Discovery of a Highly Potent and Selective HDAC8 Degrader: Advancing the Functional Understanding and Therapeutic Potential of HDAC8.

HDAC8 plays crucial roles in biological processes, from gene regulation to cell motility, making it a highly desirable target for therapeutic intervention. HDAC8 also has deacetylase-independent activity which cannot be blocked by a conventional inhibitor. In this study, we report the discovery of YX862, a highly potent and selective hydrazide-based HDAC8-proteolysis targeting chimera (PROTAC) degrader. The selectivity is achieved through rational design of the warhead to spare HDAC3 activity from the previous HDAC3/8 dual degrader YX968. We demonstrate that the degradation of HDAC8 by YX862 increases acetylation levels of its nonhistone substrates such as SMC3 without significantly triggering histone PTM, supporting HDAC8's major role in nonhistone PTM regulation. YX862 exhibits promising on-target antiproliferative activity against DLBCL cells with higher potency than the HDAC8 selective inhibitor PCI-34051. As a selective HDAC8 degrader that avoids pan-HDAC inhibition, YX862 represents a valuable tool for exploring the biological and therapeutic potential of HDAC8.

Genome-wide CRISPR screening uncovers potential targets and mechanisms of vincristine resistance in DLBCL.

In this issue, Rovsing et al. employ unbiased genome-wide CRISPR screening and functional cellular assays to investigate the cellular response to vincristine, an important component of the front-line DLBCL treatment R-CHOP. Their findings reveal intriguing targets and mechanisms that hold promise for enhancing DLBCL treatment and provide a foundation for the development of future drug regimens. This research prompts further exploration of the translational potential to advance more effective and individualized approaches in the clinical management of DLBCL. Commentary on: Rovsing et al. Resistance to vincristine in DLBCL by disruption of p53-induced cell cycle arrest and apoptosis mediated by KIF18B and USP28. Br J Haematol 2023;202:825-839.

Do Epilepsy Patients with Cognitive Impairment Have Alzheimer's Disease-like Brain Metabolism?

Although not classically considered together, there is emerging evidence that Alzheimer's disease (AD) and epilepsy share a number of features and that each disease predisposes patients to developing the other. Using machine learning, we have previously developed an automated fluorodeoxyglucose positron emission tomography (FDG-PET) reading program (i.e., MAD), and demonstrated good sensitivity (84%) and specificity (95%) for differentiating AD patients versus healthy controls. In this retrospective chart review study, we investigated if epilepsy patients with/without mild cognitive symptoms also show AD-like metabolic patterns determined by the MAD algorithm. Scans from a total of 20 patients with epilepsy were included in this study. Because AD diagnoses are made late in life, only patients aged ≥40 years were considered. For the cognitively impaired patients, four of six were identified as MAD+ (i.e., the FDG-PET image is classified as AD-like by the MAD algorithm), while none of the five cognitively normal patients was identified as MAD+ (χ2 = 8.148, p = 0.017). These results potentially suggest the usability of FDG-PET in prognosticating later dementia development in non-demented epilepsy patients, especially when combined with machine learning algorithms. A longitudinal follow-up study is warranted to assess the effectiveness of this approach.

Implementation of Pre-Admission Caregiver Testing for COVID-19.

