bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3617: Investigating a role for PIKfyve in cell migration and invasion of clear cell renal cell carcinoma
Authors: Cormier Jolène , Turcotte Sandra
Published: April 2023

Introduction. Clear cell renal cell carcinoma (ccRCC) is the most frequent type of kidney cancer. These highly vascularized tumors are characterized by mutations that inactivate the von Hippel-Lindau (VHL) tumor suppressor gene. About 30 % of patients present metastasis at diagnosis and 30-40% of patients with localized tumors relapse after surgery. Unfortunately, metastatic ccRCC remains incurable and are resistant to standard therapies. Our studies demonstrated that ccRCC with a loss of VHL can be targeted using a small molecule named STF-62247. This molecule blocks the autophagic flux causing enlargement of endolysosomes leading to cell death. More recently, we identified the lipid kinase PIKfyve as a target of STF-62247. A central complex assuring the functionality of the lysosome is formed by the lipid kinase PIKfyve, the scaffold protein ArPIKfyve (Vac14), and the phosphatase Sac3 (Fig4). Interestingly, PIKfyve has shown to play a role in cell migration but the mechanisms are not well understood.

Objectives. Our project aims to i) assess a role for PIKfyve in ccRCC migration/invasion, ii) evaluate the potential of PIKfyve inhibitors on angiogenesis, and iii) assess the effect of PIKfyve inhibitors or genetic knockdown of PIKfyve, Vac14 or Fig4 on tumor growth in vivo.

Methods and Results. PIKfyve, Vac14 and Fig4 gene expression were modified using CRISPR/Cas9 (Cr) or the SMARTvectorTM Inducible Lentiviral shRNA system. qRT-PCR and western blot validated our models. Results obtained in Cr.PIKfyve and Cr.Vac14 indicated that loss of Vac14 decreased survival of VHL-deficient ccRCC. Moreover, our recent results show that migration measured by wound healing assay is reduced in CRISPR cells compared to control. To investigate a role for PIKfyve in angiogenesis, tube formation assay was performed in treated cells with PIKfyve inhibitors. Our results indicated a reduction in tube formation in STF-62247 and apilimod treated cells. Furthermore, we will use the proteome profiler human angiogenesis array to identify angiogenic proteins linked to PIKfyve activity.

Conclusion. Understanding how PIKfyve is involved in the process of angiogenesis would play a major role in the knowledge for new targeted therapies inkidney cancer.

Citation Format: Jolène Cormier, Sandra Turcotte. Investigating a role for PIKfyve in cell migration and invasion of clear cell renal cell carcinoma. [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 3617.
Abstract 3728: A novel, simplified, streamlined workflow for high-throughput whole transcriptome RNA-seq library preparation for a variety of sample types
Authors: Pinegar Chelsea , Posfai Dora , Naishadham Gautam , Langhorst Bradley W. , Krishnan Keerthana
Published: April 2023

RNA sequencing (RNA-seq) has become an invaluable tool in the study of biology, but often requires multiple days to process samples from total RNA to final libraries that can be loaded on to a sequencer. Researchers use RNA-seq to gain insight into whole tumor gene expression, and in combination with other omics data make treatment decisions. In cancer research, time taken to yield sequencing results can be critical for clinical samples. Within oncology research, libraries are often prepared from RNA sourced from formalin-fixed, paraffin-embedded (FFPE) tissues which can be particularly challenging and is not compatible with all library preparation methods.

Here, we present a new protocol that allows users to easily generate robust RNA-seq libraries from a variety of sample types in a single day from 25-250 ng of total RNA. Compatible with poly(A) enrichment or ribosomal RNA depletion, this new workflow has reduced preparation time by about 40% compared to current library prep kits without sacrificing quality of libraries or any sequencing metrics, including 5’-3’ coverage, GC bias. Our new method provides equivalent transcript expression in FFPE samples when compared with existing protocols. In addition to master mixed components and reduced incubation times, the reduction in bead cleanup steps significantly reduces hands-on time for library preparation, simplifying and streamlining the process.

This new library preparation method is also ideal for processing samples of various inputs with a single condition at every step throughout the protocol, without modifying cycling conditions or dilutions based on input amount. This workflow provides a great advantage in efficiently generating RNA-seq libraries while simultaneously maintaining high quality sequencing results and compatibility with both high- and low-quality RNA such as FFPE.

Citation Format: Chelsea Pinegar, Dora Posfai, Gautam Naishadham, Bradley W. Langhorst, Keerthana Krishnan. A novel, simplified, streamlined workflow for high-throughput whole transcriptome RNA-seq library preparation for a variety of sample types. [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 3728.
Abstract 3738: A novel mechanism of STAT3 activation by genotoxic chemotherapy
Authors: Venkatesh Jaganathan , Muthu Magesh , Acharige Nuwan C. , Pflum Mary K. , Rishi Arun K.
Published: April 2023

