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DOE OSTI2025

An in vitro BRAF activation assay elucidates molecular mechanisms driving disassembly of the autoinhibited BRAF state

The RAF kinases (ARAF, BRAF, and CRAF) are essential components of the RAS-ERK signaling pathway, which controls vital cellular processes and is frequently dysregulated in human disease. Notably, mutations that alter BRAF function are prominent drivers of human cancer and certain RASopathy disorders, making BRAF an important target for therapeutic intervention. Despite extensive research, several aspects of BRAF regulation remain unclear. In this study, we developed an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-3 2 :MEK complexes. Our results show that fully processed, active-state KRAS alone can promote dimer-dependent BRAF activation. Moreover, we found that phosphatidylserine (PS)-containing liposomes synergized with KRAS to promote BRAF activation, achieving activity levels comparable to those observed with BRAF proteins that constitutively dimerize. In contrast, the SMP phosphatase complex had only a minimal effect on BRAF catalytic activity in this system but mediated the dephosphorylation of the negative regulatory pS365 14-3-3 binding site in a manner that was accelerated by the presence of KRAS alone or KRAS and 30% PS liposomes. Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential.

BRAF↗

DOE OSTI2026

Discovery of BBO-11818, a Potent and Selective Noncovalent Inhibitor of (ON) and (OFF) KRAS with Activity against Multiple Oncogenic Mutants

Although KRAS G12C -specific inhibitors have been introduced, no approved targeted therapies exist for other clinically significant KRAS mutants, including KRAS G12D and KRAS G12V . We discovered BBO-11818, a potent, selective, orally bioavailable noncovalent pan-KRAS inhibitor capable of targeting multiple KRAS mutants in both the inactive GDP-bound (OFF) and active GTP-bound (ON) states. BBO-11818 binds in the Switch-II/Helix 3 pocket, inducing conformational changes incompatible with effector binding, and demonstrates high-affinity binding to mutant KRAS with strong selectivity over NRAS and HRAS. BBO-11818 potently inhibited MAPK signaling and cellular viability specifically in KRAS-driven lines and produced tumor regressions in KRAS-mutant xenograft models. Combination studies with anti–PD-1, anti-EGFR antibodies, and a RAS:PI3Kα breaker compound showed enhanced efficacy. BBO-11818 has entered phase I clinical trials for patients with various KRAS mutations in colorectal, pancreatic, and lung cancers (NCT06917079).

Stahlhut, Carlos [BridgeBio Oncology Therapeutics,↗

DOE OSTI2023

Radiation biology workforce in the United States

In recent decades, the principal goals of participants in the field of radiation biologists have included defining dose thresholds for cancer and non-cancer endpoints to be used by regulators, clinicians and industry, as well as informing on best practice radiation utilization and protection applications. Importantly, much of this work has required an intimate relationship between “bench” radiation biology scientists and their target audiences (such as physicists, medical practitioners and epidemiologists) in order to ensure that the requisite gaps in knowledge are adequately addressed. However, despite the growing risk for public exposure to higher-than-background levels of radiation, e.g. from long-distance travel, the increasing use of ionizing radiation during medical procedures, the threat from geopolitical instability, and so forth, there has been a dramatic decline in the number of qualified radiation biologists in the U.S. Contributing factors are thought to include the loss of applicable training programs, loss of jobs, and declining opportunities for advancement. This report was undertaken in order to begin addressing this situation since inaction may threaten the viability of radiation biology as a scientific discipline.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

