bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2880: CD133-targeting silicate prodrug nanoparticles for effective cancer stem cell targeting
Authors: Bhatnagar Shubhmita , Jin Mengyuan , Hoye Thomas , Panyam Jayanth
Published: June 2022

Cancer stem cells (CSCs) are considered to be key contributors to drug resistance and tumor recurrence in many solid tumors. Therapeutic approaches that specifically target these cells are needed to improve treatment outcomes in these tumor types. We are investigating CD133, a widely used surface marker to identify CSCs in colon, brain, and breast tumors. Here, we report the use of polymeric nanoparticles targeting CD133 by conjugating an anti-CD133 scFv-Fc to poly(L-lactide)-PEG-maleimide nanoparticles (anti-CD133 NPs) for effective CSC targeting. The Fc tagged anti-CD133 scFv was prepared and purified using an Expi293 expression system. Nanoparticles were prepared using flash nanoprecipitation and loaded with a hydrophobic, hydrolytically labile, silicate prodrug of paclitaxel. Flash nanoprecipitation allowed for generation of particles with a size of 232 ± 17 nm (increased to 289 ± 23 nm upon scFv conjugation), a zeta potential of -11.5 mV and a drug loading of 57 ± 8 wt% measured by HPLC. The anti-CD133 scFv-Fc was thiolated using 2-iminothiolane (2-IT) and conjugated to nanoparticles using thiol-maleimide chemistry. Thiolation of the antibody was optimized using different molar excesses of 2-IT. A binding assay using Caco-2 cells (which overexpress CD133) confirmed that thiolation of the antibody did not affect its binding, even at the highest molar excess of 2-IT used. With 200x molar excess of 2-IT, 2.9 thiols per antibody were added. Antibody conjugation was confirmed using gel electrophoresis; bands corresponding to the heavy and light chains of antibody were observed for anti-CD133 NPs after reduction but were not seen for non-reduced anti-CD133 NPs or for unconjugated particles. Cytotoxicity studies in MDA-MB-231-LM2 cells revealed maximum kill in drug loaded anti-CD133 NPs, which was significantly higher than for either non-targeted, drug-loaded nanoparticles or blank nanoparticles (p<0.05). Overall, these studies indicate the potential for using anti-CD133 scFv-Fc in conjunction with FNP-produced silicate prodrug nanoparticles of paclitaxel for efficient targeting of CSCs.

Citation Format: Shubhmita Bhatnagar, Mengyuan Jin, Thomas Hoye, Jayanth Panyam. CD133-targeting silicate prodrug nanoparticles for effective cancer stem cell targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2880.
Abstract 289: A novel ex-vivo organotypic culture platform for functional interrogation of human appendiceal neoplasms
Authors: Weitz Jonathan , de Mendoza Tatiana Hurtado , Tiriac Herve , Baumgartner Joel , Kelly Kaitlyn , Veerapong Jula , Lowy Andrew
Published: June 2022

Appendiceal neoplasms are rare and often clinically present with peritoneal metastasis. While surgical tumor resection is effective for some types of primary appendiceal cancers, patients with metastatic disease have poor prognostic outcomes. Models to study appendix cancer biology are limited, given that 1) no mouse models exist and 2) reliable in vitro models are unavailable. As such, we have developed an ex-vivo organotypic slice model to examine cellular interactions between tumor cells and their local microenvironment. Tumor specimens from human appendiceal cancer patients were cut using a vibratome to make 200 μm organotypic slices. Slices were cultured on transwell inserts and tested for changes in morphological, cellular and functional characteristics over a seven-day period. Organotypic slices maintained their cellular composition in regard to the proportion of epithelial, immune cells and fibroblasts. Live cell [Ca2+]i imaging of long term cultured slices confirmed that immune cells remain functionally active when stimulated with extracellular ATP. Lasty, using tumor biopsies from human donors, we have identified a diverse immunological profile of appendiceal tumors not previously identified. Our study illustrates a novel approach for studying the pathophysiology of appendiceal cancer, a notoriously difficult disease to model.

Citation Format: Jonathan Weitz, Tatiana Hurtado de Mendoza, Herve Tiriac, Joel Baumgartner, Kaitlyn Kelly, Jula Veerapong, Andrew Lowy. A novel ex-vivo organotypic culture platform for functional interrogation of human appendiceal neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 289.
Abstract 2890: The development of an Adaptable Drug Affinity Conjugate (ADAC) targeting CD40 for a flexible therapeutic peptide cargo delivery to dendritic cells
Authors: Laurén Ida , Eltahir Mohamed , Mebrahtu Aman , Veerman Rosanne , Astorga Juan , Saleh Aljona , Ytterberg Jimmy , Lindqvist Annika , Dahllund Leif , Olsson Anders , Andersson Oskar , Rockberg Johan , Persson Helena , Mangsbo Sara
Published: June 2022

