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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Inflammation-driven colon neoplasmatogenesis in uPA-deficient mice is associated with an increased expression of Runx transcriptional regulators

Highlights: • Inflammation-induced cancer-prone uPA{sup -/-} mouse colon has increased Runx expression. • Immune cells and colonic preneoplastic epithelia are major cellular sources of Runx. • Runx1/2 in uPA{sup -/-} mouse preneoplastic colonic epithelia correlates with cancer risk. • Snai2, Bim and Claudin1 are overexpressed in uPA{sup -/-} mouse proneoplastic colitis. Deregulation of the bone morphogenetic protein (BMP) pathway has been documented in colorectal cancer (CRC). Previously, we investigated possible associations between urokinase-type plasminogen activator (uPA) deficiency and expression of extracellular constituents of BMP signaling in a newly developed mouse model of inflammation-driven intestinal neoplasmatogenesis, in which chronic colitis and CRC are induced using dextran sodium sulfate (DSS). In this report, we explored the contribution of intracellular components of Smad-mediated BMP signal transduction using the same model. Interestingly, upon DSS treatment, we noticed an overexpression of Runx1/2/3 transcription factors in both wild-type and uPA-deficient mice. Moreover, Runx1 and Runx2 expression levels exhibited an even higher increase in DSS-treated/uPA-deficient mice as compared to DSS-treated/wild-type animals. In all experimental conditions, in situ investigation of Runx-expressing cell types, revealed detection of all three Runx in the immune cells, yet in the DSS-treated/uPA-deficient mice Runx1 and Runx2 were also identified in the preneoplastic epithelium of advanced high-grade dysplasia and carcinoma in-situ colonic lesions. Finally, the uPA-deficient pro-tumorigenic colitic microenvironment exhibited increased levels of the Runx-induced target genes Snai2, Bim and Claudin1, known to have a role in tumor development and progression. These findings suggest that the absence of uPA correlates with increased levels of Runx transcriptional regulators in a way that promotes inflammation-associated carcinogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Human papillomavirus (HPV) oncoprotein E6 facilitates Calcineurin-Nuclear factor for activated T cells 2 (NFAT2) signaling to promote cellular proliferation in cervical cell carcinoma

Highlights: • Cervical cancer cells display hyperactive Calcineurin-NFAT2 signaling. • Calcineurin-NFAT2 signaling promotes cell proliferation in cervical cancer cells. • HPV oncoprotein E6 regulate NFAT2 expression and activity in cervical cancer cells. The calcineurin-NFAT signaling pathway regulates cell proliferation, differentiation, and development in diverse cell types and organ systems. Deregulation of calcineurin-NFAT signaling has been reported in leukaemias and few solid tumors such as breast and colon. In the present study, we found elevated calcineurin protein levels and phosphatase activity in cervical cancer cell lines and depletion of the same attenuated cell proliferation. Additionally, nuclear levels of NFAT2, a downstream target of calcineurin, viz, was found elevated in human papillomavirus (HPV) infected cells, HeLa and SiHa, compared to the HPV negative cells, HaCaT and C33A, indicative of its higher DNA binding activity. The nuclear levels of both NFAT1 and NFAT3 remain unaltered implicating they have little role in cervical carcinogenesis. Similar to the in vitro studies, the HPV infected human squamous cell carcinoma specimens showed higher NFAT2 levels compared to the normal cervical epithelium. Depletion of NFAT2 by RNAi attenuated growth of SiHa cells. Overexpression of HPV16 oncoproteins viz, E6 and E7 increased NFAT2 expression levels and DNA binding activity, while knockdown of E6 by RNAi decreased the same. Briefly, we now report an activation of calcineurin-NFAT2 axis in cervical cancer and a novel role of HPV oncoprotein in facilitating NFAT2 dependent cell proliferation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MiR-138/SIRT1 axis is implicated in impaired learning and memory abilities of cerebral ischemia/reperfusion injured rats

The present study aimed to explore whether deregulated miR-138 is implicated in cerebral I/R injury-impaired learning and memory abilities. Rats were subjected to bilateral common carotid occlusion followed by reperfusion to induce cerebral I/R injury. A model of oxygen-glucose deprivation and reperfusion (OGD/R) was conducted to mimic cerebral I/R conditions in vitro. MiR-138 expression levels were reduced in the hippocampus of cerebral I/R injured rats. Inhibition of miR-138 ameliorated the impaired learning and memory abilities of rats, and promoted autophagy and thus attenuated apoptosis in the OGD/R-treated hippocampal neurons. Moreover, miR-138 targets the 3′-UTR of SIRT1 and repressed its expression. These results showed that miR-138 could improve the learning and memory abilities via promoting autophagy under cerebral I/R injured conditions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Up-regulation of miR-122 protects against neuronal cell death in ischemic stroke through the heat shock protein 70-dependent NF-κB pathway by targeting FOXO3

