequent necroptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2529.
Abstract 2545: NNMT is a novel effector of EGFR driven glioblastoma malignancy
Authors: Ma Lianping , Li Ming
Published: April 2023

Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the conversion of methyl groups from S-adenosyl-L-methionine (SAM) to nicotinamide (NAM) to generate S-adenosine hyperblastoids (SAH) and 1-methylnicotinamide (MNA). Upregulation of NNMT has been observed in a variety of cancers, including glioblastoma. The mechanism by which NNMT is overexpressed in glioblastoma remains largely unknown. Epidermal growth factor receptor (EGFR) gene amplification or mutation is one of the most common genetic lesions in glioblastoma, and EGFRvIII is the most common mutant contributing to glioblastoma cell proliferation, invasion, therapeutic resistance, metastasis, and stemness. In this study, we show that activation of wild-type EGFR by EGF or the constitutively active mutant EGFRvIII promoted NNMT expression in established glioblastoma cell lines and glioblastoma stem-like cells via the p38 mitogen-activated protein kinase pathway. Furthermore, we identified STAT1 as a downstream transcription factor required for EGFR/EGFRvIII-induced NNMT upregulation. Knockdown of NNMT by RNA interference in glioblastoma cells significantly inhibited EGFRvIII-promoted glioblastoma cell invasion and proliferation in vitro. Targeting NNMT inhibited EGFRvIII-driven glioblastoma tumorigenicity and prolonged survival of tumor-bearing mice. Clinically, NNMT was highly expressed and positively correlated with EGFR expression in human glioblastoma tumors. Taken together, these findings establish NNMT as a previously unknown downstream target gene of EGFR/EGFRvIII expression and nominate it as a new potential therapeutic target for inhibiting EGFRvIII-driven glioblastoma.

Citation Format: Lianping Ma, Ming Li. NNMT is a novel effector of EGFR driven glioblastoma malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2545.
Abstract 2551: CASC4 drives anoikis resistance in high grade serous ovarian cancer through recycling of EGFR
Authors: Bapat Jaidev , Brubaker Lindsay W. , Watson Zachary L. , Yamamoto Tomomi M. , Bitler Benjamin G.
Published: April 2023

Introduction: Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian cancer (HGSOC) subtype accounts for almost 60% of ovarian cancers and is the deadliest. HGSOC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. Survival of tumor cells in the peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). CRISPR/Cas9 and transcriptomic screens identified the Golgi protein CASC4 (GOLM2, H63) as a “driver” of anoikis resistance. As CASC4 is highly uncharacterized in literature, we sought to determine how CASC4 confers anoikis resistance to HGSOC cells.

Methods: Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse clinical outcomes, such as worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we used HGSOC cell lines PEO1, CaOV3, and OVCAR3 with shRNA-mediated CASC4 knockdowns (CASC4 KD), cultured in forced suspension.

Results: Culturing cells in suspension, to recapitulate the peritoneal fluid environment in vitro, showed that CASC4 KD hampers cell proliferation and colony formation ability, and increases apoptosis. Additionally, a Reverse Phase Protein Assay (RPPA) showed that CASC4 KD results in significant changes in proteins known to be localized to the cell surface or secreted, including decreased protein levels of the receptor tyrosine kinase (RTK) Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways. A paralog of CASC4, GOLM1, is known to drive hepatocellular carcinoma through various mechanisms, such as the recycling of internalized RTKs, including EGFR. Indeed, our experiments showed that CASC4 KD in ovarian cancer cells leads to decreased EGFR levels and decreased EGFR recycling.

Conclusion: Knocking down CASC4 results in decreased survivability in suspension, decreased EGFR levels at the plasma membrane, and decreased recycling of EGFR. Further elucidating mechanisms of CASC4-dependent anoikis resistance could lead to the development of novel therapeutic approaches, such as inhibitory peptides, and may assist in developing newer biomarkers for predicting ovarian cancer malignancy.

Citation Format: Jaidev Bapat, Lindsay W. Brubaker, Zachary L. Watson, Tomomi M. Yamamoto, Benjamin G. Bitler. CASC4 drives anoikis resistance in high grade serous ovarian cancer through recycling of EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2551.
Abstract 2553: Elucidation of the downstream signaling of PIK3CA genomic alterations in serous ovarian cancer
Authors: Zhang Shibo , Hong Hei Ip , Mak Victor CY , Zhou Yuan , Wang Panpan , Cheung Lydia Wai Ting
Published: April 2023

