Citations

Abstract Primary liver cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), arise from the neoplastic transformation of hepatocytes and cholangiocytes, respectively. Loss or downregulation of PTEN, a tumor suppressor negatively regulating the PI3K/AKT pathway, is frequently observed in CCA and HCC. Notably, PTEN mutations are observed at nearly twice the frequency in combined CCA-HCC tumors than either HCC or CCA alone. Using lineage-specific liver-targeted PTEN-deficient mouse models, we demonstrate that PTEN loss drives cellular dedifferentiation and tumorigenesis, a process that is critically dependent on AKT2. Mechanistically, PTEN deficiency induces activation of NOTCH and upregulation of transcriptional factor SOX9, which plays a central role in tumor cell transformation. In parallel, PTEN loss increases SMAD4 expression and sensitizes the tumor cells to TGFβ signaling, with TGFβ treatment repressing SOX9 expression in tumor cells lacking PTEN. Together, our study defined a critical role for PTEN-AKT2 signaling in maintaining liver epithelial lineage fidelity and revealed how its disruption promotes the conversion of mature hepatocytes or cholangiocytes into liver cancer stem-like cells (LCSCs). Furthermore, we identify a PTEN-dependent crosstalk between NOTCH and TGFβ pathways that governs liver tumor development. Together, this work provides mechanistic insight into lineage plasticity in liver cancer with implications for pathway-directed therapy.

Abstract Hemophagocytic lymphohistiocytosis (HLH) is a rare but critical hyperinflammatory condition that affects around 1.5% of liver transplant patients, with mortality rates reaching up to 33%. This syndrome is characterized by intense, dysregulated activation of macrophages and T-cells, sparking a cytokine surge that leads to severe systemic inflammation and rapid organ failure. Prompt identification and early management are crucial to improve outcomes. Case Presentation: A 61-year-old man with Hepatitis C-induced end-stage liver disease (MELD score 20) underwent a deceased-donor liver transplant. Postoperative complications included atrial fibrillation, stress cardiomyopathy, and a right-sided pleural effusion treated with thoracentesis and extended course of antibiotics. Two weeks later, he presented with fever, hypotension, and nausea, prompting the initiation of IV Zosyn, Vancomycin and Posaconazole. Despite fluid resuscitation, he became hemodynamically unstable, necessitating ICU transfer for vasopressor support. While in the ICU, his condition worsened, with increased fever, tachycardia, anuria and hypoxic respiratory failure that required intubation and continuous renal replacement therapy. Marked hyperferritinemia (ferritin 62,000 µg/L) and hypofibrinogenemia (91 mg/dL) raised suspicion for HLH, with an H score of 244. Comprehensive infectious workup ruled out CMV, EBV, HSV, and other pathogens. Bone marrow biopsy confirmed hemophagocytosis (Figure 1), and flow cytometry ruled out malignant infiltration. Given the strong suspicion for HLH and no identifiable infectious trigger, he was initiated on Dexamethasone, Etoposide, and Ruxolitinib, which lowered ferritin levels and improved vasopressor requirements. However, mentation declined, and imaging revealed a right frontal subarachnoid hemorrhage, likely from disseminated intravascular coagulation, precluding surgical intervention. Despite transient improvement, he developed ventilator associated pneumonia secondary to Stenotrophomonas infections and vancomycin-sensitive Enterococcus bacteremia, leading to refractory hypoxemia. After family and multi-disciplinary team discussions, care was withdrawn. Discussion: HLH remains an exceedingly rare, rapidly fatal syndrome in liver transplant recipients, with only 11 cases documented. Early recognition of HLH can be challenging, as its features, such as fever, cytopenias, and organ dysfunction, overlap with common post-transplant complications, which can delay diagnosis. HLH can be differentiated into primary (genetic) and secondary (acquired) forms, with the latter more common among adults, often triggered by infection, malignancies and autoimmune disorders. Immunosuppressants may inadvertently modulate HLH risk by tempering macrophage and T-cell activation, yet, paradoxically, also predisposing patients to secondary triggers. Clinicians should maintain vigilance for HLH in post-transplant patients with unexplained systemic inflammation, as early intervention is critical, often with Etoposide based regimens to modulate hypercytokinemia, though prognosis remains guarded due to multiorgan failure.

