ZBTB11 Depletion Targets Metabolic Vulnerabilities in K-Ras Inhibitor Resistant PDAC.

Over 95% of pancreatic ductal adenocarcinomas (PDAC) harbor oncogenic mutations in K-Ras. Upon treatment with K-Ras inhibitors, PDAC cancer cells undergo metabolic reprogramming towards an oxidative phosphorylation-dependent, drug-resistant state. However, direct inhibition of complex I is poorly tolerated in patients due to on-target induction of peripheral neuropathy. In this work, we develop molecular glue degraders against ZBTB11, a C2H2 zinc finger transcription factor that regulates the nuclear transcription of components of the mitoribosome and electron transport chain. Our ZBTB11 degraders leverage the differences in demand for biogenesis of mitochondrial components between human neurons and rapidly-dividing pancreatic cancer cells, to selectively target the K-Ras inhibitor resistant state in PDAC. Combination treatment of both K-Ras inhibitor-resistant cell lines and multidrug resistant patient-derived organoids resulted in superior anti-cancer activity compared to single agent treatment, while sparing hiPSC-derived neurons. Proteomic and stable isotope tracing studies revealed mitoribosome depletion and impairment of the TCA cycle as key events that mediate this response. Together, this work validates ZBTB11 as a vulnerability in K-Ras inhibitor-resistant PDAC and provides a suite of molecular glue degrader tool compounds to investigate its function.

Uterine cancer among Asian Americans - Disparities & clinical characteristics.

OBJECTIVE: To evaluate the patterns and trends of uterine cancer among Asian subgroups living in the U.S. METHODS: Data were obtained from United States Cancer Statistics (2001-2017), National Cancer Database (2004-2015), and World Population Review (2023). SEER*Stat version 8.3.9.2, Joinpoint regression program 4.9.0.0, and SAS v 9.4 were employed for statistical analysis. RESULTS: Based on data from 778,891 women in the United States Cancer Statistics database, Asians had a 3.4-fold higher rate of incident uterine cancer compared to White populations (2.14% vs. 0.63%; p < 0.001). Using the National Cancer Database, 7,641 Asian women from six subgroups were analyzed: Filipino, Korean, Indian/Pakistani, Vietnamese, Chinese, and Japanese. Indian and Pakistani women had the greatest increase in the proportion of cancer diagnoses (5.0% to 14.4%; p = 0.0003). Additionally, Indian and Pakistani patients had higher comorbidity scores while Koreans had the lowest (22.7% vs. 10.7%, p < 0.0001). Regarding stage of disease, 25.3% of Filipinos presented with advanced stage disease compared to 19.2% of Indians and Pakistanis (p = 0.0001). Furthermore, Filipinos had the highest proportion of non-endometrioid cancers at 18.4% compared to other subgroups (p = 0.0003). Using the World Population Review, female obesity was highest in Pakistan (8.6%) and the Philippines (7.5%) and lowest in Vietnam (2.6%). CONCLUSION: Uterine cancer incidence increased at higher rates among Asians compared to White populations. Specifically, Indian and Pakistani uterine cancer patients were more likely to have higher comorbidity rates and Filipino patients had more advanced stage cancer with non-endometrioid histologies than other Asian subgroups. Further research is warranted to better understand these trends.

The role of perceived health in retention disparity: A HIV-testing-related behavioral intervention among African American and Latinx men who have sex with men in the United States.

Retention in healthcare and health behavior remains a critical issue, contributing to inequitable distribution of intervention benefits. In diseases such as HIV, where half of the new infections occur among racial and sexual minorities, it is important that interventions do not enlarge pre-existing health disparities. To effectively combat this public health issue, it is crucial that we quantify the magnitude of racial/ethnic disparity in retention. Further, there is a need to identify mediating factors to this relationship to inform equitable intervention design. In the present study, we assess the racial/ethnic disparity in retention in a peer-led online behavioral intervention to increase HIV self-testing behavior and identify explanatory factors. The research used data collected from the Harnessing Online Peer Education (HOPE) HIV Study that included 899 primarily African American and Latinx men who have sex with men (MSM) in the United States. Results show that African American participants had higher lost-to-follow-up rates at 12-week follow-up compared to Latinx participants (11.1% and 5.8% respectively, Odds Ratio = 2.18, 95% confidence interval: 1.12 - 4.11, p = 0.02), which is substantially mediated by participants' self-rated health score (14.1% of the variation in the African American v.s. Latinx difference in lost-follow-up, p = 0.006). Thus, how MSM perceive their health may play an important role in their retention in HIV-related behavioral intervention programs and its racial/ethnic disparity.