Highlights: • miR-122 was decreased in mouse N2A neuroblastoma (N2A) cells after OGD and mouse brain after MCAO. • Gain-of-miR-122 function significantly suppressed OGD- and MCAO-induced injures in vitro and in vivo. • miR-122 was validated to directly bind to the predicted 3′-untranslated region (3′-UTR) of FOXO3. • The inhibitory effects of miR-122 on ischemic injury in vitro and in vivo were overturned by FOXO3 overexpression and miR-122-FOXO3 axis functioned via the heat shock protein 70 (HSP-70)-mediated NF-κB pathway. • miR-122 inhibits ischemic neuronal death through HSP-70-dependent NF-κB pathway by targeting FOXO3. Dysfunction of the microRNA (miRNA) network has been emerging as a main regulator in ischemic stroke. Recently, studies have linked the deregulation of miR-122 to ischemic stroke. However, the specific role and molecular mechanism of miR-122 in ischemic stroke remain to be further investigated. Here, we found that miR-122 was decreased in mouse N2A neuroblastoma (N2A) cells after oxygen-glucose deprivation (OGD) and mouse brain after transient middle cerebral artery occlusion (MCAO). OGD treatment significantly increased N2A cell death and Caspase-3 activity, and decreased Bcl-2 protein expression. In addition, MCAO treatment induced severe mouse brain infarction, mitochondrial reactive oxygen species (ROS) production, and long-term neurological deficit. Gain-of-miR-122 function significantly suppressed OGD- and MCAO-induced injures in vitro and in vivo. Subsequently, miR-122 was validated to directly bind to the predicted 3′-untranslated region (3′-UTR) of FOXO3 gene, and the inhibitory effects of miR-122 on ischemic injury in vitro and in vivo were overturned by FOXO3 overexpression. Moreover, our results further revealed that miR-122-FOXO3 axis functioned via the heat shock protein 70 (HSP-70)-mediated NF-κB pathway. Collectively, our data suggest that miR-122 inhibits ischemic neuronal death through the HSP-70-dependent NF-κB pathway by targeting FOXO3. These findings raise the possibility that this regulatory net may contribute to the pathogenesis of the ischemic brain injury in stroke.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

MicroRNA-92a -mediated endothelial to mesenchymal transition controls vein graft neointimal lesion formation

Highlights: • TGF-β triggers microRNA-92a mediated EndMT by targeting ITGA5 is key role in vein graft vascular remodeling. • Silencing of microRNA-92a-mediated endothelial to mesenchymal transition inhibits vein graft neointimal hyperplasia. • Local adeno-associated virus delivery gene therapy deregulation of microRNA-92a-mediated EndMT may represent promising therapeutic approach for vein grafts failure. Long-term failure of vein grafts due to neointimal hyperplasia remains an important problem in coronary artery bypass graft surgery. Endothelial to mesenchymal transition (EndMT) contributes to vein graft vascular remodeling. However, there is little study on microRNA-mediated EndMT contributions to neointimal formation in vein graft. We hypothesized that microRNA-92a (miR-92a) might play an important role in determining EndMT contributions to neointimal formation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Roles of miR-204 in retinal development and maintenance

Highlights: • miR-204 plays an important role in the development, maintenance, and diseases of the retina. • Various layers of the retina including RPE express miR-204. • Retinoblastoma, age-related macular degeneration, glaucoma and optic nerve injury are among the most common retinopathies caused by miR-204 dysfunction. • miR-204 target genes are involved in diverse signaling pathways crucial for retinal physiology. The retina is the innermost part of the eye of most vertebrates and it is essential for vision. The development, maintenance, and function of this laminated structure is tightly regulated by numerous genes. Deficiencies in the expression of these genes as well as deregulation of various molecular mechanisms can cause retinopathies and blindness. MicroRNAs (miRNAs) are one of the most important and effective molecular regulatory mechanisms that underlie the biology of the retina. miRNAs have specific functional roles in the development and maintenance of different retinal layers and retinal cell types. While previous studies have reported a large number of miRNAs linked to development, maintenance and diseases of the retina, no comprehensive study has properly discussed and integrated data from these studies. Given the particular importance of miR-204 in retinal biology, we intend to critically discuss the expression and functional significance of this miRNA in the development, maintenance, and pathologies of the retina. Moreover, we explore biological processes through which miR-204 influences retinal pathophysiology. This review highlights the crucial functions of miR-204 in the retina and suggests the putative mechanism of miR-204 action in retinal biology.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Isoform-specific functions of synaptopodin-2 variants in cytoskeleton stabilization and autophagy regulation in muscle under mechanical stress