CD40 agonistic antibodies targeting antigen-presenting cells rely on simultaneous antigen presentation for optimal efficacy, as the co-stimulatory signal alone will not lead to T cell activation. Herein we have developed a novel Adaptable Drug Affinity Conjugate (ADAC), a refinement of a traditional Antibody Drug Conjugate (ADC) with a focus on tailored drug design. The cargo for ADAC is synthetic long peptides that can be modified with the profiled genetic data for each patient. The ADAC technology relies on a high-affinity interaction between a short non-immunogenic peptide-tag (pTag) and a single-chain fragment (scFv) and the targeting of CD40 expressing antigen-presenting cells. As a proof-of-concept study, we have generated data supporting a ~20 times improved half-life of peptide therapeutics bound up by the ADAC technology (in vitro), translating to improved peptide half-life in vivo along with a superior expansion of T cells in vitro and in vivo, compared to peptide and antibody mixed but not interacting physically. Through phage-display selection screening of novel CD40 agonistic antibodies, we identified a unique clone (STRIKE-1001). STRIKE-1001 binds a defined structural epitope (HDX-MS) of CD40 in a low nM range and is agonistic in an IgG2 format. Similar to selicrelumab, STRIKE-1001 does not block CD40L binding to Fc-CD40. STRIKE-1001 shows a dose-dependent agonistic activity (EC50 of ~28 nM based on measured induction of HLA-DR expression) on human DCs and can be designed into a bispecific antibody without loss of agonistic activity. STRIKE-1001 is currently incorporated into the ADAC platform to be used as the first-in-class candidate for flexible peptide cargo delivery ensuring T cell priming and expansion in vivo as a means to transform CD40 agonistic antibody to clinically relevant drug candidates.

Citation Format: Ida Laurén, Mohamed Eltahir, Aman Mebrahtu, Rosanne Veerman, Juan Astorga, Aljona Saleh, Jimmy Ytterberg, Annika Lindqvist, Leif Dahllund, Anders Olsson, Oskar Andersson, Johan Rockberg, Helena Persson, Sara Mangsbo. The development of an Adaptable Drug Affinity Conjugate (ADAC) targeting CD40 for a flexible therapeutic peptide cargo delivery to dendritic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2890.
Abstract 2893: Exploring the combinatorial potential of bispecific T-cell engagers in high throughput format
Authors: Castro Carla N. , Feger Daniel , Ulrich Sarah , Siedentopf Oliver , Ehlert Jan E.
Published: June 2022

Bispecific antibodies are being explored as a means to modulate responses of the immune system to tumor infiltration. They are emerging as a growing class of immunotherapies with potential to further improve clinical efficacy and safety. Besides tumor-targeted and dual immunomodulators, the most important class of therapeutically relevant bispecific antibodies are engagers of cytotoxic immune cells to kill tumor cells. These are mainly engager of T-cells (BiTE´s), but also NK-cell engager several of which have entered clinics with Blinatumomab (anti-CD3/anti-CD19) being FDA-approved for the treatment of Acute Lymphoblastic Leukemia. Several studies are under way to address potential combinatorial effects with other compounds in clinics. In order to facilitate analysis of the cytotoxic impact of BiTE´s in high throughput, we developed a 384-well format assay system based on Luciferase-labeled tumor cells. This detection technology has many advantages. As only the tumor cells are labeled, the specific detection of luciferase exclusively correlates to the number of viable tumor cells, allowing for co-cultures with high excess of effector cells without them interfering in the detection. Due to high sensitivity, only few tumor cells are required for a decent signal/noise ratio, keeping the overall need for primary effector cells to the lowest, even at high effector/target cell ratios. That can be of great advantage when working with rare cytotoxic subpopulations (e.g. NK-cells), precious samples from patients, or simply saves resources when performing large exploratory studies. In addition, combination of low 384-well format volumes with nanodrop agent dispensing technology minimizes amounts of expensive antibodies and other agents. Applying this technology, we evaluated the combinatorial effects of Blinatumomab with immune modulators (e.g. Lenalidomide), kinase inhibitors (e.g. MEK-inhibitor, Selumetinib) and other clinically relevant agents (e.g. MCL1-inhibitor, S63845) on the Luciferase-labeled GCB-like DLBCL cell line OCI-LY1. Depending on compound combination, we observe synergistic but also antagonistic effects. Our data support the outstanding usefulness of this methodological approach for the exploration of bispecific cytotoxic immune cell engager agents.

Citation Format: Carla N. Castro, Daniel Feger, Sarah Ulrich, Oliver Siedentopf, Jan E. Ehlert. Exploring the combinatorial potential of bispecific T-cell engagers in high throughput format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2893.
Abstract 2925: Expanding the library of small molecule imaging probes for heparanase
Authors: Schleyer Kelton , Liu Jun , Wang Zhishen , Guo Hua , Cui Lina
Published: June 2022

Heparanase is an enzyme highly involved in the progression of many types of cancer through its enzymatic processing of heparan sulfate proteoglycans. Unfortunately, research and clinical investigation of this target is hampered by the lack of simple, effective tools to detect its enzymatic activity. Despite the natural preference of heparanase to cleave polysaccharide substrates, we previously developed the first disaccharide-based fluorogenic probe for heparanase enzymatic activity, overcoming this deficiency in existing tools. However, the chemistry of the probe synthesis required installation of electron-withdrawing fluorine atoms on the fluorophore to facilitate activation by heparanase, a design strategy that excludes the use of many common imaging reporters. To accommodate a wider variety of imaging modalities for heparanase detection, we experimented with the addition of a collapsible linker with suitable electronegativity that can be easily tuned. Our results show that a tetra-fluorinated benzyl alcohol linker enables heparanase activation of a probe bearing any reporter, giving comparable results to our original fluorinated coumarin probe. This disaccharide-linker scaffold holds potential for accessing a broader scope of heparanase imaging probes for laboratory and clinical applications of this burgeoning biomarker.