Genomic aberrations of PIK3CA, which encodes the catalytic subunit p110α of the class IA phosphatidylinositol 3-kinase (PI3K), is frequently observed in cancers. In ovarian cancer of the serous subtype (the most common histotype of the disease), PIK3CA amplification is dominant over single nucleotide mutations in multiple patient cohorts, suggesting the possibility that PIK3CA amplification is an alternative to PIK3CA “hot spot” mutation for serous ovarian tumorigenesis. However, whether PIK3CA amplification and mutation exert the same functional consequences on serous ovarian tumorigenesis remains uncertain. This study aims to explore and compare the functional roles between the two types of PIK3CA gene aberrations on tumorigenicity, cellular signaling, and therapeutic responses in serous ovarian cancer. We confirmed that both PIK3CA amplification and E545K mutation could induce multiple tumorigenic phenotypes in CRISPR/Cas9-mediated E545K knock-in or wild-type PIK3CA overexpressing serous ovarian cancer cell lines. Signaling pathway analyses showed that E545K knock-in cells, but not PIK3CA-overexpressing cells, had the MAPK signaling pathway activated and were sensitive to MAPK pathway inhibition. An induction of AKT phosphorylation and PDK1 phosphorylation was observed in both E545K knock-in cells and wild-type PIK3CA-overexpressing cells. Accordingly, these cells displayed higher sensitivity towards p110α or AKT inhibitors. Intriguingly, in line with previous report which demonstrated YAP as AKT substrate, the activation of AKT associated with an increase in YAP S127 phosphorylation in the knock-in or PIK3CA-overexpressing cells. AKT inhibition suppressed YAP phosphorylation. Corroborating with the role of S127 in nuclear exclusion of YAP, YAP accumulated in the cytoplasm of these cells, leading to reduced transcriptional activity of YAP. While the functional significance of YAP cytoplasmic accumulation is yet to be investigated, our results reveal the common and distinct downstream pathways between PIK3CA amplification and mutation. These findings may be applicable to p110α-targeting therapies in serous ovarian cancer.

Citation Format: Shibo Zhang, Hei Ip Hong, Victor CY Mak, Yuan Zhou, Panpan Wang, Lydia Wai Ting Cheung. Elucidation of the downstream signaling of PIK3CA genomic alterations in serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2553.
Abstract 2556: Phosphatase nanozyme combination with kinase inhibitor for decreasing prostate cancer cell viability
Authors: Myrzagaliyeva Arailym , Nazarbek Guldan , Myrzagali Sandugash , Amin Amr , Xie Yingqiu
Published: April 2023

Background: Phosphatase has been well studied in anti-cancer research. The dietary amino acid cysteine plays essential roles in protein structure by disulfide bonds, metal ion binding, detoxification, and many metabolic functions. Cysteine has been shown in inducing human bladder cancer cells apoptosis. However, the cysteine mediated nanoparticles enzyme (nanozyme) activity and their function, signaling pathways are yet unknown. We aim to test whether the cysteine mediated nanoparticles nanozyme activity of phosphatase could enhance targeting of kinases.

Methods: Fresh L-cysteine were applied for synthesis of less than 200 nm sized nanozyme. The phosphatase enzyme activity was measured using NBT/BCIP substrate. Scanning electron microscopy, transmission electron microscopy were used for nanoparticle analysis. Cell viability was measured by crystal violet staining of cells.

Results: L-cysteine at 0.1mg/mL combined with MET inhibitor (1µm or 5µm) showed the better effect of enhanced efficacy and decreased the viability of prostate cancer cells compared to inhibitor alone or cysteine. Combination did not affect the phosphatase activity of cysteine nanozyme.

Conclusion: L-cysteine may be applied to be combined with kinase inhibitors in drug delivery for increased efficacy.

Citation Format: Arailym Myrzagaliyeva, Guldan Nazarbek, Sandugash Myrzagali, Amr Amin, Yingqiu Xie. Phosphatase nanozyme combination with kinase inhibitor for decreasing prostate cancer cell viability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2556.
Abstract 2558: Loss of TC-PTP in keratinocytes leads to increased UVB-induced autophagy
Authors: Asare Obed , Zavala Klarissa , Shim Lindsey , Hafeez Bilal , Kim Dae
Published: April 2023