Dual IKZF2 and CK1α degrader targets acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a hematologic malignancy for which several epigenetic regulators have been identified as therapeutic targets. Here we report the development of cereblon-dependent degraders of IKZF2 and casein kinase 1α (CK1α), termed DEG-35 and DEG-77. We utilized a structure-guided approach to develop DEG-35 as a nanomolar degrader of IKZF2, a hematopoietic-specific transcription factor that contributes to myeloid leukemogenesis. DEG-35 possesses additional substrate specificity for the therapeutically relevant target CK1α, which was identified through unbiased proteomics and a PRISM screen assay. Degradation of IKZF2 and CK1α blocks cell growth and induces myeloid differentiation in AML cells through CK1α-p53- and IKZF2-dependent pathways. Target degradation by DEG-35 or a more soluble analog, DEG-77, delays leukemia progression in murine and human AML mouse models. Overall, we provide a strategy for multitargeted degradation of IKZF2 and CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Targeted Protein Degradation: Design Considerations for PROTAC Development.

Targeted protein degradation has recently gained widespread interest as both a novel therapeutic strategy and a useful tool in biomedical research. Targeted protein degraders are often sub-stoichiometric and do not require strong binding affinity for their targets, enabling access to previously inaccessible targets. Proteolysis-targeting chimeras (PROTACs) are one class of targeted protein degraders that promote degradation by recruiting a target protein to an E3-ligase complex via a heterobifunctional molecule. The modular nature of PROTACs allows for their rational design and systematic optimization. Here we suggest resources and methodologies for developing PROTAC degraders for researchers that may be new to the field. © 2022 Wiley Periodicals LLC.

Revisiting social MPE: an integration of molecular pathological epidemiology and social science in the new era of precision medicine.

INTRODUCTION: Molecular pathological epidemiology (MPE) is an integrative transdisciplinary area examining the relationships between various exposures and pathogenic signatures of diseases. In line with the accelerating advancements in MPE, social science and its health-related interdisciplinary areas have also developed rapidly. Accumulating evidence indicates the pathological role of social-demographic factors. We therefore initially proposed social MPE in 2015, which aims to elucidate etiological roles of social-demographic factors and address health inequalities globally. With the ubiquity of molecular diagnosis, there are ample opportunities for researchers to utilize and develop the social MPE framework. AREAS COVERED: Molecular subtypes of breast cancer have been investigated rigorously for understanding its etiologies rooted from social factors. Emerging evidence indicates pathogenic heterogeneity of neurological disorders such as Alzheimer's disease. Presenting specific patterns of social-demographic factors across different molecular subtypes should be promising for advancing the screening, prevention, and treatment strategies of those heterogeneous diseases. This article rigorously reviewed literatures investigating differences of race/ethnicity and socioeconomic status across molecular subtypes of breast cancer and Alzheimer's disease to date. EXPERT OPINION: With advancements of the multi-omics technologies, we foresee a blooming of social MPE studies, which can address health disparities, advance personalized molecular medicine, and enhance public health.

Diverse changes in microglia morphology and axonal pathology during the course of 1 year after mild traumatic brain injury in pigs.

Over 2.8 million people experience mild traumatic brain injury (TBI) in the United States each year, which may lead to long-term neurological dysfunction. The mechanical forces that are caused by TBI propagate through the brain to produce diffuse axonal injury (DAI) and trigger secondary neuroinflammatory cascades. The cascades may persist from acute to chronic time points after injury, altering the homeostasis of the brain. However, the relationship between the hallmark axonal pathology of diffuse TBI and potential changes in glial cell activation or morphology have not been established in a clinically relevant large animal model at chronic time points. In this study, we assessed the tissue from pigs subjected to rapid head rotation in the coronal plane to generate mild TBI. Neuropathological assessments for axonal pathology, microglial morphological changes, and astrocyte reactivity were conducted in specimens out to 1-year post-injury. We detected an increase in overall amyloid precursor protein pathology, as well as periventricular white matter and fimbria/fornix pathology after a single mild TBI. We did not detect the changes in corpus callosum integrity or astrocyte reactivity. However, detailed microglial skeletal analysis revealed changes in morphology, most notably increases in the number of microglial branches, junctions, and endpoints. These subtle changes were most evident in periventricular white matter and certain hippocampal subfields, and were observed out to 1-year post-injury in some cases. These ongoing morphological alterations suggest persistent change in neuroimmune homeostasis. Additional studies are needed to characterize the underlying molecular and neurophysiological alterations, as well as potential contributions to neurological deficits.