ABSTRACT Objective To estimate the incidence, prevalence, patient characteristics and healthcare utilization patterns of moderate to severe thyroid eye disease (TED) in the United States. Design Retrospective review of medical and prescription claims data from the Inovalon deidentified claims database. Patients Patients with moderate to severe TED were identified based on specific and sensitive definitions using International Classification of Diseases (ICD) codes selected by a group of TED specialists. Results The incident moderate to severe TED population included 3364 and 1730 patients using the sensitive and specific definitions, respectively. After adjusting for age and gender, corresponding incidence rates in the sensitive and specific populations were 8.97 (13.49 in women and 4.27 in men) and 4.37 (6.59 in women and 2.05 in men) per 100,000 person‐years, respectively. The prevalent population included 16,310 and 8018 patients using the sensitive and specific definitions, respectively, corresponding to adjusted prevalence rates of 44.13 (66.00 in women and 21.39 in men) and 20.55 (31.18 in women and 9.50 in men) per 100,000 persons. A preponderance of women was observed, and comorbidity rates were high, with > 40% of prevalent patients experiencing hypertension. Steroids were the most frequently prescribed medication. The impact of teprotumumab was not captured, since this study only included claims data through 2019. Conclusions The estimated incidence and prevalence rates of TED demonstrate the considerable impact of this disease in the United States, making the creation of a specific ICD code imperative for effective healthcare planning and management.

Introduction: Intracellular calcium handling plays a central role in cardiac contractility and relaxation. Genes such as the Ca 2+ -ATPase pump (SERCA2a) and the Dihydropteridine reductase (DHPR) enzyme are especially important in the cycling of intracellular calcium. As prior research has established a link between treatment with our cardiac targeting peptide (CTP: a 12-amino acid, cardiomyocyte-specific cell penetrating peptide) and upregulation of calcium-handling genes such as SERCA2a and DHPR, we hypothesized that treatment with our novel hybrid cardiac targeting peptide (hCTP: positions #3 and 11 replaced with the D-enantiomer form of the amino acids) will improve intracellular calcium handling in human cardiomyocytes (CMCs) by increasing expression of key calcium handling genes instrumental in sequestering calcium in diastole and lowering cytosolic levels. Methods: To test hCTP’s calcium handling abilities, CMCs were plated on day 1 and allowed to settle for 24 hours, after which they received daily treatment with various concentrations of hCTP (1-25µM) for three days. On day 5, cells underwent either intracellular calcium measurements using the FLIPR 6 Calcium assay or were prepared for western blotting. Western blotting was performed to determine the protein concentrations of SERCA2a and DHPR, as compared to a GAPDH control. To test hCTP’s intrinsic calcium handling abilities, various concentrations of hCTP were mixed with a constant amount of elemental calcium dissolved in water. After 20 min at 37 o C, the concentration of free Ca 2+ was measured with an ABL90 Flex Plus radiometer. Results: In CMCs, treatment with hCTP led to a decrease in cytosolic calcium in a dose dependent fashion; concentrations as low as 1µM led to a significant decrease in cytosolic calcium (p< 0.0001 for all groups). Western blots performed on the treated CMCs revealed an increase in expression of both DHPR and SERCA2a, normalized to GAPDH control. Outside of the cellular environment, hCTP did not appear to display inherent calcium sequestring properties, suggesting that improvements in calcium handling are due to upregulation of gene expression. Conclusion: hCTP appears to lower cytosolic calcium and improve calcium handling through upregulation of SERCA2a and DHPR. Our data indicates that hCTP is worth pursuing as a stand-alone, cardio-selective therapy for cardiomyopathies involving calcium mishandling.