Amongst the deteriorating effects of the sun’s ultraviolet B (UVB) radiation on cellular homeostasis is the formation of photoproducts in the DNA, such as cyclobutene pyrimidine dimers. Photoproducts cause conformational alterations in the DNA structure which may evade cellular repair machinery and persist through DNA replication processes, leading to potential gene mutations and ultimately leading to skin cancer. Photoproducts are typically repaired by nucleotide excision repair. Notwithstanding, when the repair mechanism fails, apoptosis ensues to prevent the accumulation of mutations and to restore cellular homeostasis. T-cell protein tyrosine phosphatase (TC-PTP) is one of the members of PTP family that has been shown to increase UVB-induced apoptosis in keratinocytes via the downregulation of Flk-1/JNK signaling. This implies that loss of TC-PTP causes resistance to apoptosis in UVB-damaged keratinocytes. In the present work, we report that this resistance is accompanied by an increase in UVB-induced autophagy in the absence of TC-PTP. We generated human TC-PTP-deficient (TC-PTP/KO) HaCaT keratinocytes using CRISPR/Cas9 system. The expression of microtubule-associated protein light chain 3 (LC3) was significantly increased in TC-PTP/KO HaCaT keratinocytes compared to engineered controls (TC-PTP/Mock) following treatment with UVB irradiation. Increased expression of LC3 in TC-PTP/KO keratinocytes was accompanied by a significant decrease in the expression of the p62. We also observed that treatment of TC-PTP KO keratinocytes with chloroquine (CQ), a late-phase autophagy inhibitor, caused a remarkable increase in LC3 expression following UVB exposure in comparison with untreated TC-PTP/KO keratinocytes, implying that loss of TC-PTP increases autophagy in response to UVB. With this regard, TC-PTP deficiency in human keratinocytes significantly increased cell proliferation following UVB exposure compared to similarly treated control keratinocytes. Pretreatment of CQ before UVB irradiation in TC-PTP KO keratinocytes significantly reduced cell viability compared to untreated TC-PTP KO keratinocytes. These results suggest that TC-PTP protects keratinocytes from UVB-induced damage by negatively regulating autophagy signaling.

Citation Format: Obed Asare, Klarissa Zavala, Lindsey Shim, Bilal Hafeez, Dae Kim. Loss of TC-PTP in keratinocytes leads to increased UVB-induced autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2558.
Abstract 2605: LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells
Authors: Laurentino Talita S. , Soares Roseli S. , Lerario Antonio M. , Marie Suely K. , Oba-Shinjo Sueli Mieko
Published: April 2023

Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family of copper-dependent amino oxidase, is responsible for the crosslinking of collagen and elastin. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among the tumors with the highest LOXL3 expression levels. Moreover, LOXL3 expression increased with malignancy grade amongst diffusely infiltrative astrocytomas (from grade 2 to 4). In the present work, CRISPR-Cas9 gene editing was used to knock out LOXL3 in the human glioma U87MG cell line to evaluate the role of LOXL3 in glioblastoma. Analyses of protein expression level and genomic mutation mediated by CRISPR-Cas9 by two different sgRNA (denominated sgRNA1 and 2) were performed and the knock out efficiency and genome editing were confirmed. Transcriptome analysis of these cells revealed a downregulation of genes that coded for proteins involved in microtubule organization, cell division, and vesicle trafficking. On the other hand, upregulated genes when LOXL3 was knocked out in U87MG cells were involved in actin cytoskeleton, negative regulation of gene transcription, including chromatin remodeling, and cell-cell adhesion. Functional in vitro assays showed that LOXL3 knocked out cells presented decreased cell proliferation due to longer cell cycles with increased G1 and G2/M checkpoints compared to control cells. Additionally, LOXL3 knocked out U87MG cells presented a higher percentage of cells in the early phase of apoptosis with an increment after temozolomide treatment. Our results suggest an important role of LOXL3 in glioblastoma possibly mediated by cytoskeleton regulation, affecting cell proliferation, cell cycle, and cell death. These results reinforced the importance of LOXL3 in tumors, especially in glioblastoma, highlighting it as a potential therapeutic target.

Citation Format: Talita S. Laurentino, Roseli S. Soares, Antonio M. Lerario, Suely K. Marie, Sueli Mieko Oba-Shinjo. LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2605.
Abstract 2607: Homogenous TP53mut-associated tumor biology across mutation and cancer types revealed by comprehensive mRNA expression analysis
Authors: Budczies Jan , Romanovsky Eva , Kluck Klaus , Ourailidis Iordanis , Menzel Michael , Beck Susanne , Ball Markus , Kazdal Daniel , Christopoulos Petros , Schirmacher Peter , Stiewe Thorsten , Stenzinger Albrecht
Published: April 2023