Citation Format: Kelton Schleyer, Jun Liu, Zhishen Wang, Hua Guo, Lina Cui. Expanding the library of small molecule imaging probes for heparanase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2925.
Abstract 2932: The 6-color naica® system: a digital PCR platform and workflow for high multiplex genetic detection and analysis
Authors: Hoskins Andrew , Primus Shekerah , Yazdani Mary , Gutierrez Kimberley , Foret Benjamin , Bernard Fabien , Dangla Rémi
Published: June 2022

There are several molecular platforms routinely used to investigate genomic material. Quantitative polymerase chain reaction (qPCR) is ubiquitously used because of the ease of use and the precision however, it does not offer high multiplexing. While digital PCR (dPCR) is known for increased precision and sensitivity, most digital PCR platforms provide the same level of multiplexing as qPCR. Next-generation sequencing (NGS) is less precise than qPCR or dPCR and but has the highest multiplexing capability of the three technologies. Conventional digital PCR systems have been limited by the number of instruments required to conduct the workflow, time-to-result compared to qPCR, and level of multiplexing as compared to next-generation sequencing (NGS).

Crystal Digital PCR™ and the naica system is a next-generation digital PCR platform that simplifies the number of instruments in the workflow, speeds up the time of results to less than 3 hours, and increases the number of detection colors to 6 spectrally distinct channels. By using a combination of dual-labelled fluorescent probes, multiplexed digital PCR provides a simple solution that makes it possible to use less of a precious sample when faced with limited quantity without sacrificing sensitivity.

The naica system workflow utilizes microfluidic consumables, such as the Sapphire chip for high sensitivity applications, and the Opal chip for higher throughput studies. All reagents including primers and probes, master mix, and sample are inputted into the chips in one single loading step. Chips are transferred to the Geode instrument, where the sample is partitioned into a 2D-monolayer of droplet crystals and where all targets are amplified. After the PCR program has been completed, the same chips are then loaded into the Prism6 imager which detects all targets across 6 fluorescent channels and outputs an image of all droplets in a sample. Finally, Crystal Miner analysis software can be used to analyze and decipher the quantifications and expression of all targets in the assay.

The 6-color naica system allows for high multiplex detection of targets previously unattainable by conventional digital PCR systems. With this advent, genomic scientists have an additional platform in the molecular toolbox that supports seamless assay development of oncology biomarker assays for gene expression, rare target detection, and liquid biopsy applications.

Citation Format: Andrew Hoskins, Shekerah Primus, Mary Yazdani, Kimberley Gutierrez, Benjamin Foret, Fabien Bernard, Rémi Dangla. The 6-color naica® system: a digital PCR platform and workflow for high multiplex genetic detection and analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2932.
Abstract 295: Superior efficacy of targeted nanoparticle therapy for brain tumors
Authors: Kang HyungGyoo , Nagy Jon , Upton Bryon , Triche Timothy
Published: June 2022

Glioblastoma Multiforme is an incurable malignant brain tumor. Despite intensive efforts over many years, the prognosis is essentially unchanged. The primary reason is failure of otherwise effective therapy to reach the tumor, due to the blood brain barrier (BBB). To circumvent this functional barrier, we have developed CD99 and CD276 targeted irinotecan (Ir) containing liposomal nanoparticles (TNS: Targeted NanoSpheres) TNSs effectively penetrate the BBB and selectively bind to GBM cells while largely bypassing normal brain tissue. CD99 or CD276 targeted Ir-TNSs prolonged survival five-fold over free Ir treated animals in bi-weekly single dose level studies. Normal tissue levels were negligible, and no treatment-related toxicity was observed. QD dose intensification induced tumor regression in animals with progressive tumor. Anti-tumor effect was equivalent for either CD99 or CD276 Ir-TNSs. These results imply that intracranial therapy with otherwise effective drugs that do not pass the BBB is both feasible and shows superior efficacy over free drug therapy. Further, widespread expression of CD276 by many malignancies implies this therapy could be effective for metastatic brain tumors and extra-cranial malignancies as well.