Introduction: Role of NF-κB driven inflammation leading to heart failure with preserved ejection fraction has received little attention. Our previous in vivo work with cardiac targeting peptide (CTP) in guinea pigs identified suppression of NF- κB pathway by CTP. In the current work we sought to further study the effects of a hybrid version of CTP (hCTP: amino acids at positions 3 and 11 substituted with D-amino acids) on TNF-α mediated NF-κB activation, as well as expression of its downstream inflammatory markers IL-1β and IL-8. Methods: Human cardiomyocytes (CMCs) were transfected using Lipofectamine 3000 with a reporter luciferase plasmid carrying an NF-κB promoter site upstream of the luciferase gene. Cells were treated with various concentrations of hCTP for 24 hours, followed by a TNF-α challenge (20ng/mL), addition of luciferin as substrate, and measurement of luminescence. Cell viability in response to above manipulations was assessed by FACS using live-dead stain. CMCs were treated as above, and cell culture supernatants analyzed for IL-8 levels using human ELISA kit. In another set of experiments, CMCs were transfected with NF-κB reporter plasmid, and after treatment for 24 hours with various concentrations of hCTP, incubated with 10nM angiotensin 2 (Ang2) and 100nM phenylephrine (PE) for 24hrs to assess levels of IL-1β in the cell culture media using an ELISA assay. Results: hCTP inhibited TNF-α mediated NF-κB activation with a dose-response curve, with maximum inhibition with hCTP seen at 10µM concentrations (p<0.001). Treatment with hCTP inhibited the TNF-α induced IL-8 secretion with significant decreases seen even at the 1µM dose. Similarly, secretion of IL-1β production by CMCs in response to Ang2/PE stimulation was suppressed by hCTP even at the lowest tested dose (1µM; p<0.5). Conclusions: Our data suggests that hCTP inhibited NF-κB pathway transcriptional activity in response to TNF-α stimulation in a dose-dependent fashion in human CMCs. Furthermore, treatment with hCTP inhibited TNF-α induced IL-8 and IL-1β secretion even at doses as low as 1µM. The ability of hCTP to efficiently targeting this inflammatory pathway suggests that it has therapeutic potential in the treatment of heart failure. Further in vivo studies are ongoing to test this hypothesis.

Introduction: Heart failure with preserved ejection fraction (HFpEF) is a form of heart failure which is rapidly rising in incidence in the US today. Despite this, no therapies with mortality benefit exist for this syndrome. Previous work with our novel hybrid cardiac targeting peptide (hCTP), a cardiomyocyte-specific cell penetrating peptide, has shown multiple beneficial biological effects (improved calcium handling, anti-inflammatory properties) beyond its transduction abilities. Therefore, we hypothesized that treatment with hCTP in a mouse model of HFpEF would ameliorate the pathophysiology of this disease. Methods: To establish a HFpEF model, 6-week-old, C57BL/6NJ mice were placed on a high-fat diet (HFD), 60% kCal from lard, and water with 0.5 g/L of an iNOS inhibitor (N-Nitro-L-arginine methylester) to induce hypertension. After 5 weeks of the diet, HFD mice and their littermate controls underwent baseline exercise testing and echocardiograms. Exercise capacity was measured after 2 days of exercise training with 2 days of rest in between; mice were placed on a treadmill at a 10-degree angle and made to run at increasingly fast speeds (5-30 m/min) until exhaustion. Echocardiograms were performed under 1.5% inhalational isoflurane. After baseline testing, the mice were treated with vehicle, or either 2.5 or 5 mg/Kg hCTP, either 3 or 5 days a week (five groups, n = 3 per group). After four weeks of treatment, the mice were again subjected to the same battery of tests and euthanized. Data was compared using two way ANOVA with Sidak’s test to compare measurements pre- and post-treatment. Results: Treatment with hCTP at either concentration for five days/week led to an increase in running distance from 39.8 m to 116.3 m (p = 0.03, n = 6), compared to changes in control distance from 208.4 m to 157.8 m (p = 0.399). Running distance did not correlate with weight (R 2 < .001). Treatment with 5 mg/Kg hCTP for 5 days/week led to a normalization of the E/e’ ratio, from 43.3 to 26.9 (p = 0.004, n = 3) compared to a change in the control of 34.7 to 26.1 (p = 0.312). Ejection fractions remained consistent (>55%) across all groups. Conclusions: Our data suggests that hCTP appears to be a promising treatment in ameliorating some of the physiological effects in a murine model of HFpEF. Gene and protein expression studies in heart tissue are underway. Further work is warranted with a larger sample size.