CARP-1, a perinuclear phospho-protein, is a biphasic regulator of cell survival and apoptosis signaling induced by genotoxic drugs. We have previously found that apoptosis induced by genotoxic drugs involved elevated levels of CARP-1. Exposure to genotoxic drugs also induced CARP-1 interaction with NF-κB kinase subunit γ (aka, NEMO) to promote NF-κB activation and cell survival. To further elucidate chemotherapy-activated, CARP-1-dependent cell survival mechanisms, we UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed STAT3 interaction with CARP-1 (614-638) peptide. Our mutagenesis and co-IP-WB experiments revealed CARP-1 (550-650) directly interacts with a 40 amino acid peptide located in the STAT3 DNA binding domain. Overexpression of mutant STAT3 with in-frame-deletion of CARP-1/CCAR1-binding epitope (CE) peptide (Gst-STAT3 (ΔCE) mutant), but not Gst-STAT3 (WT), failed to translocate to nucleus in Adriamycin-treated cells. Disruption of STAT3-CARP-1 interaction or expression of STAT3 (ΔCE) mutant abolished Adriamycin-induced STAT3 Y705 and S727 phosphorylation. Collectively our data demonstrate that CARP-1 interaction with STAT3 regulates genotoxic chemotherapy-activated STAT3 and its nuclear translocation to promote cancer cell survival and growth.

Citation Format: Jaganathan Venkatesh, Magesh Muthu, Nuwan C. Acharige, Mary K. Pflum, Arun K. Rishi. A novel mechanism of STAT3 activation by genotoxic chemotherapy. [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 3738.
Abstract 3865: Multiple cancer types rapidly escape from multiple MAPK inhibitors to generate mutagenesis-prone subpopulations
Authors: Hoffman Timothy E. , Yang Chen , Nangia Varuna , Ill Christopher Ryland , Spencer Sabrina L.
Published: April 2023

Many cancers harbor pro-proliferative mutations of the mitogen-activated protein kinase (MAPK) pathway and many targeted inhibitors now exist for clinical use, but drug resistance remains a major issue. We previously showed that BRAF-driven melanoma cells treated with BRAF inhibitors can non-genetically adapt to drug within 3-4 days to escape quiescence and resume slow proliferation. Here we show that this phenomenon is not unique to melanomas treated with BRAF inhibitors but rather is widespread across many clinical MAPK inhibitors and cancer types driven by EGFR, KRAS, and BRAF mutations. In all treatment contexts examined, a subset of cells can escape drug-induced quiescence within four days to resume proliferation. These escapee cells broadly experience aberrant DNA replication, accumulate DNA lesions, spend longer in G2-M cell cycle phases, and mount an ATR-dependent stress response. We further identify the Fanconi anemia (FA) DNA repair pathway as critical for successful mitotic completion in escapees. Long-term cultures, patient samples, and clinical data demonstrate a broad dependency on ATR- and FA-mediated stress tolerance. Together, these results highlight the pervasiveness with which MAPK-mutant cancers are able to rapidly escape drug and the importance of suppressing early stress tolerance pathways to potentially achieve more durable clinical responses to targeted MAPK pathway inhibitors.

Citation Format: Timothy E. Hoffman, Chen Yang, Varuna Nangia, Christopher Ryland Ill, Sabrina L. Spencer. Multiple cancer types rapidly escape from multiple MAPK inhibitors to generate mutagenesis-prone subpopulations. [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 3865.
Abstract 3870: TNF-MK2 signaling drives protective autophagy following MAPK pathway inhibition in pancreatic cancer
Authors: Khawar Iftikhar Ali , Wei Qing , Chen Timothy Hung-Po , Lin Lin , Grierson Patrick M. , Lim Kian-Huat
Published: April 2023

Targeting the oncogenic KRAS-RAF-MEK-ERK (MAPK) pathway has remain unsuccessful in the clinic for patients with pancreatic ductal adenocarcinoma (PDAC), a highly lethal cancer with few effective treatment options. To identify novel, effective therapeutic strategies, we investigated the impact of MEK and ERK inhibitors on production of inflammatory cytokines in PDAC cells. We found that both MEK and ERK inhibitors dramatically upregulated production of TNF, leading to activation of multiple TNF signaling pathways which include the pro-death and pro-survival NF-kB and p38.MK2 cascades. Silencing of TNF receptor 1 (TNFR1) abrogated the pro-apoptotic effect of MAPK inhibitors, suggesting that pro-death effect driven by TNF signaling is required for the therapeutic effect of MAPK inhibitors. However, targeting the NF-kB and MK2 pathways greatly augment the suppressive effect of MAPK inhibitors. Notably, the mechanisms via which MK2 promotes PDAC cell survival are mediated through phospho-activation of Heat shock protein 27 (Hsp27) and Beclin1, a critical mediator of autophagy. Intriguingly, silencing of TNFR1 abrogated induction of proactive autophagy following MAPK inhibition, strongly suggesting autocrine signaling as the inciting event of autophagy. Mechanistically, we showed that TNF signaling upregulate unfolded protein response (UPR) in PDAC cells, which is required for autophagy induction. Targeting MK2 abrogated transcription of key UPR genes and blocks subsequent autophagy. The combination of MK2 inhibitor ATI-450 and ERK inhibitor ulixertinib was more effective in curbing the growth of PDAC patient-derived xenograft in vivo and prolonged the survival of autochthonous PDAC mice (KPC model). Overall, our study provided novel insights on the mechanisms that drive protective autophagy following MAPK pathway inhibition and a rationale and feasible therapeutic combination that can be tested in clinical trials for PDAC patients.