Highlights: • Muscle cells express different synaptopodin-2 isoforms with individual functions. • Reduced SYNPO2 expression results in enhanced vulnerability of myofibrils. • SYNPO2e recruites BAG3 to mechanically stressed myofibrillar areas. • SYNPO2 is an organizer and regulator of BAG3-containing proteostasis systems. • BAG3 forms functionally distinct protein complexes with different SYNPO2 isoforms. Protein homeostasis (proteostasis) in multicellular organisms depends on the maintenance of force-bearing and force-generating cellular structures. Within myofibrillar Z-discs of striated muscle, isoforms of synaptopodin-2 (SYNPO2/myopodin) act as adapter proteins that are engaged in proteostasis of the actin-crosslinking protein filamin C (FLNc) under mechanical stress. SYNPO2 directly binds F-actin, FLNc and α-actinin and thus contributes to the architectural features of the actin cytoskeleton. By its association with autophagy mediating proteins, i.e. BAG3 and VPS18, SYNPO2 is also engaged in protein quality control and helps to target mechanical unfolded and damaged FLNc for degradation. Here we show that deficiency of all SYNPO2-isoforms in myotubes leads to decreased myofibrillar stability and deregulated autophagy under mechanical stress. In addition, isoform-specific proteostasis functions were revealed. The PDZ-domain containing variant SYNPO2b and the shorter, PDZ-less isoform SYNPO2e both localize to Z-discs. Yet, SYNPO2e is less stably associated with the Z-disc than SYNPO2b, and is dynamically transferred into FLNc-containing myofibrillar lesions under mechanical stress. SYNPO2e also recruits BAG3 into these lesions via interaction with the WW domain of BAG3. Our data provide evidence for a role of myofibrillar lesions as a transient quality control compartment essential to prevent and repair contraction-induced myofibril damage in muscle and indicate an important coordinating activity for SYNPO2 therein.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Engineering improved ethylene production: Leveraging systems biology and adaptive laboratory evolution

Ethylene is a small hydrocarbon gas widely used in the chemical industry. Annual worldwide production currently exceeds 150 million tons, producing considerable amounts of CO 2 contributing to climate change. The need for a sustainable alternative is therefore imperative. Ethylene is natively produced by several different microorganisms, including Pseudomonas syringae pv. phaseolicola via a process catalyzed by the ethylene forming enzyme (EFE), subsequent heterologous expression of EFE has led to ethylene production in non-native bacterial hosts including E. coli and cyanobacteria. However, solubility of EFE and substrate availability remain rate limiting steps in biological ethylene production. We employed a combination of genome scale metabolic modelling, continuous fermentation, and protein evolution to enable the accelerated development of a high efficiency ethylene producing E. coli strain, yielding a 49-fold increase in production, the most significant improvement reported to date. Furthermore, we have clearly demonstrated that this increased yield resulted from metabolic adaptations that were uniquely linked to the EFE enzyme (WT vs mutant). Our findings provide a novel solution to deregulate metabolic bottlenecks in key pathways, which can be readily applied to address other engineering challenges.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Biochemical Characterization and Structure-Based Mutational Analysis Provide Insight into the Binding and Mechanism of Action of Novel Aspartate Aminotransferase Inhibitors

Pancreatic cancer cells are characterized by deregulated metabolic programs that facilitate growth and resistance to oxidative stress. Among these programs, pancreatic cancers preferentially utilize a metabolic pathway through the enzyme aspartate aminotransferase 1 [also known as glutamate oxaloacetate transaminase 1 (GOT1)] to support cellular redox homeostasis. As such, small molecule inhibitors that target GOT1 could serve as starting points for the development of new therapies for pancreatic cancer. We ran a high-throughput screen for inhibitors of GOT1 and identified a small molecule, iGOT1-01, with in vitro GOT1 inhibitor activity. Application in pancreatic cancer cells revealed metabolic and growth inhibitory activity reflecting a promiscuous inhibitory profile. We then performed an in silico docking analysis to study inhibitor–GOT1 interactions with iGOT1-01 analogues that possess improved solubility and potency properties. Here, these results suggested that the GOT1 inhibitor competed for binding to the pyridoxal 5-phosphate (PLP) cofactor site of GOT1. To analyze how the GOT1 inhibitor bound to GOT1, a series of GOT1 mutant enzymes that abolished PLP binding were generated. Application of the mutants in X-ray crystallography and thermal shift assays again suggested but were unable to formally conclude that the GOT1 inhibitor bound to the PLP site. Mutational studies revealed the relationship between PLP binding and the thermal stability of GOT1 while highlighting the essential nature of several residues for GOT1 catalytic activity. Insight into the mode of action of GOT1 inhibitors may provide leads to the development of drugs that target redox balance in pancreatic cancer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling

Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). In this paper, we report on structure–activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at submicromolar concentrations in vitro, increase levels of active RAS–GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase–extracellular regulated kinase (MAPK–ERK) pathway, resulting in a decrease in pERK1/2 T202/Y204 protein levels at higher compound concentrations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Coupling High-Throughput and Targeted Screening for Identification of Nonobvious Metabolic Engineering Targets

Identification of metabolic engineering targets is a fundamental challenge in strain development programs. While high-throughput (HTP) genetic engineering methodologies capable of generating vast diversity are being developed at a rapid rate, a majority of industrially interesting molecules cannot be screened at sufficient throughput to leverage these techniques. We propose a workflow that couples HTP screening of common precursors (e.g., amino acids) that can be screened either directly or by artificial biosensors, with low-throughput targeted validation of the molecule of interest to uncover nonintuitive beneficial metabolic engineering targets and combinations hereof. Using this workflow, we identified several nonobvious novel targets for improving p-coumaric acid (p-CA) and L -DOPA production from two large 4k gRNA libraries each deregulating 1000 metabolic genes in the yeast Saccharomyces cerevisiae. We initially screened yeast cells transformed with gRNA library plasmids for individual regulatory targets improving the production of l-tyrosine-derived betaxanthins, identifying 30 targets that increased intracellular betaxanthin content 3.5–5.7 fold. Hereafter, we screened the targets individually in a high-producing p-CA strain, narrowing down the targets to six that increased the secreted titer by up to 15%. To investigate whether any of the six targets could be additively combined to improve p-CA production further, we created a gRNA multiplexing library and subjected it to our proposed coupled workflow. The combination of regulating PYC1 and NTH2 simultaneously resulted in the highest (threefold) improvement of the betaxanthin content, and an additive trend was also observed in the p-CA strain. Lastly, we tested the initial 30 targets in a L -DOPA producing strain, identifying 10 targets that increased the secreted titer by up to 89%, further validating our screening by proxy workflow. This coupled approach is useful for strain development in the absence of direct HTP screening assays for products of interest.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling

Deregulation of the Ras–mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes. Kinase suppressor of Ras (KSR) is a MAPK scaffold that is subject to allosteric regulation through dimerization with RAF. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. In conclusion, these results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras–MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A

S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Dynamics of the human gut microbiome in inflammatory bowel disease

Inflammatory bowel disease (IBD) is characterized by flares of inflammation with periodic need for increased medication and sometimes even surgery. IBD etiology is partly attributed to a deregulated immune response to gut microbiome dysbiosis. Cross-sectional studies have revealed microbial signatures for different IBD diseases, including ulcerative colitis (UC), colonic Crohn’s Disease (CCD), and ileal CD (ICD). Although IBD is dynamic, microbiome studies have primarily focused on single timepoints or few individuals. Here we dissect the long-term dynamic behavior of the gut microbiome in IBD and differentiate this from normal variation. Microbiomes of IBD subjects fluctuate more than healthy individuals, based on deviation from a newly-defined healthy plane (HP). ICD subjects deviated most from the HP, especially subjects with surgical resection. Intriguingly, the microbiomes of some IBD subjects periodically visited the HP then deviated away from it. Inflammation was not directly correlated with distance to the healthy plane, but there was some correlation between observed dramatic fluctuations in the gut microbiome and intensified medication due to a flare of the disease. These results help guide therapies that will re-direct the gut microbiome towards a healthy state and maintain remission in IBD.

gut, human↗
DOE OSTI2022

Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro

Abstract In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like protease (3CLpro) can cleave human immune signaling proteins, like NF-κB Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.50 Å resolution crystal structure of 3CLpro C145S bound to NEMO 226–234 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro-NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for, in the pathology of COVID-19.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Targeting of intracellular oncoproteins with peptide-centric CARs

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

60 APPLIED LIFE SCIENCES↗