Citation Format: HyungGyoo Kang, Jon Nagy, Bryon Upton, Timothy Triche. Superior efficacy of targeted nanoparticle therapy for brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 295.
Abstract 2955: SMYD2 regulates chromatin modifier KMT2D in ER+/PIK3CA mutant breast cancer
Authors: Blawski Ryan , Sallaku Mirna , Guo Xinyu , Kittane Srushti , Scaltriti Maurizio , Luo Minkui , Toska Eneda
Published: June 2022

Breast cancer leads current cancer diagnoses worldwide, accounting for 1 in 4 cancer cases and 1 in 6 cancer deaths in women. Over 70% of breast tumors are hormone receptor positive and can be treated with endocrine therapy, however tumor recurrence remains a major obstacle. Oncogenic mutations in the catalytic subunit of PI3K occur in up to 40% of ER+/HER2- negative breast tumors, making it a promising therapeutic target. Clinically, PI3K inhibitors have shown limited efficacy as monotherapy due to a number of resistance mechanisms, including an upregulation of ER-dependent signaling. Identification of the ER-PI3K signaling crosstalk led to clinical trials which resulted in the approval of the PI3K inhibitor alpelisib in combination with fulvestrant in patients with hormone-refractory ER+/PIK3CA-mutant breast cancer. Further work uncovered a key role for the chromatin modifier KMT2D in regulating ER-dependent transcription upon PI3K inhibition. PI3K effectors AKT1/SGK1 negatively regulate KMT2D through direct S1331 phosphorylation. When PI3K is inhibited, this downstream regulatory event is lost, leading to increased chromatin recruitment of KMT2D and an upregulation of ER-dependent transcription. We have identified a novel methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. Knockdown of SMYD2 sensitizes ER+/PIK3CA mutant breast cancer cell lines to PI3K inhibition. The combination of SMYD2 knockdown and alpelisib leads to a significant reduction in tumor growth compared to alpelisib alone in a xenograft model. Mechanistically, SMYD2 knockdown is able to attenuate alpelisib-induced KMT2D chromatin binding globally and at ER loci. Accordingly, loss of SMYD2 abrogates alpelisib-induced changes in gene expression, including ER-dependent transcription. Altogether our results demonstrate a role for epigenetic therapy using SMYD2 inhibitors in combination with alpelisib for the treatment of ER+/PIK3CA mutant breast cancer.

Citation Format: Ryan Blawski, Mirna Sallaku, Xinyu Guo, Srushti Kittane, Maurizio Scaltriti, Minkui Luo, Eneda Toska. SMYD2 regulates chromatin modifier KMT2D in ER+/PIK3CA mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2955.
Abstract 2958: Chromatin repressor complex as a converge point of RAS and PP2A activities in lung cancer
Authors: Sharma Mukund , Aakula Anna , Tabaro Francesco , Pavic Karolina , Huhtaniemi Riikka , Nykter Matti , Westermarck Jukka
Published: June 2022

Protein phosphatase 2A (PP2A) is a tumor suppressor while RAS proteins are potent oncogenes in different cancer types. It is well established that RAS-mediated oncogenic transformation requires simultaneous inhibition of PP2A, but the molecular basis of their interaction is poorly understood. To address the target mechanisms co-regulated by RAS and PP2A we performed a phosphoproteomics screen upon RAS or PP2A manipulation. Overall the results indicated that RAS and PP2A regulate overlapping cellular processes, and that their activities might in particular converge at epigenetic machineries.

To study the RAS and PP2A synergy on transcriptional and epigenome regulation we performed multi-omics analysis of cancer cells in which RAS and PP2A activities were modulated. PP2A inhibition resulted in global gene activation while RAS inhibition lead predominantly to gene repression. Use of GFP reporter indicative of epigenetic silencing further confirmed the role of PP2A as a transcriptional repressor and RAS as a transcriptional activator. To investigate the DNA methylation changes and chromatin remodeling effects upon PP2A modulation we used RRBS (Reduced representation bisulfite sequencing) & ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) respectively. PP2A inhibition resulted in global DNA hypomethylation and an open state of chromatin confirming its role as a global repressor of epigenetic processes.

To delineate the molecular mechanism behind the transcriptional regulation by RAS and PP2A we monitored the chromatin recruitment of transcriptional repressors upon PP2A or RAS manipulation. Either pharmacological or siRNA mediated PP2A or RAS modulation significantly affected gene repressor complex recruitment to the chromatin. Role of phosphorylation as a critical switch for chromatin repressor complex recruitment was demonstrated by CRISPR/Cas9-mediated mutagenesis of PP2A target phosphorylation site, which also led to a clear phenotype in in vivo tumorigenesis and oncogenic transcription.

Collectively these results identify a novel phosphorylation switch on chromatin repressor complex as a converge point of KRAS and PP2A activities in lung cancer. Generally the results provide first indications to global importance of PP2A-mediated phosphorylation regulation in epigenetic gene regulation in cancer.

Citation Format: Mukund Sharma, Anna Aakula, Francesco Tabaro, Karolina Pavic, Riikka Huhtaniemi, Matti Nykter, Jukka Westermarck. Chromatin repressor complex as a converge point of RAS and PP2A activities in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2958.
Abstract 2959: Investigating the oncogenic effects and mechanisms of the SMYD3 methyltransferase in HPV-negative head and neck squamous cell carcinoma
Authors: Murali Madhavi , Tsai Daniel , Nigam Nupur , Burkitt Kyunghee , Kim Sohyoung , Cheng Hui , Saloura Vassiliki
Published: June 2022

Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world and is associated with an overall poor prognosis. The protein methyltransferase SET and MYND domain-containing 3 (SMYD3), which trimethylates H3K4 and H4K20, can stimulate gene transcription and activate several oncogenic pathways, including epithelial-mesenchymal transition, and cell cycle related pathways. Additionally, its expression is associated with a poor prognosis in various cancer types, such as colon and ovarian cancer. This study aims to decipher whether SMYD3 has direct oncogenic effects and relevant mechanisms in HPV-negative HNSCC. Our preliminary results have demonstrated that SMYD3 is significantly overexpressed in HPV-negative HNSCC compared to normal and dysplastic epithelium. SMYD3 knockdown decreased cellular proliferation and clonal capacity in CFAs after siRNA-mediated SMYD3 knockdown in several HPV-negative HNSCC cell lines or CRISPR-mediated SMYD3 knockout. Cell cycle analysis of CRISPR SMYD3 KO cell lines showed a significant decrease in the S phase, supporting that cell cycle arrest may be a mechanism through which this decreased cellular proliferation occurs. Nuclear/cytoplasmic dissociation and western blotting of 6 HPV-negative HNSCC cell lines, and immunohistochemistry for SMYD3 in primary and recurrent/metastatic HPV-negative HNSCC tumor tissues have identified the expression of SMYD3 in both the cytoplasm and nucleus, with greater expression in the cytoplasm than the nucleus. Ongoing experiments aim to investigate the effects of SMYD3 knockdown on the global proteome, as well as its effects on histone post-translational modifications in HPV-negative HNSCC cell lines. Genome-wide mapping for SMYD3, H3K4me3 and H4K20me3 in HPV- negative HNSCC cells using CUT&RUN assays are ongoing with the goal to identify direct downstream gene targets regulated by human SMYD3. Finally, we plan to investigate if SMYD3 knockout decreases tumor growth in NSG xenografts. This study may provide the biological rational to target SMYD3 as a novel therapeutic avenue in HPV-negative HNSCC.

Citation Format: Madhavi Murali, Daniel Tsai, Nupur Nigam, Kyunghee Burkitt, Sohyoung Kim, Hui Cheng, Vassiliki Saloura. Investigating the oncogenic effects and mechanisms of the SMYD3 methyltransferase in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2959.
Abstract 2970: Oncometabolites confer vulnerabilities in NAD biosynthesis via NAPRT gene silencing
Authors: Noronha Katelyn , Liang Jiayu , Bindra Ranjit
Published: June 2022

Cancer associated mutations in citric acid cycle enzymes cause overproduction of 2-hydroxyglutarate (2HG), fumarate, or succinate, commonly referred to as oncometabolites. These oncometabolite producing mutations are prevalent in 70% of gliomas, >76% of hereditary leiomyomatosis and renal cell carcinoma, and >25% of paraganglioma and pheochromocytomas, respectively. These oncometabolite producing cancers harbor vulnerabilities in multiple cellular pathways because oncometabolites competitively inhibit alpha ketoglutarate dependent dioxygenases. This group of enzymes includes the ten eleven translocation (TET) family of enzymes, which catalyze locus specific DNA demethylation. Thus, oncometabolite producing cancers have been shown to exhibit global DNA hypermethylation, which drastically alters gene expression in the cell and is thought to occur via TET inhibition. Cells with isocitrate dehydrogenase 1 (IDH1) mutations, which produce 2HG, have loss of expression of a critical NAD biosynthesis enzyme, nicotinate phosphoribosyltransferase (NAPRT). We show that in addition to IDH1 mutant cells, fumarate hydratase (FH)-deficient isogenic cell lines, which overproduce fumarate, show loss of NAPRT expression, which can be targeted by nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. The goal of our work is to determine if this NAPRT silencing is present in many oncometabolite producing cancers, elucidate the mechanism of silencing, and test therapies to target this vulnerability in NAD biosynthesis. FH-deficient cancer cell lines show reduction of NAPRT mRNA levels by qRT-PCR and absence of protein by western blot, suggesting a reduction in NAPRT transcription. In addition, FH-deficient cell lines exhibit synthetic lethality in short term cell viability assays when treated with NAMPT inhibitor, FK866, which inhibits a parallel NAD biosynthesis pathway. Cell viability is not rescued by nicotinic acid supplementation in FH-deficient lines while parental lines are rescuable, validating the loss of functional NAPRT in only FH-deficient lines. Furthermore, patient samples of FH-deficient renal cell carcinomas show higher NAPRT promoter methylation compared to healthy tissue via methylation array. Ongoing studies are investigating the role of TET1/2/3 enzymes in NAPRT promoter methylation. Together, these results suggest that hypermethylation of the NAPRT promoter is associated with silencing of NAPRT expression in FH-deficient cancers, which can be therapeutically targeted through NAMPT inhibition.