Citation Format: Iftikhar Ali Khawar, Qing Wei, Timothy Hung-Po Chen, Lin Lin, Patrick M. Grierson, Kian-Huat Lim. TNF-MK2 signaling drives protective autophagy following MAPK pathway inhibition in pancreatic 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 3870.
Abstract 3871: KRAS G12C mutant allele amplification drives resistance to sotorasib in vitro
Authors: Mohn Deanna , Saiki Anne Y. , Achanta Pragathi , Stapper Andres Plata , Lipford J. Russell , Verma Rati
Published: April 2023

Sotorasib is an approved KRASG12C-selective inhibitor for the treatment of KRAS p.G12C-mutant advanced and previously treated non-small cell lung cancers (NSCLC). Acquired resistance due to genomic alterations following sotorasib treatment has been observed in 28% of lung cancer patients (CodeBreaK100, ASCO 2022) and can include bypass signaling, inactivation of negative feedback loops, or compensatory mutations in KRASG12C including amplification of KRAS.

Preclinical studies on the mechanisms of acquired resistance are critical for understanding these resistance patterns and can reveal new therapeutic or combination strategies in the clinic. For that reason, we developed an NCI-H358 lung cancer model of acquired resistance to KRASG12C inhibition in vitro. NCI-H358 cells were continuously cultured in the presence of elevated concentrations of sotorasib (IC90) until resistance to KRASG12C inhibitor was achieved. Expression analyses of the resistant cells by ddPCR and immunoblotting showed increases in both KRAS gene and protein levels, resulting in enhanced MAPK signaling. siRNA knockdown of KRAS G12C expression re-sensitized the resistant cells to an analog of sotorasib. Further characterization of this cell line by whole exome sequencing (WES) and fluorescence in situ hybridization (FISH) showed that resistance was not due to genetic co-alterations but amplification of the mutant allele specifically. In comparison to DMSO-treated and parental controls, the sotorasib-resistant NCI-H358 cells contained an approximately 50-fold increase in KRAS G12C mutant allele copy numbers.

Preclinical data generated using a KRASG12C inhibitor-resistant lung cancer cell line is consistent with clinical observations of acquired KRAS amplification following KRASG12C inhibitor treatment as a mechanism of resistance. This model further provides an opportunity to study acquired KRAS amplification and investigate combination treatment options in vitro.

Citation Format: Deanna Mohn, Anne Y. Saiki, Pragathi Achanta, Andres Plata Stapper, J. Russell Lipford, Rati Verma. KRAS G12C mutant allele amplification drives resistance to sotorasib in vitro. [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 3871.
Abstract 3876: Kinetics of RTK activation determine ERK signaling dynamics and resistance to BRAF and MEK inhibitors
Authors: Kim Sungsoo , Carvajal Richard , Kim Minah , Yang Hee Won
Published: April 2023

Combination treatment of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) produces remarkable response rates and is expected to prevent ERK activation in BRAF-mutant melanoma. However, sporadic ERK reactivation occurs in melanoma models, and efficacy is severely limited by the emergence of drug resistance. The underlying molecular mechanisms that drive ERK reactivation and adaptation to BRAFi/MEKi remain elusive. Here, using single-cell imaging and optogenetic approaches, we show that the strength and duration of receptor tyrosine kinase (RTK) activation determines ERK reactivation and the development of BRAFi/MEKi-resistant cells. Single-cell data reveal that despite global RTK activation by the same external stimuli, only small subsets of melanoma cells induce effective RTK and ERK activation to develop BRAFi/MEKi resistance. Furthermore, by directly controlling RTK activity, we demonstrate that the kinetics of RTK activation determine ERK signaling dynamics and the percentage of drug-resistant cells. While these drug-resistant cells are rare during early stages of drug treatment, they eventually become the dominant population through sustained RTK-mediated ERK activation that drives cell proliferation. Thus, RTK activation in established drug-resistant cells causes effective ERK activation and cell proliferation. Importantly, limiting RTK signaling activation reverses the BRAFi/MEKi-resistant state. We show that targeting SHP2, a mediator of RTK signaling, effectively suppresses RTK-mediated ERK activation and cell proliferation in BRAFi/MEKi-resistant cells. Our results provide mechanistic insights into the contribution of heterogeneity in RTK activation kinetics to ERK reactivation and BRAFi/MEKi resistance, also highlighting SHP2 activity as a promising target to overcome drug resistance in BRAF-mutant melanoma.

Citation Format: Sungsoo Kim, Richard Carvajal, Minah Kim, Hee Won Yang. Kinetics of RTK activation determine ERK signaling dynamics and resistance to BRAF and MEK inhibitors. [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 3876.
Abstract 390: A comparison of the activity of the quadruplex-targeting experimental drugs QN-302 and CX-5461 (pidnarulex) in wild-type and gemcitabine-resistant pancreatic cancer cell lines
Authors: Ahmed Ahmed , Arshad Tariq , Neidle Stephen
Published: April 2023