TP53 mutations are the most common single gene alteration in human cancer with diagnostic and prognostic implications in some cancer types. While no TP53-targeting therapeutics have been approved in the USA or Europe yet, drugs tailored to specific TP53 mutations, restoring the functionality of mutated TP53 (TP53mut), and protecting TP53 from negative regulation are being explored in preclinical studies and clinical trials. We performed a comprehensive mRNA expression analysis in 24 cancer types of the TCGA to extract (i) a consensus expression signature shared across TP53 mutation types and cancer types, (ii) differential gene expression patterns between different TP53 mutation types such as LOF, GOF, as well as dominant-negative mutations, and (iii) cancer types specific gene expression patterns. Mutational hotspots showed a similar pattern across cancer types, but at the same time prevalence was significantly different between cancer types for about half of the most prevalent hotspots. Both can be explained in part by the mutational processes operational for example the clock-like process behind SBS1 that is operational across cancer types and aflatoxin exposure assocated with SBS24 that ist operational in liver hepatocellular carcinoma. Virtually no genes were differential between tumors harboring different types of TP53 mutations in none of the cancer types, while hundreds of genes were over- and underexpressed in TP53mut compared to TP53wt tumors. A consensus gene list of 178 common over- and 32 common underexpressed genes was shared between at least two-thirds of the 24 cancer types. Analysis of the immune tumor microenvironment revealed exclusively decreased immune cell populations in the TP53mut tumors in six, a mixed pattern in four, exclusively increased immune cell populations in two, and no significant alterations in 20 cancer subtypes. The analysis of a large cohort of human tumors complements results from experimental studies and support the development of novel strategies for therapeutic targeting of TP53mut.

Citation Format: Jan Budczies, Eva Romanovsky, Klaus Kluck, Iordanis Ourailidis, Michael Menzel, Susanne Beck, Markus Ball, Daniel Kazdal, Petros Christopoulos, Peter Schirmacher, Thorsten Stiewe, Albrecht Stenzinger. Homogenous TP53mut-associated tumor biology across mutation and cancer types revealed by comprehensive mRNA expression analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2607.
Abstract 2608: Defining the function of tumor suppressor p53 arginine 181 residue in tumorigenesis
Authors: Moses Renyta , Kelly Gregory , Indeglia Alexandra , Miller Sven , Karanicolas John , Murphy Maureen , Maxwell Kara
Published: April 2023

The gene Tp53 encodes for the protein p53 which has many tumor suppressive functions. Li-Fraumeni syndrome is characterized by germline Tp53 mutations that significantly increase the risk of cancer. The p53 amino acid residue arginine 181 is one such mutation site that promotes tumorigenesis by impacting a phenomenon known as p53 cooperativity. This is the ability of one p53 molecule to interact with another in order to efficiently bind specific sites on DNA to enact specific p53 functional pathways. Two p53 molecules interact with each other through the formation of a salt bridge between H1 helices found in respective DNA binding domains. The two key amino acid residues in this salt bridge are positively charged arginine (R) 181 and negatively charged glutamic acid (E) 180 which form electrostatic interactions with the residues on the opposite p53 H1 helix. Cooperativity mutants such as R181H and R181C show a hypomorphic behavior wherein much of p53’s canonical functions remain intact. Colony suppression data show that H1299 cells with p53 R181H and R181C yield a similar number of colonies as p53 wild type, which is significantly fewer than the yield of p53 hotspot mutation R175H and the absence of p53. Despite retaining some wild-type behavior, R181 mutants are found in LFS patients with cancer. Discovering the importance of the R181 residue for p53’s role in defending against tumorigenesis can lead to unique treatment opportunities for LFS patients with cooperativity mutants.

Citation Format: Renyta Moses, Gregory Kelly, Alexandra Indeglia, Sven Miller, John Karanicolas, Maureen Murphy, Kara Maxwell. Defining the function of tumor suppressor p53 arginine 181 residue in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2608.
Abstract 2619: Characterization of RAS assembly architectures in living cells
Authors: Zhou Zhiwei , Li Xing-Xiao , Meyer Cynthia , Zhang Yu-An , Angira Deekshi , Westover Kenneth
Published: April 2023

Assembly of RAS proteins and RAS effectors into “nanoclusters” at the cell membrane regulates RAS signaling output. The structure and composition of membrane-bound RAS complexes is dynamic, but defined states appear to exist. We previously found biological and biophysical evidence for two different models of membrane-bound RAS assemblies where RAS proteins form symmetric or asymmetric RAS-RAS interactions. It is unknown what functions these two architectures might serve and what determinants these nanoclusters might be subject to. To further characterize which architectural states occur in various biological scenarios, we developed a plate-based assay to detect RAS-RAS interactions in cells. The system controls for RAS expression and can be used to discriminate symmetric and asymmetric RAS-RAS interactions. It can also be used to assess the relative contributions of the G domain (1-166) and hypervariable region (HVR) in mediating RAS-RAS interactions. We found that RAS isoforms can homo- and hetero-dimerize and observed that the G domain contributes to RAS-RAS interactions, but that the HVR does not. We also found that the primary determinant of HVR interaction with the membrane is electrostatic charge. Regarding cancer-causing mutations, we found that in the KRAS G12D context, RAS-RAS interactions appear to favor asymmetric RAS-RAS interactions while KRAS Q61H favors symmetric RAS-RAS interactions.