DOE OSTI2023

Restrictive spirometry pattern among construction trade workers

Spirometry-based studies of occupational lung disease have mostly focused on obstructive or mixed obstructive/restrictive outcomes. We wanted to determine if restrictive spirometry pattern (RSP) is associated with occupation and increased mortality. Study participants included 18,145 workers with demographic and smoking data and repeatable spirometry. The mortality analysis cohort included 15,445 workers with known vital status and cause of death through December 31, 2016. Stratified analyses explored RSP prevalence by demographic and clinical variables and trade. Log-binomial regression models explored RSP risk factors while controlling for important confounders such as smoking, obesity, and comorbidities. Cox regression models explored mortality risk by spirometry category. Prevalence of RSP was very high (28.6%). Mortality hazard ratios for RSP were 1.50 for all causes, 1.86 for cardiovascular diseases, 2.31 for respiratory diseases, and 1.66 for lung cancer. All construction trades except painters, machinists, and roofers had significantly elevated risk for RSP compared to our internal reference group. RSP was significantly associated with both parenchymal and pleural changes seen by chest X-ray. Construction trade workers are at significantly increased risk for RSP independent of obesity. Individuals with RSP are at increased risk for all-cause mortality as well as mortality attributable to respiratory diseases, cardiovascular diseases, and lung cancer. RSP deserves greater attention in occupational medicine and epidemiology.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Codelivery of Paclitaxel and Parthenolide in Discoidal Bicelles for a Synergistic Anticancer Effect: Structure Matters

Nanotechnology has long been used to improve the tumor targeting of chemotherapeutics. The structure of these nanocarriers greatly affects their efficacy and toxicity. Herein, structurally stable nanodiscoidal phospholipid bicelles, which are capable of encapsulating anticancer drugs, paclitaxel (PTX) and parthenolide (PTL), are designed. This nanodiscoidal bicelle structure is shown previously to exhibit longer blood circulation, deeper tumor penetration, and increased tumor cell uptake compared with spherical particles of similar compositions. Structural characterization and loading capacity of the PTX‐ and PTL‐ loaded nanodiscs are conducted along with an investigation into the cytotoxicity of the bicelles based on the drug‐to‐lipid ratios of both drugs loaded separately and in combination. A significant synergistic effect between both loaded drugs is also observed in A549 lung cancer and drug‐resistant MDA‐MB‐231 cell lines. An in vivo study using mice bearing subcutaneous A549 tumors shows a long circulation time of the intravenously injected DiR‐loaded bicelles and significant antitumor efficacy of the PTX‐ and PTL‐loaded bicelles. This efficacious self‐assembled nanoparticle system displays great potential as a nanoplatform to codeliver multiple therapeutics or diagnostics for improving cancer therapy.

Rad, Armin Tahmasbi↗

DOE OSTI2023

Crystal Structure of an i‐Motif from the HRAS Oncogene Promoter

Abstract An i‐motif is a non‐canonical DNA structure implicated in gene regulation and linked to cancers. The C‐rich strand of the HRAS oncogene, 5′‐CG CCC GTG CCC TGCG CCC GCAA CCC GA‐3′ (herein referred to as iHRAS), forms an i‐motif in vitro but its exact structure was unknown. HRAS is a member of the RAS proto‐oncogene family. About 19 % of US cancer patients carry mutations in RAS genes. We solved the structure of iHRAS at 1.77 Å resolution. The structure reveals that iHRAS folds into a double hairpin. The two double hairpins associate in an antiparallel fashion, forming an i‐motif dimer capped by two loops on each end and linked by a connecting region. Six C−C + base pairs form each i‐motif core, and the core regions are extended by a G−G base pair and a cytosine stacking. Extensive canonical and non‐canonical base pairing and stacking stabilizes the connecting region and loops. The iHRAS structure is the first atomic resolution structure of an i‐motif from a human oncogene. This structure sheds light on i‐motifs folding and function in the cell.

Li, Kevin S.↗

DOE OSTI2023

Crystal Structure of an i‐Motif from the HRAS Oncogene Promoter

Li, Kevin S.↗

DOE OSTI2023

Tumor‐pH‐value responsive non‐peripheral substituted phthalocyanines: Synthesis, investigation of photophysical and photochemical properties