Abstract Clinical and preclinical research has demonstrated that iPSC-derived NK (iNK) cells have a high therapeutic potential, yet poor understanding of the detailed process of their differentiation in vitro and their counterpart cell development in vivo has hindered therapeutic iNK cell production and engineering. Here we dissect the crucial differentiation of both fetal liver NK cells and iNK cells to enable the rational design of advanced iNK production protocols. We use a comparative analysis of single-cell RNA-seq (scRNA-seq) to pinpoint key factors lacking in the induced setting which we hypothesized would hinder iNK differentiation and/ or functionality. By analyzing key transcription factor regulatory networks, we discovered the importance of TBX21, EOMES, and STAT5A in the differentiation timeline. This analysis provides a blueprint for further engineering new iPSC lines to obtain iNK cells with enhanced functions. We validated this approach by creating a new line of STAT5A-iPSCs which can be differentiated to STAT5A-expressing macrophages with both NK cell and macrophage features such as perforin production, phagocytosis, and anti-tumor functions.

One of the bottlenecks to bringing new therapies to the clinic has been a lack of vectors for delivering novel therapeutics in a targeted manner. Cell penetrating peptides (CPPs) have received a lot of attention and have been the subject of numerous developments since their identification nearly three decades ago. Known for their transduction abilities, they have generally been considered inert vectors. In this review, we present a schema for their classification, highlight what is known about their mechanism of transduction, and outline the existing literature as well as our own experience, vis a vis the intrinsic anti-inflammatory properties that certain CPPs exhibit. Given the inflammatory responses associated with viral vectors, CPPs represent a viable alternative to such vectors; furthermore, the anti-inflammatory properties of CPPs, mostly through inhibition of the NF-κB pathway, are encouraging. Much more work in relevant animal models, toxicity studies in large animal models, and ultimately human trials are needed before their potential is fully realized.

Background: Deeper depth of response (DpR) after induction therapy, especially gain of negative minimal residual disease (MRD), has been linked to prolonged survival in multiple myeloma (MM). However, flow-MRD examination focuses on the numbers but not on the biological characteristics of residual plasma cells (PCs). Objectives: To explore whether the genetic features of residual tumor cells affect the survival time of patients with MM. Design: A retrospective cohort study. Methods: We investigated the clonality of cytogenetic abnormalities (CAs) of the residual PCs using interphase fluorescence in situ hybridization (iFISH) in the National Longitudinal Cohort of Hematological Diseases in China (NCT04645199). Here, a longitudinal cohort of 269 patients with patient-paired diagnostic and post-induction iFISH results was analyzed. Results: Persistent CAs after induction therapy were detected in about half of the patients (118/269, 43%), and patients with undetectable CAs showed significantly improved survival compared with those with genetically detectable MRD [median progression-free survival (mPFS): 59.7 versus 35.7 months, p < 0.001; median overall survival (mOS): 97.1 versus 68.8 months, p = 0.011]. In addition, different patterns of therapy-induced clonal evolution were observed by comparing the clonal structure of residual PCs with paired baseline samples. Patients who maintained at a high risk during follow-up had the worst survival (mPFS: 30.5 months; mOS: 54.4 months), while those who returned to lower risk or had iFISH− at both time points had the best survival (mPFS: 62.0 months, mOS: not reached). Conclusion: These findings highlighted the prognostic value of genetic testing in residual tumor cells, which may provide a deep understanding of clonal evolution and guide clinical therapeutic strategies.