In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases.

Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synthases. We employ apocytochrome c and peptide analogs containing CXXCH as substrates, examining recognition determinants, thioether attachment, and subsequent release and folding of cytochrome c. Peptide analogs reveal very different recognition requirements between HCCS and CcsBA. For HCCS, a minimal 16-mer peptide is required, comprised of CXXCH and adjacent alpha helix 1, yet neither thiol is critical for recognition. For bacterial CcsBA, both thiols and histidine are required, but not alpha helix 1. Heme attached peptide analogs are not released from the HCCS active site; thus, folding is important in the release mechanism. Peptide analogs behave as inhibitors of cytochrome c biogenesis, paving the way for targeted control.

From tiny bacteria to the tallest trees, most life on Earth carries a protein called cytochrome c, which helps to create the energy that powers up cells. Cytochrome c does so thanks to its heme, a molecule that enables the chemical reactions required for the energy-creating process. Despite both relying on cytochrome c, animals and bacteria differ in the enzyme they use to attach the heme to the cytochrome. Spotting variations in how this 'cytochrome c synthase' works would help to find compounds that deactivate the enzyme in bacteria, but not in humans. However, studying cytochrome c synthase in living cells is challenging. To bypass this issue, Sutherland, Mendez, Babbitt et al. successfully reconstituted cytochrome c synthases from humans and bacteria in test tubes. This allowed them to examine in detail which structures the enzymes recognize to spot where to attach the heme onto their target. The experiments revealed that human and bacterial synthases actually rely on different parts of the cytochrome c to orient themselves. Different short compounds could also block either the human or bacterial enzyme. Variations between human and bacterial cytochrome c synthase could lead to new antibiotics which deactivate the cytochrome and kill bacteria while sparing patients. The next step is to identify molecules that specifically interfere with cytochrome c synthase in bacteria, and could be tested in clinical trials.

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba.

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain's frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen's high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

Structure-Function Analysis of the Bifunctional CcsBA Heme Exporter and Cytochrome c Synthetase.

Although intracellular heme trafficking must occur for heme protein assembly, only a few heme transporters have been unequivocally discovered and nothing is known about their structure or mechanisms. Cytochrome c biogenesis in prokaryotes requires the transport of heme from inside to outside for stereospecific attachment to cytochrome c via two thioether bonds (at CXXCH). The CcsBA integral membrane protein was shown to transport and attach heme (and thus is a cytochrome c synthetase), but the structure and mechanisms underlying these two activities are poorly understood. We employed a new cysteine/heme crosslinking tool that traps endogenous heme in heme binding sites. We combined these data with a comprehensive imidazole correction approach (for heme ligand interrogation) to map heme binding sites. Results illuminate the process of heme transfer through the membrane to an external binding site (called the WWD domain). Using meta-genomic data (GREMLIN) and Rosetta modeling programs, a structural model of the transmembrane (TM) regions in CcsBA were determined. The heme mapping data were then incorporated to model the TM heme binding site (with TM-His1 and TM-His2 as ligands) and the external heme binding WWD domain (with P-His1 and P-His2 as ligands). Other periplasmic structure/function studies facilitated modeling of the full CcsBA protein as a framework for understanding the mechanisms. Mechanisms are proposed for heme transport from TM-His to WWD/P-His and subsequent stereospecific attachment of heme. A ligand exchange of the P-His1 for histidine of CXXCH at the synthetase active site is suggested.IMPORTANCE The movement or trafficking of heme is critical for cellular functions (e.g., oxygen transport and energy production); however, intracellular heme is tightly regulated due to its inherent cytotoxicity. These factors, combined with the transient nature of transport, have resulted in a lack of direct knowledge on the mechanisms of heme binding and trafficking. Here, we used the cytochrome c biogenesis system II pathway as a model to study heme trafficking. System II is composed of two integral membrane proteins (CcsBA) which function to transport heme across the membrane and stereospecifically position it for covalent attachment to apocytochrome c We mapped two heme binding domains in CcsBA and suggest a path for heme trafficking. These data, in combination with metagenomic coevolution data, are used to determine a structural model of CcsBA, leading to increased understanding of the mechanisms for heme transport and the cytochrome c synthetase function of CcsBA.