Citation Format: Katelyn Noronha, Jiayu Liang, Ranjit Bindra. Oncometabolites confer vulnerabilities in NAD biosynthesis via NAPRT gene silencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2970.
Abstract 2977: Profiling oncogenic Ras mutant drugs with homogeneous bioluminescent immunoassays
Authors: Zegzouti Hicham , Engel Laurie , Swiatnicki Matthew , Alves Juliano , Goueli Said A.
Published: June 2022

Activating KRAS mutations present in many cancers are difficult to target with drugs until recently with the FDA approval of sotorasib (AMG-510), a KRAS G12C selective inhibitor. In an effort to study Ras inhibitors in cells and identify new compounds that inhibit Ras and its downstream signaling, assays such as western or ELISA are commonly used. These traditional immunoassays can be tedious, require multiple washing steps, and are not easily adaptable to a high throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. We applied this bioluminescent and homogeneous “Add and Read” assay to analyze Ras signaling pathway activation and inhibition through multiple nodes of the pathway including detection of phosphorylated ERK. The assay was used to profile different allele specific inhibitors with cell lines harboring different activating KRAS mutations and obtained the predicted pharmacology and selectivity. The potency of PROTAC compound targeting selective degradation of KRAS G12C was also tested using Lumit and the result showed that maximal decrease in RAS signaling was achieved. Unlike current assays that require lengthy sample preparation and multiple washing steps, the Lumit immunoassay provides an alternative platform because it is homogeneous and allows to decipher signaling pathways activities in a fast and simple manner. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest such as Ras, and identify much needed inhibitors of its mutants.

Citation Format: Hicham Zegzouti, Laurie Engel, Matthew Swiatnicki, Juliano Alves, Said A. Goueli. Profiling oncogenic Ras mutant drugs with homogeneous bioluminescent immunoassays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2977.
Abstract 2981: Loss of the small GTPase RHOA enhances lymphomagenesis and impairs the activity of lenalidomide in mantle cell lymphoma
Authors: Santos Juliana C. , Ribeiro Marcelo L. , Roue Gael
Published: June 2022

The small GTPase Ras homolog family member A (RHOA) is one of the most extensively investigated members of the Rho GTPase family, that acts as a molecular switch controlling a wide variety of signal transduction pathways. Although RHOA has long been implicated in malignant transformation in solid tumors, recent evidences have demonstrated its tumor suppressor activity in different subgroups of B-cell non-Hodgkin lymphoma (B-NHL). Here, using a panel of 11 cell lines covering the most common and/or aggressive B-NHL subtypes, we observed that the lowest mRNA and protein levels of RHOA are found in mantle cell lymphoma (MCL), the most aggressive entity with a median overall survival of 5-7 years. Depletion of RHOA expression and activity was carried out in the two RHOA+ MCL cell lines, REC-1 and Z-138, by CRISPR/Cas9-mediated gene edition followed by RHOA pulldown activation assay. In RHOA knockout (KO) subclones, cell proliferation was increased by 40%, in association with a 10-fold increase in mitotic index and a 3-fold potentiation of cell migration, as assessed respectively by CellTitter-Glo assay, histone H3-pSer10 labeling and Transwell assay, thus suggesting a tumor suppressor role of RHOA in MCL malignancy. Comparative RNA-seq analysis of RHOAWT and RHOAKO MCL subclones then highlighted a crucial role of RHOA and its downstream signaling in the control of MCL cell proliferation, cell cycle and cell migration. Interestingly, the immunomodulatory drug lenalidomide has previously shown to regulate such processes and to be clinically active in patients with relapsed/refractory MCL. In a co-culture system associating RHOAWT MCL cells with peripheral blood-derived effector cells, we observed that lenalidomide treatment facilitated the formation of immunological synapse, as revealed by simultaneous immunodetection of effector T cells, target B cells and perforin, and by quantification of lactate dehydrogenase (LDH) release. Conversely, lenalidomide exhibited minimal effect in the RHOAKO MCL co-cultures, as shown by a 30-40% decrease in the formation of immune synapse and a 4-fold decrease in LDH secretion. Taken together, these data provide the first insight into the tumor suppressive activity of RHOA in MCL and into its requirement for the immune-mediated mechanism of action of lenalidomide in this disease. Ongoing in vivo validation studies will be presented.

Citation Format: Juliana C. Santos, Marcelo L. Ribeiro, Gael Roue. Loss of the small GTPase RHOA enhances lymphomagenesis and impairs the activity of lenalidomide in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2981.
Abstract 2987: A versatile assay suite for the discovery of new KRAS pathway inhibitors
Authors: Kouznetsova Ekaterina , McCauley Lauren , Steinke-Neal Elaine , Sullivan Timothy , Primavera Louis , Eells Rebecca , Ma Haiching
Published: June 2022

The small GTPase, KRAS, is a known oncogene and a desirable drug target due to prevalence of mutations with poor disease prognosis. The absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful. Identification of new inhibitor target sites has led to the resurgence of interest in KRAS drug discovery. To facilitate drug discovery activities targeting KRAS/MAPK pathway, we have produced the full spectrum of pathway proteins including kinases, wild type and mutated KRAS, guanine nucleotide exchange factor and effectors. This poster will summarize our most recent assay development efforts for biochemical and biophysical assays for these targets.