Cancer is one of the diseases with the highest mortality rate worldwide. Although PDT has recently produced encouraging outcomes, there are still many areas that need to be improved. The first of these is the negative consequences faced by patients treated with the PDT method when exposed to sunlight. For this reason, a new PDT method has been developed in recent years, and it is aimed at using photosensitizer molecules that can be active in acidic conditions. Since the pH values of tumor tissues are more acidic than normal tissues, preparing molecules that act effectively in acidic conditions will allow for more effective results in treating cancer with PDT. In this context, within the scope of this study, 3‐(4‐propionylphenoxy)phthalonitrile ( 1 ) and its non‐peripheral tetra‐substituted phthalocyanine derivatives [( 2 ), ( 3 ), and ( 4 )] were prepared. With these phthalocyanine derivatives, the novel compounds ( 5 ), ( 6 ), and ( 7 ) were synthesized for the first time. The aggregation tendencies of newly synthesized phthalocyanines ( 5–7 ) were investigated in solvent media. The effects of pH changes upon UV–Vis and fluorescence spectra were performed. The electronic and emission spectra of synthesized phthalocyanine derivatives are highly sensitive to pH changes. Formation constant (Log K) values of mono‐ and di‐protonated phthalocyanine forms were calculated by the Henderson–Hasselback equation. The mono‐ and di‐protonated species' equilibrium constants (logK 1 and logK 2 ) were calculated as ~5.0. This value may be promising for pH‐sensitizing photosensitizers. Also, the photophysical and photochemical properties of synthesized metallophthalocyanine derivatives ( 2 ) and ( 5 ) were studied at different pH values. The singlet oxygen quantum yield of ( 2 ) and ( 5 ) was calculated to be 0.78 and 0.81 in DMSO, respectively. When pH = 6.4, that is, tumor‐pH‐values, this value for ( 5 ) has increased to 0.92. The newly synthesized phthalocyanines are suitable photosensitizers for PDT applications, especially with high singlet oxygen quantum yield at pH 6.4.

Genc Bilgicli, Hayriye↗

DOE OSTI2022

Development of a prediction model for radiotherapy response among patients with head and neck squamous cell carcinoma based on the tumor immune microenvironment and hypoxia signature

Abstract Introduction The immune system and hypoxia are major factors influencing radiosensitivity in patients with different cancer types. This study aimed at developing a model to predict radiotherapy response in patients with head and neck squamous cell carcinoma (HNSCC) based on the tumor immune microenvironment and hypoxia signature. Materials and Methods We first evaluated the hypoxia status and tumor immune microenvironment in the Cancer Genome Atlas (TCGA) cohort by using transcriptomic data. Differentially expressed genes (DEGs) were identified between the “high immunity and low hypoxia” and “low immunity and high hypoxia” groups and those DEGs significantly associated with disease‐specific survival in the univariate Cox regression analysis were selected as the prognostic DEGs. We selected the immune hypoxia–related genes (IHRGs) by intersecting prognostic DEGs with immune and hypoxia gene sets. We used the IHRGs to train a multivariate Cox regression model in the TCGA cohort, based on which we calculated the IHRG prognostic index (IHRGPI) for each patient and validated its efficacy in predicting radiotherapy response in the Gene Expression Omnibus cohorts. Furthermore, we explored potential mechanisms and effective combinational treatment strategies for different IHRGPI groups. Results Five IHRGs were used to construct the IHRGPI, which was used to dichotomize the cohorts. The patients with lower IHRGPI showed a better radiotherapy response across different cohorts and endpoints, including overall survival, progression‐free survival, and recurrence‐free survival ( p < 0.05). Patients with higher IHRGPI showed greater hypoxia and lesser immune cell infiltration. A lower IHRGPI indicated a better immunotherapy response, while a higher IHRGPI indicated a better chemotherapy response. Conclusions IHRGPI is promising for predicting radiotherapy response and guiding combinational treatment strategies in patients with HNSCC.

Zhu, Guang‐Li↗

DOE OSTI2020

Validation of Stage N3 of the Eighth Edition AJCC Staging System for Nasopharyngeal Carcinoma

Objectives To validate stage nodal (N)3 of the 8th edition American Joint Committee on Cancer (AJCC) staging system for nasopharyngeal carcinoma (NPC). Methods This retrospective cohort study extracted NPC patients from the Surveillance, Epidemiology, and End Results database between 2004 and 2016. Pathologically confirmed patients with complete data of level IV, N3a, and N3b lymph node metastasis were investigated. The included patients were divided into level IV, N3a, and N3b groups. Five‐year overall survival (OS) and cancer‐specific survival (CSS) were compared among the three groups. Results A total of 693 patients were included: 285 (41.13%) patients in the level IV group, 124 (17.89%) patients in the N3a group, and 284 (40.98%) patients in the N3b group. The 5‐year OS (57.1%, 55.0%, and 55.2%) and CSS (64.4%, 63.5%, and 64.4%) were not different among the level IV, N3a, and N3b groups. Multivariate regression analysis revealed that N stage was not an independent prognostic factor for OS (hazard ratio [HR] = 1.03, 95% confidence interval [CI]: 0.91–1.17; P = .65) or CSS (HR = 1.03, 95% CI: 0.89–1.19; P = .70). Conclusion Stage N3 of the 8th edition AJCC staging system for NPC is reasonable. Level of Evidence III Laryngoscope , 131:535–540, 2021