BACKGROUND: Caregivers are often at the bedside of hospitalized children posing an additional risk for coronavirus disease 2019 (COVID-19) transmission. We describe the implementation of caregiver COVID-19 testing before inpatient pediatric admissions and the effect on patient cohorting and bed capacity. METHODS: We implemented an ordering pathway to facilitate COVID-19 testing of caregivers of patients admitted to the inpatient units from the pediatric emergency department, elective procedural admissions, or direct admissions at a tertiary children's hospital in the Northeastern United States in August 2021. Testing was expedited by the clinical laboratory, and caregiver results were used to inform cohorting, infection prevention, and bed management decisions. RESULTS: From August 2021 to January 2022, 2558 caregiver tests were ordered through this pathway, and 83 (3.2%) were positive. Of the positive tests, 72 (86.7%) occurred after December 18, 2021, coinciding with the local Omicron variant wave. Among positives, 67 caregiver or child pairs were identified, and 36 positive caregivers had a COVID-19 negative child leading to use of isolation precautions. Reintroduction of patient cohorting increased overall bed capacity from 74% to 100% of available beds. CONCLUSIONS: The overall incidence of COVID-19 among caregivers before admission correlated well with rates of COVID-19 positivity among asymptomatic adults in the community during the study period. Implementation of caregiver testing increased bed capacity by reintroducing cohorting of patients and identified patients needing isolation that would have been missed by patient testing alone. More research is necessary to determine the extent that routine caregiver testing mitigates the risk of nosocomial severe acute respiratory syndrome coronavirus 2 transmission.

Longitudinal Assessment of Global and Regional Left Ventricular Strain in Patients with Multisystem Inflammatory Syndrome in Children (MIS-C).

Multisystem inflammatory syndrome in children (MIS-C) is one of the most significant sequela of coronavirus disease 2019 (COVID-19) in children. Emerging literature has described myocardial dysfunction in MIS-C patients using traditional and two-dimensional speckle tracking echocardiography in the acute phase. However, data regarding persistence of subclinical myocardial injury after recovery is limited. We aimed to detect these changes with deformation imaging, hypothesizing that left ventricular global longitudinal (GLS) and circumferential strain (GCS) would remain impaired in the chronic phase despite normalization of ventricular function parameters assessed by two-dimensional echocardiography. A retrospective, single-institution review of 22 patients with MIS-C was performed. Fractional shortening, GLS, and GCS, along with regional longitudinal (RLS) and circumferential strain (RCS) were compared across the acute, subacute, and chronic timepoints (presentation, 14-42, and > 42 days, respectively). Mean GLS improved from - 18.4% in the acute phase to - 20.1% in the chronic phase (p = 0.4). Mean GCS improved from - 19.4% in the acute phase to - 23.5% in the chronic phase (p = 0.03). RCS and RLS were impaired in the acute phase and showed a trend towards recovery by the chronic phase, with the exception of the basal anterolateral segment. In our longitudinal study of MIS-C patients, GLS and GCS were lower in the acute phase, corroborating with left ventricular dysfunction by traditional measures. Additionally, as function globally recovers, GLS and GCS also normalize. However, some regional segments continue to have decreased strain values which may be an important subclinical marker for future adverse events.

Three-dimensional CRISPR screening reveals epigenetic interaction with anti-angiogenic therapy.

Angiogenesis underlies development, physiology and pathogenesis of cancer, eye and cardiovascular diseases. Inhibiting aberrant angiogenesis using anti-angiogenic therapy (AAT) has been successful in the clinical treatment of cancer and eye diseases. However, resistance to AAT inevitably occurs and its molecular basis remains poorly understood. Here, we uncover molecular modifiers of the blood endothelial cell (EC) response to a widely used AAT bevacizumab by performing a pooled genetic screen using three-dimensional microcarrier-based cell culture and CRISPR-Cas9. Functional inhibition of the epigenetic reader BET family of proteins BRD2/3/4 shows unexpected mitigating effects on EC survival and/or proliferation upon VEGFA blockade. Moreover, transcriptomic and pathway analyses reveal an interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs via the cell cycle regulator CDC25B phosphatase. Collectively, our findings provide insight into epigenetic regulation of the EC response to VEGFA blockade and may facilitate development of quality biomarkers and strategies for overcoming resistance to AAT.

Combined EZH2 Inhibition and IKAROS Degradation Leads to Enhanced Antitumor Activity in Diffuse Large B-cell Lymphoma.