Citation Format: Ekaterina Kouznetsova, Lauren McCauley, Elaine Steinke-Neal, Timothy Sullivan, Louis Primavera, Rebecca Eells, Haiching Ma. A versatile assay suite for the discovery of new KRAS pathway inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2987.
Abstract 299: Self-assembling nanoparticles: A novel approach for targeted cancer treatment using tumor treating fields
Authors: Desai Preshita Prafulla , Prabhu Sunil
Published: June 2022

Tumor Treating Fields (TTFields) are used in cancer treatment as they offer increased efficacy, safety, and low adverse effects. They have shown significant potential in pancreatic cancer (PC) clinical trials when combined with chemotherapy. It is pertinent to know that although TTFields-chemotherapy combination definitely enhances the treatment efficacy, it also results in chemotherapy induced severe adverse effects. To resolve this, we earlier reported development of Gemcitabine loaded self-assembling cationic-anionic polymer nanoparticles (S-CAP NPs) capable of achieving TTFields-triggered targeted drug release at the PC tumor site. Initially, four S-CAP NPs [C4 and C7 from chitosan- bovine serum albumin (Chitosan-BSA) S-CAP NPs; P5 and P8 from polyethylenimine- bovine serum albumin (PEI-BSA) S-CAP NPs] were shortlisted [Table 1 - particle size: ~ 200 nm; % encapsulation efficiency (EE): 55-65%]. For proof-of-concept studies, effect of TTFields on the % EE and % cumulative drug release of the S-CAP-NPs was investigated. The studies revealed (Table 1) significant reduction (p value < 0.05) in the % EE of S-CAP NPs within 48h TTFields treatment, confirming that the TTFields destabilize the NPs triggering targeted drug release. Similarly, In vitro drug release studies showed significant increase (p< 0.01) in rate of drug release from S-Cap NPs in presence of TTFields. The cell inhibition assay confirmed the synergism in cell apoptosis wherein PEI-BSA S-CAP NPs showed higher efficacy compared to chitosan-BSA S-CAP NPs. Further, additional stability studies continued over a period of 18 months confirmed stable formulations. In vivo acute and chronic toxicity studies are underway to identify the most optimum dose of S-CAP NPs for further in vivo efficacy studies. To summarize, S-CAP-NPs present a promising avenue to achieve targeted chemotherapy in conjunction with TTFields. This work is supported by AACR-Novocure tumor treating fields research fellowship.

Table 1. Effect of TTFields on % encapsulation efficiency (n=3). Data represented as Mean ± SD. Batch Particle size (nm) Encapsulation efficiency (%) - 0h Encapsulation efficiency (%) - 24h - 150 KHZ Encapsulation efficiency (%) - 48h - 150 KHZ Chitosan-BSA S-CAP NPs C4 210.54 ± 38.96 61.26 ± 5.11 36.21 ± 3.53 24.86 ± 3.19 C7 215.67 ± 32.55 65.31 ± 5.84 32.19 ± 2.68 21.86 ± 4.64 PEI-BSA S-CAP NPs P5 198.29 ± 41.05 58.83 ± 3.33 27.66 ± 3.31 18.96 ± 3.96 P8 209.92 ± 31.33 64.31 ± 5.13 24.83 ± 4.29 19.42 ± 2.73

Citation Format: Preshita Prafulla Desai, Sunil Prabhu. Self-assembling nanoparticles: A novel approach for targeted cancer treatment using tumor treating fields [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 299.
Abstract 2990: Role of CCR6-CCL20 chemokine axis in colon cancer
Authors: Brock Briana A. , Singh Shailesh , Mir Hina A.
Published: June 2022

Colon cancer remains the third leading cause of cancer-related deaths because chemotherapeutics currently offered to treat early, and advanced colon cancer fail to achieve optimal clinical response. Therefore, development of more efficacious treatment option is crucial to improve clinical outcomes. We have used TCGA and CPTAC analysis from UALCAN data base for the expression of CCR6 and CCL20 in colon cancer tissue compared to adjacent normal. RT-qPCR and proteomics approach were used for analysis of CCR6 and CCL20 expression in colon cancer cell lines. Our data shows higher expression of CCR6 and its ligand CCL20 in colon cancer tissue and cell lines. We further investigated involvement of CCR6/CCL20 on therapeutic response of 5-Fluorouracil and Oxaliplatin commonly used to treat colon cancer. Our data suggest that colon cancer cell lines respond poorly to 5-Fluorouracil and Oxaliplatin when treated with CCL20 and blocking CCR6 with anti-CCR6 improves cytotoxic effect of 5-Fluorouracil and Oxaliplatin. Poor response to chemotherapeutic after CCR6 activation was primarily due to activation molecular and cellular phenotypes and alteration in cell cycle. In conclusion, our data suggest that CCR6/CCL20 promotes colon cancer progression and CCR6-mediates signals contributed to poor response to conventional therapeutic such as 5-Fluorouracil and Oxaliplatin. Hence, targeting CCR6 in early stage will prevent metastasis, enhance therapeutic efficacy and reduce toxicity and will be a promising approach to improve clinical outcome.