Citation Format: Zhiwei Zhou, Xing-Xiao Li, Cynthia Meyer, Yu-An Zhang, Deekshi Angira, Kenneth Westover. Characterization of RAS assembly architectures in living cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2619.
Abstract 2646: Screening the membrane proteome to determine antibody specificity and de-risk CAR-T cell development
Authors: Rucker Joseph , Fong Rachel H. , Sullivan Tabb , Navia Carmen , Screnci Brad , Wilf Rona , Doranz Benjamin J.
Published: April 2023

Rigorous specificity analysis is critical for the development of antibody-based therapies, as even minimal off-target binding can lead to toxicity and clinical failures. Long believed to be exquisitely specific, recent preclinical data and our own work indicate that monoclonal antibodies (MAbs) frequently (~25%) display cross-reactivity. In many cases, off-target interactions occur with unrelated proteins that cannot be predicted by protein sequence homology. Cross-reactivity can lead to serious or even life-threatening consequences especially when MAbs are configured as bispecifics, antibody-drug conjugates (ADC) or CAR-T cell therapies, and IND applications for biotherapeutics require cross-reactivity assessment to prevent adverse events. Tissue cross-reactivity (TCR) studies have traditionally been used to screen for off-target binding, however, with poor predictive value for in vivo safety and toxicity. We developed the Membrane Proteome Array (MPA) platform to de-risk MAb-based therapeutics by testing for specificity across 6,000 human membrane proteins expressed in live cells. In contrast to TCR studies, proteins in the MPA exist in their native conformations and are not altered by fixation. The MPA assesses binding interactions by high-throughput flow cytometry allowing for high sensitivity detection and rapid analysis. We will discuss the importance of early-stage specificity testing to expose possible toxicities and present case studies for antibody and cell therapy profiling. MPA data have been used in numerous successful regulatory applications and may be used to replace or complement other cross-reactivity studies. In particular, MPA profiling has been used in IND applications for CAR-T and cell-therapies where conventional immunohistochemical binding assays did not suffice.

Citation Format: Joseph Rucker, Rachel H. Fong, Tabb Sullivan, Carmen Navia, Brad Screnci, Rona Wilf, Benjamin J. Doranz. Screening the membrane proteome to determine antibody specificity and de-risk CAR-T cell development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2646.
Abstract 2686: The combination of Cucurbitacin B and Evodiamine inhibits cell migration and propagation, and induces apoptosis in in vitro glioma cells
Authors: Mehta Vrutti V. , Benzeroual Kenza E.
Published: April 2023

Glioblastoma is a lethal brain tumor. Despite aggressive treatment, the median survival time is 6 to 12 months. These tumors acquire resistance against many therapeutic regimens. There is a need to screen for potential new drugs and drug combinations. Cucurbitacin B (CuB), a compound obtained from plants, and evodiamine (Evo), a supplement, have shown promising results in various neoplasms but have not been well studied in glioblastoma and especially, as a combination treatment. This study investigated the combined effects of CuB and Evo on U-87MG human glioma cell line. Results demonstrated that CuB and Evo individually decreased the U-87MG cell viability in time and dose dependent manners. The effect was significantly pronounced with the combined treatment. The combination treatment of CuB and Evo induced cellular apoptosis most likely by interfering with the PI3K/AKT and JNK/ERK pathways. In addition, the flow cytometry analysis showed that the combination treatment arrested the cell cycle at the G2M phase and inhibited cell propagation. Moreover, CuB and Evo combined effect successfully inhibited cellular migration. The cytotoxic effect of combination treatment of CuB and Evo was three times lower in neuronal PC-12 cells compared to U-87MG cells. Overall, a combination of cucurbitacin B and evodiamine proved to be a superior potential anti-neoplastic treatment in U-87MG cells as compared to individual treatment, and may be considered as a potential adjuvant therapy for GBM.