PURPOSE: The efficacy of EZH2 inhibition has been modest in the initial clinical exploration of diffuse large B-cell lymphoma (DLBCL), yet EZH2 inhibitors are well tolerated. Herein, we aimed to uncover genetic and pharmacologic opportunities to enhance the clinical efficacy of EZH2 inhibitors in DLBCL. EXPERIMENTAL DESIGN: We conducted a genome-wide sensitizing CRISPR/Cas9 screen with tazemetostat, a catalytic inhibitor of EZH2. The sensitizing effect of IKZF1 loss of function was then validated and leveraged for combination treatment with lenalidomide. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing analyses were performed to elucidate transcriptomic and epigenetic changes underlying synergy. RESULTS: We identified IKZF1 knockout as the top candidate for sensitizing DLBCL cells to tazemetostat. Treating cells with tazemetostat and lenalidomide, an immunomodulatory drug that selectively degrades IKAROS and AIOLOS, phenocopied the effects of the CRISPR/Cas9 screen. The combined drug treatment triggered either cell-cycle arrest or apoptosis in a broad range of DLBCL cell lines, regardless of EZH2 mutational status. Cell-line-based xenografts also showed slower tumor growth and prolonged survival in the combination treatment group. RNA-seq analysis revealed strong upregulation of interferon signaling and antiviral immune response signatures. Gene expression of key immune response factors such as IRF7 and DDX58 were induced in cells treated with lenalidomide and tazemetostat, with a concomitant increase of H3K27 acetylation at their promoters. Furthermore, transcriptome analysis demonstrated derepression of endogenous retroviruses after combination treatment. CONCLUSIONS: Our data underscore the synergistic interplay between IKAROS degradation and EZH2 inhibition on modulating epigenetic changes and ultimately enhancing antitumor effects in DLBCL.

Treatment resistance in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous disease and represents the most common subtype of lymphoma. Although 60-70% of all patients can be cured by the current standard of care in the frontline setting, the majority of the remaining patients will experience treatment resistance and have a poor clinical outcome. Numerous efforts have been made to improve the efficacy of the standard regimen by, for example, dose intensification or adding novel agents. However, these results generally failed to demonstrate significant clinical benefits. Hence, understanding treatment resistance is a pressing need to optimize the outcome of those patients. In this Review, we first describe the conceptual sources of treatment resistance in DLBCL and then provide detailed and up-to-date molecular insight into the mechanisms of resistance to the current treatment options in DLBCL. We lastly highlight the potential strategies for rationally managing treatment resistance from both the preventive and interventional perspectives.

Underutilization of Cardiac Therapies in Patients with Acute Ischemic Stroke and Elevated Troponin.

INTRODUCTION: Recent findings have shown that in Acute Ischemic Stroke (AIS) patients, elevated troponin is associated with increased mortality. However, due to concerns of cerebral hypoperfusion and hemorrhagic transformation, current practice has been slow to apply proven cardiac therapies to these patients. This study aims to determine this rate of utilization. MATERIALS AND METHODS: A single-center review of 83 patients with AIS and measured troponin was conducted. Patients were stratified based on elevated and non-elevated troponin. Between groups, we measured the utilization of evidence-based cardiac therapies and used a univariate logistic regression to compare outcomes of mortality, re-hospitalization, recurrent acute ischemic stroke, recurrent acute myocardial infarction, and a composite of these outcomes. RESULTS: Of 83 patients, 25 had elevated troponin and 58 had non-elevated troponin. There was no statistical difference in the use of cardiac therapies between the two groups. Adenosine diphosphate P2Y12 antagonists were infrequently used in both elevated and non-elevated troponin groups at 32% vs. 24% (p = 0.64), as were Angiotensin-Converting Enzyme Inhibitors (ACE-I) and angiotensin II receptor blockers (ARB) at 56% vs. 69% (p = 0.38). Those in the elevated troponin group encountered a statistically significant increase in composite endpoint 64% vs. 33% (Odds Ratio [OR] 7.28, 95% Confidence interval [CI] 2.19-28.88, p<0.01). CONCLUSION: Cardiac therapies are underutilized in patients with acute ischemic stroke and elevated troponin levels. In turn, this low usage may explain the increase in morbidity and mortality seen in these patients and the use of such therapies should be considered when treating this subset of patients as the cardio protective nature of these therapies may outweigh the risks associated with them in AIS patients.