Pan, Xin‐Bin↗

DOE OSTI2021

Assessment of task‐based performance from five clinical DBT systems using an anthropomorphic breast phantom

Purpose Digital breast tomosynthesis (DBT) is a limited‐angle tomographic breast imaging modality that can be used for breast cancer screening in conjunction with full‐field digital mammography (FFDM) or synthetic mammography (SM). Currently, there are five commercial DBT systems that have been approved by the U.S. FDA for breast cancer screening, all varying greatly in design and imaging protocol. Because the systems are different in technical specifications, there is a need for a quantitative approach for assessing them. In this study, the DBT systems are assessed using a novel methodology with an inkjet‐printed anthropomorphic phantom and four alternative forced choice (4AFC) study scheme. Method A breast phantom was fabricated using inkjet printing and parchment paper. The phantom contained 5‐mm spiculated masses fabricated with potassium iodide (KI)‐doped ink and microcalcifications (MCs) made with calcium hydroxyapatite. Images of the phantom were acquired on all five systems with DBT, FFDM, and SM modalities where available using beam settings under automatic exposure control. A 4AFC study was conducted to assess reader performance with each signal under each modality. Statistical analysis was performed on the data to determine proportion correct (PC), standard deviations, and levels of significance. Results For masses, overall detection was highest with DBT. The difference in PC was statistically significant between DBT and SM for most systems. A relationship was observed between increasing PC and greater gantry span. For MCs, performance was highest with DBT and FFDM compared to SM. The difference between PC of DBT and PC of SM was statistically significant for all manufacturers. Conclusions This methodology represents a novel approach for evaluating systems. This study is the first of its kind to use an inkjet‐printed anthropomorphic phantom with realistic signals to assess performance of clinical DBT imaging systems.

Ikejimba, Lynda C.↗

DOE OSTI2025

Integrated structural model of the palladin–actin complex using XL ‐ MS , docking, NMR , and SAXS

Abstract Palladin is an actin‐binding protein that accelerates actin polymerization and is linked to the metastasis of several types of cancer. Previously, three lysine residues in an immunoglobulin‐like domain of palladin have been identified as essential for actin binding. However, it is still unknown where palladin binds to F‐actin. Evidence that palladin binds to the sides of actin filaments to facilitate branching is supported by our previous study showing that palladin was able to compensate for Arp2/3 in the formation of Listeria actin comet tails. Here, we used chemical crosslinking to covalently link palladin and F‐actin residues based on spatial proximity. Samples were then enzymatically digested, separated by liquid chromatography, and analyzed by tandem mass spectrometry. Peptides containing the crosslinks and specific residues involved were then identified for input to the HADDOCK docking server to model the most likely binding conformation. Small‐angle x‐ray scattering was used to provide further insight into palladin flexibility and the binding interface, and NMR spectra identified potential interactions between palladin's Ig domains. Our final structural model of the F‐actin:palladin complex revealed how palladin interacts with and stabilizes F‐actin at the interface between two actin monomers. Three actin residues that were identified in this study also appear commonly in the actin‐binding interface with other proteins such as myotilin, myosin, and tropomodulin. An accurate structural representation of the complex between palladin and actin extends our understanding of palladin's role in promoting cancer metastasis through the regulation of actin dynamics.