Citation Format: Vrutti V. Mehta, Kenza E. Benzeroual. The combination of Cucurbitacin B and Evodiamine inhibits cell migration and propagation, and induces apoptosis in in vitro glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2686.
Abstract 2694: Blockade of the cyclooxygenase-2 prevents chemotherapy-induced cognitive impairments
Authors: Rashid Mohammad Abdur , Tang Jason J. , Yoo Ki-Hyun , Corujo-Ramirez Ana , Kim Sang Hoon , Oliveros Alfredo , Cole Peter , Hawse John R. , Jang Mi-Hyeon
Published: April 2023

Chemotherapy-induced cognitive impairment (CICI, also termed “chemobrain”) is a major neurotoxic side effect exhibited by a wide range of chemotherapeutic agents. These side effects affect approximately 14 million cancer survivors. More importantly, CICI persists well after cessation of therapy, severely interfering with quality of life. Unfortunately, there is no treatment and novel therapies are urgently needed. Utilizing cisplatin, a platinum-based chemotherapy, to model chemobrain mice in vivo, we have established that cisplatin accelerates the brain aging process thus leading to long-term memory impairment in mice similar to what is clinically reported. We further show that cisplatin causes oxidative DNA damage, mitochondrial defects, impaired neurogenesis, synaptic defects, and increased gliosis in the adult hippocampus, a brain region critical for learning and memory. Mechanistically, our RNA-sequencing and qRT-PCR analysis revealed that cisplatin dramatically increases COX-2 (Ptgs2) expression and its major product, prostaglandin E2 (PGE2) in adult mouse brain and human cortical neurons derived from induced pluripotent stem cells (iPSCs). Similar as cisplatin treatment, the levels of COX-2 expression began to increase at 0.1 µM methotrexate treatment, which significantly increased at 1 µM in human cortical neurons, indicating that COX-2 induction is a common pathogenic mechanism mediating cognitive impairment associated with cisplatin and methotrexate in spite of the fact that these compounds have different mechanisms of action for cancer eradication. Most importantly, NS-398, a selective COX-2 inhibitor, effectively prevents cognitive deficits in mice and reduction in cell viability of human neurons associated with these chemotherapies, without promoting tumor growth or interfering with cisplatin’s anti-tumor activity. Taken together, our findings strongly suggest that COX-2 induction is a main cause of CICI, making COX-2 inhibition a therapeutic target for CICI. Given that COX-2 inhibitors are currently being tested in cancer prevention as well as age-related memory loss in clinical trials, inhibiting COX-2 may have far-reaching therapeutic effects on CICI and cancer treatment, which can be applied quickly, safely, and effectively to clinical trials, in the effort to improve cancer survivor quality of life.

Citation Format: Mohammad Abdur Rashid, Jason J. Tang, Ki-Hyun Yoo, Ana Corujo-Ramirez, Sang Hoon Kim, Alfredo Oliveros, Peter Cole, John R. Hawse, Mi-Hyeon Jang. Blockade of the cyclooxygenase-2 prevents chemotherapy-induced cognitive impairments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2694.
Abstract 2705: Low-dose crystalline cytokine macrophage mediated Trojan horse immunotherapy of solid cancers
Authors: Jones Michael H. , Calla Nirk E. Quispe , Talbot-Cooper Callum , Zeng Hong , Best Jonathan , Smith Robert
Published: April 2023

Protein therapeutics are often compromised by sub-optimal biodistribution contributing to poor efficacy and adverse events. Drug delivery mechanisms better able to target the disease site and provide localized, sustained drug release have the potential to transform therapeutic standards. PODS crystals (PCs) are natural-mimetic, micron-scale protein co-crystals engineered to incorporate a protein cargo that is sustainably released under the action of resident proteases. PCs are efficiently taken up by phagocytic cells and their cargo protein is subsequently released in a bioactive form. Since blood-circulating phagocytic cells, including monocytes, are actively recruited into the tumor microenvironment, we postulated that monocyte/macrophage-mediated PC delivery could be used as a molecular “Trojan horse” to efficiently deliver therapeutic proteins to target cells. This could improve the pharmacodynamics and pharmacokinetics of protein drug delivery to treat systemic and disseminated diseases. Interleukin-2 (IL-2) is notoriously toxic at the high doses required for therapeutic efficacy. Here, we demonstrate the therapeutic efficacy and tolerability of low doses of PC-IL-2 administered intravenously in a mouse model of melanoma. We further demonstrate the therapeutic benefit of PCs delivering low doses of IL-2, interleukin-15 (IL-15) and interferon-gamma (IFN-γ) in a mouse model of renal cell carcinoma. In all the treated mice, cachexia, a side effect of high-dose cytokine therapy, was notably absent. This study provides proof-of-concept for the utility of intravenously administered PCs to provide a generalised and widely applicable mechanism to effectively deliver protein drugs for the therapy of cancer and other diseases.