Counting nuclei released from microcarrier-based cultures using pro-fluorescent nucleic acid stains and volumetric flow cytometry.

Counting nuclei released from intact cells is a convenient and reliable approach to assess cell density during microcarrier-based culture of adherent cells. However, commonly used methods for counting nuclei, such as crystal violet staining and quantification with a hemocytometer/automated imaging system or a Coulter counter, are imprecise, laborious and, limited in throughput. Here, we describe the use of high-affinity pro-fluorescent nucleic acid stains and volumetric flow cytometry for automated counting of nuclei released from cells attached to microcarriers with improved precision and high sample throughput. This simple procedure facilitates rapid and precise assessment of cell attachment, survival, and proliferation on microcarriers, and can provide information about the cell cycle, all without the need for cell detachment. Consequently, various microcarrier-based applications, from small-scale multi-factor experiments to large-scale functional genetic screens and clinical/industrial cultures, could be enhanced by this approach.

Is it really an abscess? An unusual case of metastatic stromal cell sarcoma of the prostate.

INTRODUCTION: Prostatic stromal sarcomas account for about 0.1% of all prostatic malignancies. Local recurrence into bladder, seminal vesicles and rectum has been documented. Distal metastasis, has so far only been reported in lung and bone. PRESENTATION OF CASE: We report the case of a 42 year old man with a subcutaneous metastatic deposit of a prostatic stromal cell sarcoma 5 years after radical prostatectomy. Additional staging with CT- and PET-scan showed lymph node involvement in the neck and left axilla. A core biopsy of the skin lesion was undertaken, of which the histology revealed a low grade spindle cell tumour that was morphologically identical to a previously diagnosed prostatic stromal sarcoma. DISCUSSION: In literature distant metastases to the lung and bone have been documented before. This is the first documented case of a subcutaneous metastasis of prostatic stromal cell sarcoma. CONCLUSION: The preferred treatment for prostatic stromal cell sarcoma is surgery by radical prostatectomy or cystoprostatectomy. There is currently not enough literature on the topic to elucidate the role of chemo- or radiotherapy in loco-regional or distant spread.

Promiscuity and the conformational rearrangement of drug-like molecules: insight from the protein data bank.

Selectivity is a central aspect of lead optimization in the drug discovery process. Medicinal chemists often try to decrease molecular flexibility to improve selectivity, given the common belief that the two are interdependent. To investigate the relationship between polypharmacology and conformational flexibility, we mined the Protein Data Bank and constructed a dataset of pharmaceutically relevant ligands that crystallized in more than one protein target while binding to each co-crystallized receptor with similar in vitro affinities. After analyzing the molecular conformations of these 100 ligands, we found that 59 ligands bound to different protein targets without significantly changing conformation, suggesting that there is no distinct correlation between conformational flexibility and polypharmacology within our dataset. Ligands crystallized in similar proteins and highly ligand-efficient compounds with five or fewer rotatable bonds were less likely to adjust conformation when binding.

Emergent retrograde tracheal intubation in a 3-year-old with stevens-johnsons syndrome.

A 3-year-old girl suffering from Stevens-Johnsons Syndrome with severe sloughing of the oropharyngeal mucosa was brought to the operating room for an emergent tracheostomy after multiple failed attempts to intubate the trachea in the pediatric intensive care unit. However, a retrograde tracheal intubation was successfully performed in the operating room to secure her airway, after which a tracheostomy was performed. Retrograde intubation can be a quick and effective method for securing the difficult airway.