The compound QN-302, a tetra-substituted naphthalene diimide (ND) derivative has been designed as a potent compound for targeting quadruplex sequences in cancer genes. It has single-digit nM anti-proliferative activity in a panel of human pancreatic cancer (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It also has significant anti-tumor activity in xenograft, orthotopic and genetic (KPC) models for PDAC. Its mode of action involves the targeting of quadruplex (G4)-forming sites in promoter regions of cancer-associated genes, where their prevalence is over-represented compared to other genes. The structurally unrelated compound CX-5461 has also been reported to be a G4-binding agent, with selective activity in BRCA1/2 deficient cells and tumors (Xu and Hurley, Bioorg Med Chem Lett. 2022; Xu et al., Nat Commun, 2017). It is currently in early-stage clinical trials in breast and other solid cancers with deficiencies in DNA repair (Hilton et al., Nat Commun. 2022). We have previously reported that a precursor to QN-302, the tri-substituted naphthalene diimide compound CM03, is highly active in both wild-type and gemcitabine-resistant PDAC cell lines (Ahmed et al, Sci Rep, 2020). This is explained by the finding using whole-genome transcriptome analysis that the CM03-sensitive genes are largely unaffected in the resistant lines. We now report that the clinical candidate compound QN-302, which is 10-fold more potent than CM03, retains its potency in both MIA-PACA2 and PANC-1 PDAC cell lines with >1000-fold increase in gemcitabine resistance. This is also the case for CX-5461, although this compound is 50-100 times less potent, with GI50 values of 83-95 nM in cell proliferation assays treated for 96 h. Gemcitabine either alone or in combination is still a mainstay of current PDAC treatment. Resistance to gemcitabine is common and is a major contributor to the poor outcomes for most PDAC patients. The ability of QN-302 to retain activity in chemo-resistant PDAC cell lines suggests that it will offer significant advantage in the clinic over gemcitabine-based therapies. QN-302 is bio-available at therapeutic doses and is well tolerated at these levels in animal models. It is being developed for clinical evaluation by Qualigen Therapeutics Inc and is currently at the pre-IND stage.

Citation Format: Ahmed Ahmed, Tariq Arshad, Stephen Neidle. A comparison of the activity of the quadruplex-targeting experimental drugs QN-302 and CX-5461 (pidnarulex) in wild-type and gemcitabine-resistant pancreatic cancer cell lines [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 390.
Abstract 3900: In vivo inhibition of metastatic HER2 positive trastuzumab resistant breast cancer using engineered destabilized 3UTR ARE of HER2 improves survival outcomes
Authors: Awah Chidiebere U. , Mun Joo Sun , Boylu Baris , Paragodaarachchi Alooka , Ochu Chika , Matsui Hiroshi , Ogunwobi Olorunseun O.
Published: April 2023

Breast cancer is the most common form of cancer in women with mortality of about 58% in developing countries. HER2+ breast cancers are treated with trastuzumab in various forms in combination with chemotherapy and has been successful in targeting HER2 overexpression in HER2+ breast cancer and improving survival as a standard first line therapy for more than a decade. However, about 25% of early and 75% of late stage HER2 driven breast cancers are resistant to trastuzumab. For these patients, the clinical outcomes are grim. We report the destabilization of HER2 3‘UTR ARE which degraded oncogene HER2 transcript, protein expression, HER2 dependent kinases and interactome. In vitro, we transfected the engineered destabilizing HER2 constructs into BT474 clone 5 trastuzumab resistant breast cancer and within days, we inhibited cancer cell growth and degraded HER2 transcript and protein. The degradation of HER2 leads to loss of many kinases especially YES1 and WNK1 that are known causes of chemoresistance in HER2 positive trastuzumab resistant breast cancer. In vivo, we administered the destabilizing constructs as naked constructs or as complexed with nanocages to mice bearing tumors of BT474 clone 5 trastuzumab resistant cells. We found that our constructs, both the naked and nanocage-delivered, conferred significant survival (p values: 0.0178 and 0.00492) to about 60% of the exceptionally surviving mice compared to the controls. Taken together, we have developed a new therapy to target trastuzumab resistant HER2+ metastatic breast cancers with increased survival outcomes.

Citation Format: Chidiebere U. Awah, Joo Sun Mun, Baris Boylu, Alooka Paragodaarachchi, Chika Ochu, Hiroshi Matsui, Olorunseun O. Ogunwobi. In vivo inhibition of metastatic HER2 positive trastuzumab resistant breast cancer using engineered destabilized 3UTR ARE of HER2 improves survival outcomes. [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 3900.
Abstract 3937: An intracellular variant of zona pellucida glycoprotein 3 is expressed in cancer
Authors: Schultz Iman J. , Zimmerman Yvette , Moelans Cathy B. , Chrusciel Marcin , Krijgh Jan , van Diest Paul J. , Huhtaniemi Ilpo T. , Bennink Herjan J. Coelingh
Published: April 2023