Citation Format: Michael H. Jones, Nirk E. Quispe Calla, Callum Talbot-Cooper, Hong Zeng, Jonathan Best, Robert Smith. Low-dose crystalline cytokine macrophage mediated Trojan horse immunotherapy of solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2705.
Abstract 2710: Developing a novel doppel-targeting antibody-drug conjugate (ADC) for solid cancer
Authors: Lee Ha Kyeong , Kim Byoungmo , Kweon Seho , Choi Jeong Uk , Kim Sang Yoon , Byun Youngro
Published: April 2023

The development of anticancer therapeutics has two main objectives: enhancing efficacy and lowering toxicity. Given these goals, an antibody-drug conjugate (ADC), which delivers highly cytotoxic payloads to tumor tissue specifically, is one of the most favorable treatments. ADC is a growing drug delivery technology, with twelve FDA-approved ADCs as of 2022. Most of ADCs in use today target particular molecules for hematologic malignancies. ADCs for solid tumors are relatively in the early stages of development due to the lack of well-defined solid tumor-specific target molecules. To put it another way, the market has an enormous unmet demand for solid cancer-specific ADCs. As seen by recently approved ADCs and ADCs in clinical trials, finding novel targets for solid cancer has drawn a lot of attention in current development trends. Doppel, a prion-like protein, is transiently expressed in the neonatal brain endothelium; in adults, it is exclusively present in testicular cells. In our previous study, we found that Doppel is specifically expressed only in tumor tissues and tumor blood vessels, but not in normal adult tissues; its expression promoted cancerous blood vessel formation. Then, we generated several monoclonal antibodies which target Doppel; the most effective clone was selected. We observed that angiogenesis-related factors were suppressed in the tumor endothelial cells treated with the Doppel-targeting antibody. In addition, we injected the antibody into mouse models and visualized the tumor-targeting efficacy using in vivo imaging system (IVIS); the antibody accumulates in the tumor significantly higher compared to non-targeting IgG. We suggest what we believe to be a novel strategy to target a molecular marker that is altered and overexpressed specifically in tumor tissues and tumor endothelial cells. Based on the above discoveries, we have developed the Doppel-targeting ADC; this drug, according to our hypothesis, will target only cancer cells that express Doppel and result in apoptotic cell death. The ADC has demonstrated remarkable anticancer efficacy in both cell and mouse models, although it is still in the early stages of research. We believe that the enhanced specificity for targeting tumors, the high tumor-to-organ ratio, and the bystander killing effect would make this Doppel-targeting ADC a successful “solid cancer-specific” one.

Citation Format: Ha Kyeong Lee, Byoungmo Kim, Seho Kweon, Jeong Uk Choi, Sang Yoon Kim, Youngro Byun. Developing a novel doppel-targeting antibody-drug conjugate (ADC) for solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2710.
Abstract 2752: Targeting intracellular tumor antigens using fully human TCR mimic antibodies derived from HLA transgenic RenMiceTM
Authors: Du Jun , Liu Taolin , Tang Wanbo , Zhang Yue , Zhao Limin , Jiao Xin , Sun Chao , Du Pengfei , Zhang Yuqi , Liu Baihong , Lin Qingcong , Yang Yi
Published: April 2023

Therapeutic antibodies have ushered in a new age of cancer immunotherapy. Historically, these therapies have targeted a limited subset of soluble and cell surface tumor-associated antigens (TAAs). T cell receptors (TCRs) on cytotoxic CD8+ T cells recognize peptide antigens bound to major histocompatibility class I (MHC-I) proteins, called HLA-A/B/C in humans. By this pathway, antigen is regularly sampled from the intracellular peptidome, processed, and presented to cytotoxic T cells. Expanding TCR-based recognition of soluble, intracellular TAAs presented on the surface of malignant cells by this mechanism is a propitious therapeutic strategy. Here we describe a novel platform for generating T cell receptor mimic (TCRm) antibodies using our humanized immunoglobulin (RenMabTM) mice engineered to express HLA. TCRm antibodies have the same binding properties as endogenous TCRs and recognize processed, HLA-bound peptides including intracellular tumor-associated antigens, viral oncoproteins, and cancer-testis antigen (CTA). TCRm antibodies bind peptide-HLA with high specificity and up to nanomolar affinity. Our optimized immunization protocols and high-throughput screening methods allow for one-step TCRm antibody generation. TCRm antibodies can also be used to assemble bispecific T cell engaging antibodies (BiTEs) to enhance tumor targeting of cytotoxic T cells. Biocytogen’s TCRm antibodies are a flexible and powerful tool for cancer immunotherapy. By enabling TCR-mediated recognition of an unrestricted repertoire of cancer neoantigens, TCRm antibodies may find broad clinical application.