Citation Format: Briana A. Brock, Shailesh Singh, Hina A. Mir. Role of CCR6-CCL20 chemokine axis in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2990.
Abstract 2997: Combination treatment with cIAP and WEE1 inhibitors exhibits synergism in HPV-positive and HPV-negative head and neck squamous carcinoma cells
Authors: Toni Tiffany , Morgan Ethan , Viswanathan Ramya , Yang Xinping , Cheng Hui , Van Waes Carter
Published: June 2022

Head and neck squamous cell carcinoma (HNSCC) remains a lethal and prevalent diagnosis with limited treatment options for recurrent metastatic cases, particularly in patients with sporadic, human papillomavirus (HPV) negative disease. Recently, the Human Cancer Genome Project identified cell death and NF-κB signaling alterations in a subset of HPV- and HPV+ HNSCC. Co-amplification of Fas-associated death domain (FADD) and cellular inhibitor of apoptosis protein 1 (cIAP1) was identified in HPV- HNSCC, whereas Tumor Necrosis Factor receptor-associated factor 3 (TRAF3) deletion was linked with HPV+ HNSCC. Birinapant, a cIAP inhibitor with primary affinity for cIAP1, functions as a SMAC mimetic to modulate downstream TNF death signaling and promote apoptosis. Clinical trials with Birinapant have demonstrated tolerability and favorable pharmacokinetics but limited activity as a single agent. Our lab recently demonstrated a key interaction between TNF-NF-κB signaling and the G2/M checkpoint kinase WEE1, providing a possible rationale for combination treatment targeting these pathways. We hypothesize that dual-antagonist therapy has the potential to synergistically inhibit TNF-induced canonical NF- κB pro-survival signaling, while enhancing sensitization to TNF-caspase and G2/M mitotic cell death. To investigate this, in vitro studies of Birinapant in combination with Adavosertib, a potent WEE1 inhibitor, were performed. Birinapant and Adavosertib demonstrated drug synergism to varying degrees in all HPV- and HPV+ cell lines tested, both in the presence and absence of tumor necrosis factor alpha (TNF-α), according to the Chou-Talalay method. In the majority of cell lines, synergistic drug activity, as indicated by a low combination index, was positively correlated with percent inhibition. These results were confirmed by increased levels of apoptosis as demonstrated by flow cytometry and both early and sustained cell growth inhibition over time in impedance assays. Ongoing studies include additional characterization of the downstream effects of these agents on NF-κB pro-survival signaling and the cell cycle, along with evaluation in a preclinical murine xenograft model with combined radiotherapy.

Citation Format: Tiffany Toni, Ethan Morgan, Ramya Viswanathan, Xinping Yang, Hui Cheng, Carter Van Waes. Combination treatment with cIAP and WEE1 inhibitors exhibits synergism in HPV-positive and HPV-negative head and neck squamous carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2997.
Abstract 3023: Fatty acid synthase (FASN) inhibitors synergize with carfilzomib (CFZ) in acute myeloid leukemia (AML) and multiple myeloma (MM)
Authors: Peng Maoyu , Ghafouri Sanaz Noelle , McDermott Martina SJ , Slamon Dennis J. , Larson Sarah M.
Published: June 2022

Background: Fatty acid synthase (FASN) upregulation during conditions of oxidative stress contributes to tumor proliferation and survival, which appears to be a mechanism of proteasome inhibitor resistance. Here we demonstrate that acute myeloid leukemia (AML) cell lines resistant to carfilzomib (CFZ), a second-generation proteasome inhibitor, have higher basal FASN expression and targeting FASN with small molecule inhibitors enhances the cytotoxic effect of CFZ in both AML and multiple myeloma (MM) cell lines.

Methods: In a proliferation assay, human AML and MM cell lines were treated with a single dose of CFZ for 7 days. Inhibition of proliferation was defined using an IC50 cutoff for CFZ of 10nM for AML and 5nM for MM as the threshold for sensitivity. Sensitive and resistant cell lines were subjected to apoptosis and cell cycle analyses by flow cytometry after being exposed to CFZ for 72 hours. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). For CFZ and FASN inhibitor combination assays, AML and MM cell lines with varying sensitivities to CFZ were exposed to CFZ and FASN inhibitors, orlistat or TVB-3166, simultaneously, and the apoptosis rate were analyzed by flow cytometry. For western blots, selected AML and MM cell lines were incubated with compounds for 24 hours, and the lysates were probed for selected targets.

Results: Single-agent CFZ induced apoptosis in sensitive AML and MM cell lines, while apoptotic rates remained low in resistant cell lines. Cell cycle analysis showed increased sub-G1 population in sensitive cell lines compared to resistant cell lines. RPPA revealed that FASN, a key enzyme involved in lipogenesis, correlated with CFZ sensitivity, and CFZ resistant lines trended towards higher basal FASN levels. When CFZ was combined with FASN inhibitors, orlistat or TVB-3166, significant synergy was observed in the apoptosis assays in the AML and MM cell lines. Western blot analyses showed FASN inhibitors enhanced the anti-proliferation and pro-apoptotic effects of CFZ.

Conclusion: CFZ demonstrated single agent activity in nanomolar ranges in human AML and MM cell lines. When combined with agents targeting lipid-metabolism, CFZ showed synergistic effect in apoptosis, suggesting this combination could potentially be a new therapeutic strategy for AML and MM.

Citation Format: Maoyu Peng, Sanaz Noelle Ghafouri, Martina SJ McDermott, Dennis J. Slamon, Sarah M. Larson. Fatty acid synthase (FASN) inhibitors synergize with carfilzomib (CFZ) in acute myeloid leukemia (AML) and multiple myeloma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3023.