This study describes the expression of an intracellular variant of the Zona Pellucida glycoprotein 3 (ZP3) that is predominantly expressed in cancer. ZP3 is an important constituent of the Zona Pellucida (ZP), the extracellular layer surrounding oocytes. It is highly expressed in maturing oocytes, but not in other adult human tissues. As ZP3 protein has also been detected in cancer, it may serve as an interesting diagnostic and therapeutic target. Here, we extend the cancer-related ZP3 expression data using immunohistochemistry (IHC) of tumor biopsies, interrogating publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, as well as computational analysis and real-time quantitative PCR (qPCR) of CCLs. IHC data for several cancer types shows abundant ZP3 protein expression, which is confined to the cytoplasm, contradicting the extracellular ZP3 localization in oocytes. An alternative ZP3 mRNA variant, which we term ‘ZP3-Cancer’, is annotated in the NCBI and Ensembl databases, and lacks the genetic information encoding the N-terminal signal peptide that governs extracellular secretion. Analysis of publicly available RNA-seq data of 1339 CCLs indicates that ZP3-Cancer is the dominant variant as compared to ZP3-Oocyte, which was validated by independent computational analysis. Expression of ZP3-Cancer in CCLs was confirmed by qPCR. Publicly available RNAseq data of tumor and normal tissues confirms strongly enhanced expression of ZP3-Cancer in cancer cells. In addition, ZP3-Cancer expression is upregulated in advanced stages and shows a relationship with patient survival in a number of cancer types. The folliculogenesis specific transcription factor FIGLA, which activates transcription of ZP3-Oocyte in maturing oocytes, appears absent in cancer as inferred from publicly available transcriptome data, indicating alternative ZP3-Cancer transcriptional activation mechanisms in this disease. The cancer restricted expression of this ZP3 transcript variant renders it an attractive tumor specific antigen for the development of a therapeutic cancer vaccine, particularly using mRNA technology.

Citation Format: Iman J. Schultz, Yvette Zimmerman, Cathy B. Moelans, Marcin Chrusciel, Jan Krijgh, Paul J. van Diest, Ilpo T. Huhtaniemi, Herjan J. Coelingh Bennink. An intracellular variant of zona pellucida glycoprotein 3 is expressed in 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 3937.
Abstract 3938: In vivo genome-wide CRISPR screen in pancreatic ductal adenocarcinoma defines HSPE1 as a potential oncogene by acting through G2/M cell cycle arrest
Authors: Boudreault Julien , Wang Ni , Yan Gang , Dai Meiou , Poulet Sophie , Daliah Girija , Lebrun Jean-Jacques
Published: April 2023

Superior knowledge of cancer biology has enabled unprecedented innovations in therapies targeting mutated driver genes. Despite the attempt of targeting cancer-inducing genes such as KRAS, the life expectancy of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) has poorly improved. We performed an unbiased genome wide in-vivo loss-of-function CRISPR screen to discover novel oncogenes and identified heat-shock protein HSPE1, whose function is still unknown in PDAC. By decreasing HSPE1 expression with shRNA and CRISPR technology, we detected a slowdown in cell growth of PDAC cancer cells. HSPE1 knock-out cells injected in mice displayed a drastic reduction in tumor volume. We exploited this vulnerability by disrupting HSPE1 function by using KHS101, a validated HSPD1-HSPE1 complex inhibitor. Several PDAC cell lines cultured in vitro were sensitive upon KHS101 treatment and in vivo administration of KHS101 reduced tumorigenesis. Compiling the negative fitness scores from DepMap database revealed a co-dependency between HSPE1 and PLK1, a protein involved in regulating G2/M checkpoint. Indeed, we detected a protein-level decrease of PLK1 in our experimental model of PDAC cells having either a drug-induced HSPE1 deficiency by KHS101 exposure or a HSPE1 gene-expression reduction by shRNA. We found that the cell cycle was disrupted through G2/M arrest in PDAC cells treated with KHS101. Our findings highlight a new role underlying PDAC tumorigenesis for HSPE1 and could unlock a new area of research towards precision medicine.

Citation Format: Julien Boudreault, Ni Wang, Gang Yan, Meiou Dai, Sophie Poulet, Girija Daliah, Jean-Jacques Lebrun. In vivo genome-wide CRISPR screen in pancreatic ductal adenocarcinoma defines HSPE1 as a potential oncogene by acting through G2/M cell cycle arrest. [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 3938.
Abstract 3961: Identification of lncRNAs as potential novel therapeutic targets in triple negative breast cancer
Authors: O'Malley Megan , Rana Zohaib , Sarkar Debina , Chia Jolyn , Diermeier Sarah
Published: April 2023

Triple negative breast cancer (TNBC) lacks targeted therapies. Recently, our laboratory and other groups described long non-coding RNAs (lncRNAs) as new drivers of TNBC progression, which represent an exciting new avenue for targeted treatments. LncRNAs are the most versatile and diverse class of non-coding RNAs with roles in the progression of TNBC development, including cell proliferation, differentiation, apoptosis, metastasis and drug resistance. LncRNAs have greater tissue-specific expression compared to proteins and can be targeted using nucleotide sequence-specific RNA therapeutics, leading to reduced systemic toxicities when used as a cancer target. Using a combined approach of computational analysis of patient data and CRISPR functional screening, we identified hundreds of lncRNAs as novel drivers of TNBC. We prioritized one lncRNA target (lncTNBC3) with high translational potential. LncTNBC3 knockdown lead to reduced TNBC cell proliferation in two different loss-of-function models. To gain initial insights into the mechanism via which lncTNBC3 functions, RNA sequencing was carried out on knockdown cells, revealing that lncTNBC3 impacts cell cycle and DNA repair pathways. Cell cycle analysis confirmed an increase in G2/M cell cycle arrest upon lncTNBC3 knockdown. In conclusion, we have identified a promising novel lncRNA target with a role in TNBC growth.