Multiple identification and risks: examination of peer factors across multiracial and single-race youth.

Multiracial youth are thought to be more vulnerable to peer-related risk factors than are single-race youth. However, there have been surprisingly few well-designed studies on this topic. This study empirically investigated the extent to which multiracial youth are at higher risk for peer influenced problem behavior. Data are from a representative and longitudinal sample of youth from Washington State (N = 1,760, mean age = 14.13, 50.9% girls). Of those in the sample, 225 youth self-identified as multiracial (12.8%), 1,259 as White (71.5%), 152 as Latino (8.6%), and 124 as Asian American (7.1%). Results show that multiracial youth have higher rates of violence and alcohol use than Whites and more marijuana use than Asian Americans. Higher levels of socioeconomic disadvantage and single-parent family status partly explained the higher rates of problem behaviors among multiracial youth. Peer risk factors of substance-using or antisocial friends were higher for multiracial youth than Whites, even after socioeconomic variables were accounted for, demonstrating a higher rate of peer risks among multiracial youth. The number of substance-using friends was the most consistently significant correlate and predictor of problems and was highest among multiracial youth. However, interaction tests did not provide consistent evidence of a stronger influence of peer risks among multiracial youth. Findings underscore the importance of a differentiated understanding of vulnerability in order to better target prevention and intervention efforts as well as the need for further research that can help identify and explain the unique experiences and vulnerabilities of multiracial youth.

Intergenerational Cultural Dissonance, Parent-Child Conflict and Bonding, and Youth Problem Behaviors among Vietnamese and Cambodian Immigrant Families.

Intergenerational cultural dissonance (ICD)-a clash between parents and children over cultural values-is a frequent issue for Asian American youth. Using longitudinal data from the Cross Cultural Families Project, this study examines the mechanisms by which ICD contributes to problem behaviors, including whether ICD predicts parent-child conflict, whether parent-child conflict then has a direct effect on youth problem behavior, and whether positive bonding with parents mediates the effects of such conflict on youth problem behaviors among Vietnamese (n = 164) and Cambodian (n = 163) families with adolescents [average age = 15.2 years (SD = 1.05)]. The results from the path analyses show that, in both groups, ICD indirectly predicts problem behaviors by increasing parent-child conflict, which in turn weakens positive parent-child bonding. Interventions that target youths' perception of intergenerational cultural gaps, help them manage conflict, and help strengthen bonds with parents may prevent problem behaviors among Cambodian and Vietnamese families. This study contributes to inform how to effectively prevent problems and difficulties among these families.

Memory T cells have gene expression patterns intermediate between naive and effector.

The biological basis underlying differentiation of naive (NAI) T cells into effector (EFFE) and memory (MEM) cells is incompletely understood. Furthermore, whether NAI T cells serially differentiate into EFFE and then MEM cells (linear differentiation) or whether they concurrently differentiate into either EFFE or MEM cells (parallel differentiation) remains unresolved. We isolated NAI, EFFE, and MEM CD8(+) T cell subsets from human peripheral blood and analyzed their gene expression by using microarrays. We identified 156 genes that strongly differentiate NAI, EFFE, and MEM CD8(+) T cells; these genes provide previously unrecognized markers to help identify each cell type. Using several statistical approaches to analyze and group the data (standard heat-map and hierarchical clustering, a unique circular representation, multivariate analyses based on principal components, and a clustering method based on phylogenetic parsimony analysis), we assessed the lineage relationships between these subsets and showed that MEM cells have gene expression patterns intermediate between NAI and EFFE T cells. Our analysis suggests a common differentiation pathway to an intermediate state followed by a split into EFFE or MEM cells, hence supporting the parallel differentiation model. As such, conditions under which NAI T cells are activated may determine the magnitude of both EFFE and MEM cells, which arise subsequently. A better understanding of these conditions may be very useful in the design of future vaccine strategies to maximize MEM cell generation.