Sargent, Rachel [Department of Chemistry and Bioch↗

DOE OSTI2025

An evolutionarily conserved tryptophan cage promotes folding of the extended RNA recognition motif in the hnRNPR ‐like protein family

Abstract The heterogeneous nuclear ribonucleoprotein (hnRNP) R‐like family is a class of RNA binding proteins in the hnRNP superfamily with diverse functions in RNA processing. Here, we present the 1.90 Å X‐ray crystal structure and solution NMR studies of the first RNA recognition motif (RRM) of human hnRNPR. We find that this domain adopts an extended RRM (eRRM1) featuring a canonical RRM with a structured N‐terminal extension (N ext ) motif that docks against the RRM and extends the β‐sheet surface. The adjoining loop is structured and forms a tryptophan cage motif to position the N ext motif for docking to the RRM. Combining mutagenesis, solution NMR spectroscopy, and thermal denaturation studies, we evaluate the importance of residues in the N ext –RRM interface and adjoining loop on eRRM folding and conformational dynamics. We find that these sites are essential for protein solubility, conformational ordering, and thermal stability. Consistent with their importance, mutations in the N ext –RRM interface and loop are associated with several cancers in a survey of somatic mutations in cancer studies. Sequence and structure comparison of the human hnRNPR eRRM1 to experimentally verified and predicted hnRNPR‐like proteins reveals conserved features in the eRRM.

Biochemistry & Molecular Biology↗

DOE OSTI2020

Cisplatin-Induced Alteration on Membrane Composition of A549 Cells Revealed by ToF-SIMS

Cisplatin has been clinically used for treatment of solid tumors such as non-small-cell lung cancer for decades. However, tumor resistance may be acquired with losing the antitumor activity of cisplatin. As cellular membrane is the first barrier that cisplatin has to overcome before its further action inside the cells, the membrane composition must play a vital role in the cisplatin uptake and excretion which further influences cisplatin sensitivity. In this work, we applied ToF-SIMS surface analysis combined with principle component analysis to distinguish the differences of cell membrane composition between non-small-cell lung cancer cells (A549) and its cisplatin resistant counterpart A549/DDP cells. The decreased phosphatidylcholine content and more abundant cholesterol were observed in the drug resistant cell surfaces, indicating the decreased membrane fluidity of A549/DDP cells. Moreover, we further compared membrane composition of A549 and A549/DDP cells after being treated with different concentrations of cisplatin. A higher composition level of proteins was discovered on all groups of A549/DDP cell membranes. The altered surface chemistry of cellular membranes induced by cisplatin indicate the significance of membrane structures in the drug resistance, which deserves further investigations to this regard.

Zhang, Yanyan↗

DOE OSTI2023

Hierarchically Templated Synthesis of 3D‐Printed Crosslinked Cyclodextrins for Lycopene Harvesting

Abstract Plants produce a wide range of bioactive phytochemicals, such as antioxidants and vitamins, which play crucial roles in aging prevention, inflammation reduction, and reducing the risk of cancer. Selectively harvesting these phytochemicals, such as lycopene, from tomatoes through the adsorption method is cost‐effective and energy efficient. In this work, a templated synthesis of 3D‐printed crosslinked cyclodextrin polymers featuring nanotubular structures for highly selective lycopene harvesting is reported. Polypseudorotaxanes formed by triethoxysilane‐based telechelic polyethylene glycols and α‐cyclodextrins (α‐CDs) are designed as the template to (1) synthetically access urethane‐based nanotubular structures at the molecular level, and (2) construct 3D‐printed architectures with designed macroscale voids. The polypseudorotaxane hydrogels showed good rheological properties for direct ink writing, and the 3D‐printed hydrogels were converted to the desired α‐CD polymer network through a three‐step postprinting transformation. The obtained urethane‐crosslinked α‐CD monoliths possess nanotubular structures and 3D‐printed voids. They selectively adsorb lycopene from raw tomato juice, protecting lycopene from photo‐ or thermo‐degradations. This work highlights the hierarchically templated synthesis approach in developing functional 3D‐printing materials by connecting the bottom‐up molecular assembly and synthesis with the top‐down 3D architecture control and fabrication.