Citation Format: Megan O'Malley, Zohaib Rana, Debina Sarkar, Jolyn Chia, Sarah Diermeier. Identification of lncRNAs as potential novel therapeutic targets in triple negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3961.
Abstract 4075: A modified FcγRI expressing-T cell, SolidT, enables antibody-mediated cytotoxicity to overcome the limitations of CAR-T cell therapy against solid tumors
Authors: Rasoulouniriana Diana , Santana-Magal Nadine , Gutwillig Amit , Farhat-Younis Leen , Shpilt Hana , Dotan Shahar , Pilpel Noam , Rider Peleg , Carmi Yaron
Published: April 2023

The pioneering design of chimeric antigen receptor T-cell (CAR-T) therapy demonstrated the potential of reprogramming the immune system. Nonetheless, T-cell exhaustion, toxicity, and suppressive microenvironment limit their efficacy in solid tumors. Recently, we characterized a novel subset of tumor-infiltrating CD4+ T-cells expressing the FcγRI receptor (Rasoulouniriana et al, 2019). Herein we detail the engineering of a novel receptor, based on the FcγRI structure, which is retrovirally transduced into PBMCs (both CD4 and CD8 T-cells). These cells, named SolidT cells, can target tumor cells using antibody intermediates. They show effective and specific cytotoxicity only when an appropriate antibody is added. Only target-bound antibodies activate these cells, while free antibodies are internalized without activation. Their cytotoxic activity is correlated to target protein density, therefore targeting tumor cells with high antigen density while sparing normal cells with low or no expression. This activation mechanism prevents premature exhaustion. Furthermore, during ADCC these cells secrete attenuated cytokine levels compared to CAR-T, thereby enhancing their safety profile. These cells eradicate established melanoma tumors, infiltrate the tumor microenvironment and facilitate host immune cell recruitment in immunocompetent mice. In NSG mice, they infiltrate, persist, and eradicate tumors. As opposed to CAR therapies, which require changing the receptor across different types of cancer, our engineered T-cells remain the same throughout tumor types, while only the injected antibody changes. As such, can be adapted to treat a wide range of solid cancers without changing the manufacturing program. Overall, SolidT cells are capable of binding tumor cells with high affinity, while preserving the cytotoxic specificity only to cells expressing a high density of tumor-associated antigens.

Citation Format: Diana Rasoulouniriana, Nadine Santana-Magal, Amit Gutwillig, Leen Farhat-Younis, Hana Shpilt, Shahar Dotan, Noam Pilpel, Peleg Rider, Yaron Carmi. A modified FcγRI expressing-T cell, SolidT, enables antibody-mediated cytotoxicity to overcome the limitations of CAR-T cell therapy against solid tumors. [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 4075.
Abstract 4126: Anti-tumor immunity of M9657, a conditional CD137 immune agonist, is correlated with mesothelin expression on tumor cells
Authors: Xu Chunxiao , Zhou Xueyuan , Yalavarthi Sireesha , Schweickhardt Rene , Blaukat Andree , Helming Laura
Published: April 2023

Background: In clinical trials, systemic administration of first-generation CD137 agonist monotherapies was suspended due to either low anti-tumor efficacy or hepatotoxicity mediated by the epitope recognized on CD137 and Fc gamma receptor (FcγR) ligand-dependent clustering. M9657 is a bispecific conditional agonist that has been developed to bind simultaneously to mesothelin (MSLN) and CD137 to stimulate an anti-tumor immune response in the tumor microenvironment. M9657 was engineered in a tetravalent bispecific antibody (mAb2™) format with a human IgG1 backbone with LALA mutations, which abrogates binding to FcγRs but retains FcRn binding for IgG-like pharmacokinetics. M9657 is expected to have enhanced anti-tumor efficacy while avoiding systemic immune activation.

Methods: MSLN surface copy number was quantified in a series of cancer cell lines with a broad range of MSLN expression. CD8+ T cell-mediated tumor cell cytotoxicity and cytokine release from CD8+ T cells were investigated in a series of in vitro functional assays. The receptor occupancy (RO) of MSLN on the tumor cell surface and of CD137 on CD8+ T cells was determined by flow cytometry. MSLN in EMT-6 cells was knocked out using CRISPR and confirmed by immunohistochemistry. The anti-tumor efficacy of FS122m, a murine-reactive surrogate of M9657, was investigated in EMT-6 parental and MSLN knockout syngeneic tumor models.

Results: M9657 displayed MSLN-dependent cytokine release and tumor cell cytotoxicity. IFNγ release by CD8+ T cells correlated with MSLN copy number on tumor cells. MSLN copy number of 3000 was found to be the minimum required to trigger CD137 agonism. CD137 RO by M9657 on activated human CD8+ T cells and MSLN RO by M9657 on tumor cells increased with increasing concentrations of M9657. M9657 caused full agonism (EC100) when CD137 RO on activated human CD8+ T cells was approximately 30-38%. The mouse surrogate FS122m demonstrated potent dose-dependent anti-tumor efficacy in the EMT-6 tumor model, while no anti-tumor efficacy was observed in MSLN knockout tumors, which further confirmed that MSLN expression is required for M9657 anti-tumor immunity.