Citation Format: Jun Du, Taolin Liu, Wanbo Tang, Yue Zhang, Limin Zhao, Xin Jiao, Chao Sun, Pengfei Du, Yuqi Zhang, Baihong Liu, Qingcong Lin, Yi Yang. Targeting intracellular tumor antigens using fully human TCR mimic antibodies derived from HLA transgenic RenMiceTM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2752.
Abstract 2758: Expanding PRISM high-throughput screening capabilities to other modalities
Authors: Smigiel Jacob , Eskra Jillian , Masciotti Antonella , Golabi Aydin , Ip Blanche , Ronan Melissa , Rees Matthew G. , Roth Jennifer
Published: April 2023

PRISM, a high-throughput drug screening platform, has been used extensively to profile DMSO-soluble compounds, including nearly all commercially available FDA-approved small molecule drugs, in pools of cancer cell lines. In addition to these highly-optimized compounds, novel small-molecule libraries comprising tens of thousands of compounds have been screened through collaborative partnerships. While PRISM has been highly effective in discovering new therapeutic targets and classes via libraries of this nature, we have not yet explored screening aqueous molecules at scale, such as antibodies, antibody-drug conjugates (ADCs), cytokines, and other biologics. There is also untapped potential for the further development of PRISM-based assays utilizing more physiologically relevant assay conditions to gain new insights into cancer biology and highlight more clinically relevant targets. Specifically, the incorporation of environmental cues such as media nutrients, extracellular matrix, and other non-cancer cell types can have a drastic impact on cancer cell state, and thus cancer cell responses to therapy. For example, PRISM pools grown as 3D spheroids, compared to 2D mono-layers, reveal models showing increased sensitivity to mutant KRAS inhibition. Similarly, the addition of immune effector cells, via co-culture methods, to the PRISM platform will enable identification of key mechanisms involved in antibody-dependent cellular cytotoxicity and measurement of immunotherapeutic-relevant phenotypes at an unprecedented scale. This coupling of aqueous screening, with the addition of relevant tumor-microenvironmental cues may help identify therapies more destined to succeed in the clinic, and delineate novel mechanisms involved in cancer cell-state that underpin the many clinical challenges we face in curing cancer.

Citation Format: Jacob Smigiel, Jillian Eskra, Antonella Masciotti, Aydin Golabi, Blanche Ip, Melissa Ronan, Matthew G. Rees, Jennifer Roth. Expanding PRISM high-throughput screening capabilities to other modalities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2758.
Abstract 2759: Real-time cellular target engagement and protein quantification for drug discovery
Authors: Babic Ivan , Bryan Nikolas , Cunningham Claire , Sampson Avery , Starczynowski Daniel , Nurmemmedov Elmar
Published: April 2023

Preclinical assessment of direct drug-target engagement and analysis of binding kinetics within the cellular environment is essential for development of safer and more effective therapeutics. Infusion of such critical data into early drug discovery would significantly reduce the failure rate of new drug candidates, accelerate drug discovery, and validate repurposing of existing drugs. Described here is a novel drug-target engagement technology that can sensitively interrogate direct binding of drugs to cellular, bacterial, or viral protein targets within the physiological environment. Micro-Tag cell target engagement technology is based on complementation of a small 15-amino acid subunit with a large subunit into an active RNA-processing enzyme. The small subunit can be cloned to any drug target for transient or stable expression using existing tools such as CRISPR. Upon complementation, the active enzyme cleaves a FRET-based oligonucleotide substrate resulting in rapid generation of fluorescent signal that can be quantified in real time. The Micro-Tag technology enables in-cell quantitation of drug target levels using qPCR systems. This is the next-generation of target engagement technology that allows for real-time monitoring of drug-target interaction in the cell. We show here data demonstrating direct engagement of several reference compounds and novel small molecules with high-profile cancer targets such as K-RAS, MTH1, EGFR, and UBE2N. Selectivity of these drugs to the targets in the cell is further delineated using their mutant counterparts as well as inactive stereoisomer compounds.

The Micro-Tag cell target engagement technology provides the power of cell target engagement to a large family of target proteins. It can be employed for high-throughput screens directed at initial on-target ranking or med-chem optimization of drug candidates. It is amenable for interrogation of drug candidates across various modalities: small molecules, peptides, antibodies and PROTACs. Importantly, this next-generation target engagement technology seamlessly integrates with qPCR systems, fluorescence microscopy, live-cell microscopy, FACS analysis, and offers multiplexing capability, thus allowing for further mechanistic insight.

Citation Format: Ivan Babic, Nikolas Bryan, Claire Cunningham, Avery Sampson, Daniel Starczynowski, Elmar Nurmemmedov. Real-time cellular target engagement and protein quantification for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2759.