Chemistry↗

DOE OSTI2021

Privacy-Preserving Knowledge Transfer with Bootstrap Aggregation of Teacher Ensembles

There is a need to transfer knowledge among institutions and organizations to save effort in annotation and labeling or in enhancing task performance. However, knowledge transfer is difficult because of restrictions that are in place to ensure data security and privacy. Institutions are not allowed to exchange data or perform any activity that may expose personal information. With the leverage of a differential privacy algorithm in a high-performance computing environment, we propose a new training protocol, Bootstrap Aggregation of Teacher Ensembles (BATE), which is applicable to various types of machine learning models. The BATE algorithm is based on and provides enhancements to the PATE algorithm, maintaining competitive task performance scores on complex datasets with underrepresented class labels.We conducted a proof-of-the-concept study of the information extraction from cancer pathology report data from four cancer registries and performed comparisons between four scenarios: no collaboration, no privacy-preserving collaboration, the PATE algorithm, and the proposed BATE algorithm. The results showed that the BATE algorithm maintained competitive macro-averaged F1 scores, demonstrating that the suggested algorithm is an effective yet privacy-preserving method for machine learning and deep learning solutions.

Yoon, Hong-Jun↗

DOE OSTI2025

Deformable phrase level attention: A flexible approach for improving AI based medical coding

Objective: Improving the AI-driven automated medical encoding of clinical text plays a vital role in gathering information on the occurrence of diseases to improve population-level health. This work presents a novel attention mechanism designed to enhance text classification models and ensure appropriate classification of medical concepts in unstructured electronic health records. Materials and Methods: We developed a deformable, phrase-level attention mechanism to identify important lexical word-level and contextual phrase-level information from clinical text documents. We evaluated conventional and transformer-based deep learning models that we extended with our attention mechanism on the extraction of critical cancer information (e.g., site, subsite, laterality, histology, behavior) from 629,908 electronic pathology reports and on the automated medical encoding of 52,722 hospital discharge summaries. Results: Transformer-based models with the deformable, phrase-level attention mechanism achieved the best performance on the extraction of critical cancer information from pathology reports. Conventional- and transformer-based models show similar or better performance than their baseline counterparts on the automated medical encoding of clinical documents. Discussion: The addition of phrase-level information allowed models extended with our proposed method to outperform standard word-level attention. Our method showed favorable properties for the real-world application in terms of model robustness and phenotyping. These results indicate that our method is promising for automated data harmonization for common data models. Conclusion: This work proposes a novel deformable, phrase-level attention mechanism that enhances text classification models in the extraction of medical concepts from clinical text documents. We demonstrate strong performances on two clinical text datasets and showcase real-world deployability of our method.

Automated medical encoding↗

DOE OSTI2023

Mitocans induce lipid flip-flop and permeabilize the membrane to signal apoptosis

Pancratistatin (PST) and narciclasine (NRC) are natural therapeutic agents that exhibit specificity toward the mitochondria of cancerous cells and initiate apoptosis. Unlike traditional cancer therapeutic agents, PST and NRC are effective, targeted, and have limited adverse effects on neighboring healthy, noncancerous cells. Currently, the mechanistic pathway of action for PST and NRC remains elusive, which in part inhibits PST and NRC from becoming efficacious therapeutic alternatives. Herein, we use neutron and x-ray scattering in combination with calcein leakage assays to characterize the effects of PST, NRC, and tamoxifen (TAM) on a biomimetic model membrane. We report an increase in lipid flip-flop half-times (t 1/2 ) (≈12.0%, ≈35.1%, and a decrease of ≈45.7%) with 2 mol percent PST, NRC, and TAM respectively. An increase in bilayer thickness (≈6.3%, ≈7.8%, and ≈7.8%) with 2 mol percent PST, NRC, and TAM, respectively, was also observed. Lastly, increases in membrane leakage (≈31.7%, ≈37.0%, and ≈34.4%) with 2 mol percent PST, NRC, and TAM, respectively, were seen. Considering the maintenance of an asymmetric lipid composition across the outer mitochondrial membrane (OMM) is crucial to eukaryotic cellular homeostasis and survival, our results suggest PST and NRC may play a role in disrupting the native distribution of lipids within the OMM. A possible mechanism of action for PST- and NRC-induced mitochondrial apoptosis is proposed via the redistribution of the native OMM lipid organization and through OMM permeabilization.

59 BASIC BIOLOGICAL SCIENCES↗