Conclusion: M9657 exhibits promising and potent MSLN-dependent conditional immune agonism, supporting its clinical investigation.

Citation Format: Chunxiao Xu, Xueyuan Zhou, Sireesha Yalavarthi, Rene Schweickhardt, Andree Blaukat, Laura Helming. Anti-tumor immunity of M9657, a conditional CD137 immune agonist, is correlated with mesothelin expression on tumor 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 4126.
Abstract 415: Metabolic reprogramming driven by IGF2BP3-COX6B2 axis promotes acquired resistance to EGFR-TKIs in non-small lung cancer
Authors: Lin Ziyou , Li Jingwei , Zhang Jian , Feng Weineng , Lu Jiaye , Ma Xiaofan , Ding Wen , Ouyang Shumin , Lu Jinjian , Yue Peibin , Wan Guohui , Liu Peiqing , Zhang Xiaolei
Published: April 2023

Acquired resistance represents a bottleneck to molecular targeted therapy in lung cancer. Metabolic adaptation is a distinct hallmark of human lung cancer, but its role in acquired resistance has not been fully understood. In this study, we discovered a novel mechanism of TKI resistance involved in IGF2BP3-dependent crosstalk between the epigenetic modifications and metabolic reprogramming through the IGF2BP3-COX6B2 axis. We demonstrated the upregulated expression of RNA binding protein IGF2BP3 reduced the sensitivity of TKI treatment and exacerbated the development of drug resistance via promoting oxidative phosphorylation (OXPHOS). Analysis of patient samples revealed a positive correlation between IGF2BP3 and COX6B2 and poor prognosis in lung cancer patients. We further confirmed that COX6B2 is a key downstream target in IGF2BP3-mediated acquired TKI-resistance. Mechanistically, IGF2BP3 bound to the 3’-untranslated region of COX6B2 in an m6A-dependent manner to increase COX6B2 mRNA stability. Moreover, IGF2BP3 significantly enhanced OXPHOS capacity in lung cancer through the IGF2BP3-COX6B2 axis by regulating nicotinamide metabolism, thus resulting in TKI resistance in lung cancer. Inhibition of OXPHOS with IACS-010759, a small-molecule inhibitor, resulted in remarkable growth inhibition in vitro and in vivo in a gefitinib-resistant patient-derived xenograft model. Collectively, our findings suggest that metabolic reprogramming directed by the IGF2BP3-COX6B2 axis plays a critical role in TKI resistance, and targeting the metabolic pathway provides a new therapeutic strategy to overcome EGFR-TKI resistance in lung cancer.

Citation Format: Ziyou Lin, Jingwei Li, Jian Zhang, Weineng Feng, Jiaye Lu, Xiaofan Ma, Wen Ding, Shumin Ouyang, Jinjian Lu, Peibin Yue, Guohui Wan, Peiqing Liu, Xiaolei Zhang. Metabolic reprogramming driven by IGF2BP3-COX6B2 axis promotes acquired resistance to EGFR-TKIs in non-small lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 415.
Abstract 4156: Site specific tumor response to combination chemotherapy and immune checkpoint inhibitor in the syngeneic Pan02 pancreatic ductal adenocarcinoma model
Authors: Wong Andrew , Fadden Patrick , Clouse Cara , Wood Patrick , Brown-Augustine Marianna , Zhao Fei , Ser Elliot , Kolb Joseph , Hall Chassidy , Rainbolt Elizabeth
Published: April 2023

Syngeneic tumor models provide a platform to assess novel immune-oncology therapeutics in fully immuno-competent mice. A subcutaneous flank tumor implant provides a convenient and accessible location to monitor the tumor growth and response to therapy. However, the tumor microenvironment, and therefore, the responsiveness to immuno-oncology therapies, is greatly impacted by the location in which the tumor develops (Ho 2021, Oliver 2018). Given the potential of different immuno-suppressive features driven by the specific tumor location, we wanted to compare the tumor infiltrating leukocyte populations in the subcutaneous flank and orthotopic pancreatic Pan02 syngeneic models, as well as the response to standard of care therapies. Pan02 cells were either implanted orthotopically in the pancreas or subcutaneously in the flank and allowed to establish before dosing with either vehicle, oxaliplatin, anti-CTLA-4 or the combination of therapies. Established tumors from untreated orthotopic and flank models were sampled for flow cytometry to assess infiltration by a broad range of immune cell populations. Response to therapy was clearly dependent on tumor location with flank tumors being resistant to both monotherapy and combination therapy, while the orthotopic tumors responded modestly to monotherapy but displayed synergy with the combination therapy. The results suggest that for a given syngeneic tumor cell line with identical mutational load and antigenicity, we can generate unique tumor microenvironments dependent on implant location leading to different outcomes when testing novel immune-oncology therapies. These types of models can help define the context in which novel immune-oncology therapeutics can be expected to drive an effective immune response.

Citation Format: Andrew Wong, Patrick Fadden, Cara Clouse, Patrick Wood, Marianna Brown-Augustine, Fei Zhao, Elliot Ser, Joseph Kolb, Chassidy Hall, Elizabeth Rainbolt. Site specific tumor response to combination chemotherapy and immune checkpoint inhibitor in the syngeneic Pan02 pancreatic ductal adenocarcinoma model. [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 4156.
Abstract 4238: Specific oral microbiome is closely associated with oral potentially malignant disorders and oral squamous cell carcinoma
Authors: Kioi Mitomu , Isono Hiroshi , Nakajima Shintaro
Published: April 2023

Background: Bacterial and virus infection may cause pre-malignant and malignant disorder such as hepatocellular carcinoma and cervical cancer. Recent studies reported that oral bacterium is associated with gastric and colon cancer. However, it is not clear yet if the oral microbiome affects the development of pre-malignant disorder in oral cavity and oral cancer. Thus, this study aims to identify the periodontal pathogen candidates involved in the onset and progression of oral squamous cell carcinoma (OSCC).

Materials and Methods: Saliva was collected from 112 patients without oral mucosal diseases (OMDs) as control, 36 patients with oral potentially malignant disorders (OPMDs), and 104 patients with OSCC, and DNA was extracted in each sample. Periodontal examinations were performed on all patients. Endpoint-PCR was performed on seven species of oral pathogens using the extracted DNA. 16S ribosomal RNA (rRNA) was utilized for analysis of oral microbiome with 20 samples in each group. 4-nitroqquinoline-1-oxide (4-NQO)-induced tongue tumor model in mice was analyzed with or without endotoxin of one of the oral bacteria, Porphyromonas gingivalis (P. gingivalis).

Results: From the statistical analysis of the clinical parameters, periodontal disease tended to worsen in the OMPD and OSCC groups compared to the control group. The number of oral bacteria was significantly higher in the OSCC group than those in other groups. From PCR results, the detection rates of P. gingivalis, Aggregatibacter actinomycetemcomitans, and Treponema denticola were significantly higher in the OSCC group than those in the control group. From 16S rRNA analysis, the relative abundance of Prevotella buccae and intermedia was significantly higher in OSCC groups than in the control group. Furthermore, the relative abundance of P. gingivalis was higher in the OSCC group compared to other groups, regardless of age and the number of teeth. In mouse model, mice treated with P. gingivalis-derived endotoxin developed oral leukoplakia at earlier time points and tumors significantly higher lesions compared to control group.

Conclusion: These results suggest that specific periodontal pathogens are present in the oral cavity of OPMDs and OSCC patients and those changes in the bacterial flora due to their presence may contribute to the onset and progression of OMDs.

Citation Format: Mitomu Kioi, Hiroshi Isono, Shintaro Nakajima. Specific oral microbiome is closely associated with oral potentially malignant disorders and oral squamous cell carcinoma. [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 4238.
Abstract 4279: Method for identifying microsatellite instability high DNA abnormality samples
Authors: Perera Dilmi , Khakabi Sahand , Nip Ka Mun , Brahmbatt Sonal , Kense Adrian , Tam Kevin , Mulder David , McConechy Melissa , Huntsman David , Miller Ruth , Aguirre-Hernández Rosalía
Published: April 2023

Introduction: Tissue samples with high microsatellite instability (MSI-H) can be indicators of cancerous tumors that are sensitive to certain types of cancer treatments (e.g., immune modulation-checkpoint inhibitor treatment). MSI-H regions can be identified with Polymerase Chain Reaction (PCR) based assays and next-generation sequencing (NGS). However, these MS regions are susceptible to PCR and sequencing errors. We developed a computational method for detecting microsatellite instability high (MSI-H) tumors using next-generation sequencing (NGS) data to accurately identify true MSI-H samples from MS-Stable samples based on an analysis of these MS regions.

Methods: We developed a method for classifying a tissue sample as being microsatellite instability high (MSI-H) without using normal tissue from the same person which doubles the sequencing cost. Furthermore, the algorithm was designed for amplicon targeted assays where it is not always feasible to choose the most predictive MS sites. The machine learning classifier (ML) algorithm is a random forest algorithm with a training set of known MSI-H and MS-Stable samples to learn the relationship between the MSI status and the distribution of repeats in microsatellite regions of genomes using 21 MS loci. A negative control was used to normalize the ML features and therefore reduce the effects of PCR and sequencing errors in noisy MS sites.

Results: The MSI detection algorithm was validated in analytical and clinical samples achieving an accuracy greater than 98%. Analytical samples consist of commercial reference standard samples and well characterized FFPE treated cell-lines. Clinical samples consists of clinical FFPE tumor samples from cancer patients that were orthogonally validated using immunohistochemistry (expression of mismatch repair genes, i.e., MMRnormal vs MMRd) on tumor tissue and/or the Promega MSI PCR using matched tumor/normal. All experiments were performed in the Imagia Canexia Health CAP, CLIA, DAP certified laboratory using the Find It assay standard operating procedures for detecting genomic mutations in solid tumor tissue.

Conclusions: The MSI detection algorithm can accurately identify samples with MSI-H tumors. When used in a clinical setting, these patients can then be directed to treatments such as immune-checkpoint inhibitors.

Citation Format: Dilmi Perera, Sahand Khakabi, Ka Mun Nip, Sonal Brahmbatt, Adrian Kense, Kevin Tam, David Mulder, Melissa McConechy, David Huntsman, Ruth Miller, Rosalía Aguirre-Hernández. Method for identifying microsatellite instability high DNA abnormality samples. [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 4279.