The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. PIM kinases act in downstream effector functions as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, and prostate, gastric, and head and neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma and to assess the role PIM function in disease progression and their potential to serve as molecular targets for therapy. One hundred thirty-seven cases of urothelial carcinoma were included in this study of surgical biopsy and resection specimens. High levels of expression of all three PIM family members were observed in both noninvasive and invasive urothelial carcinomas. The second-generation PIM inhibitor, TP-3654, displays submicromolar activity in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line. TP-3654 displays favorable human ether-à-go-go-related gene and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor, SGI-1776, and exhibits oral bioavailability. In vivo xenograft studies using a bladder cancer cell line show that PIM kinase inhibition can reduce tumor growth, suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas.

The genetic architecture of atrial fibrillation (AF) encompasses low impact, common genetic variants and high impact, rare variants. Here, we characterize a high impact AF-susceptibility allele, KCNQ1 R231H, and describe its transcontinental geographic distribution and history. Induced pluripotent stem cell-derived cardiomyocytes procured from risk allele carriers exhibit abbreviated action potential duration, consistent with a gain-of-function effect. Using identity-by-descent (IBD) networks, we estimate the broad- and fine-scale population ancestry of risk allele carriers and their relatives. Analysis of ancestral migration routes reveals ancestors who inhabited Denmark in the 1700s, migrated to the Northeastern United States in the early 1800s, and traveled across the Midwest to arrive in Utah in the late 1800s. IBD/coalescent-based allele dating analysis reveals a relatively recent origin of the AF risk allele (~5000 years). Thus, our approach broadens the scope of study for disease susceptibility alleles to the context of human migration and ancestral origins.

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.

Recently, certain studies have demonstrated in vitro that prostaglandin E2 (PGE2) promotes human cluster of differentiation (CD)34+ cell homing. However, the sub‑type receptors activated by PGE2 are unknown, as the PGE2 receptor EP1-4 subtypes (EP1-4) are expressed on the membrane of human CD34+ cells. Based on the above, the present study aimed to screen the receptor subtype activity by PGE2 to promote human CD34+ cell homing. It was observed that human CD34+ cells expressed the four PGE2 sub‑receptors, particularly EP2 and 4. PGE2 increased EP2 and 4 mRNA expression significantly, while EP1 and 3 mRNA exhibited no significant alteration. PGE2, EP2 agonist (EP2A), and EP4A upregulated C‑X‑C chemokine receptor 4 mRNA and protein expression in human CD34+ cells, and promoted stromal cell‑derived factor 1α (SDF‑1α) expression in bone marrow mesenchymal stem cells (BMMSCs). These phenomena were inhibited by the associated receptor antagonists. PGE2, EP2A, and EP4A facilitated human CD34+ cell migration towards SDF‑1α and BMMSCs. The results of the present study suggested that PGE2 promoted human CD34+ cell homing through EP2 and 4 receptors in vitro.

The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which comprise a major part of the nuclear lamina, provide mechanical support to the nucleus, and participate in diverse intracellular signaling. LMNA mutations give rise to a collection of diseases called laminopathies, including dilated cardiomyopathy (LMNA-DCM) and muscular dystrophies. Although nuclear deformities are a hallmark of LMNA-DCM, the role of nuclear abnormalities in the pathogenesis of LMNA-DCM remains incompletely understood. Using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LMNA mutant patients and healthy controls, we show that LMNA mutant iPSC-CM nuclei have altered shape or increased size compared to healthy control iPSC-CM nuclei. The LMNA mutation exhibiting the most severe nuclear deformities, R249Q, additionally caused reduced nuclear stiffness and increased nuclear fragility. Importantly, for all cell lines, the degree of nuclear abnormalities corresponded to the degree of Lamin A/C and Lamin B1 mislocalization from the nuclear envelope. The mislocalization was likely due to altered assembly of Lamin A/C. Collectively, these results point to the importance of correct lamin assembly at the nuclear envelope in providing mechanical stability to the nucleus and suggest that defects in nuclear lamina organization may contribute to the nuclear and cellular dysfunction in LMNA-DCM.

Mutations in the human LMNA gene cause a collection of diseases called laminopathies, which includes muscular dystrophy and dilated cardiomyopathy. The LMNA gene encodes lamins, filamentous proteins that form a meshwork on the inner side of the nuclear envelope. How mutant lamins cause muscle disease is not well understood, and treatment options are currently limited. To understand the pathological functions of mutant lamins so that therapies can be developed, we generated new Drosophila models and human iPS cell-derived cardiomyocytes. In the Drosophila models, muscle-specific expression of the mutant lamins caused nuclear envelope defects, cytoplasmic protein aggregation, activation of the Nrf2/Keap1 redox pathway, and reductive stress. These defects reduced larval motility and caused death at the pupal stage. Patient-derived cardiomyocytes expressing mutant lamins showed nuclear envelope deformations. The Drosophila models allowed for genetic and pharmacological manipulations at the organismal level. Genetic interventions to increase autophagy, decrease Nrf2/Keap1 signaling, or lower reducing equivalents partially suppressed the lethality caused by mutant lamins. Moreover, treatment of flies with pamoic acid, a compound that inhibits the NADPH-producing malic enzyme, partially suppressed lethality. Taken together, these studies have identified multiple new factors as potential therapeutic targets for LMNA-associated muscular dystrophy.

Genome editing in induced pluripotent stem cells is currently hampered by the laborious and expensive nature of identifying homology-directed repair (HDR)-modified cells. We present an approach where isolation of cells bearing a selectable, HDR-mediated editing event at one locus enriches for HDR-mediated edits at additional loci. This strategy, called co-targeting with selection, improves the probability of isolating cells bearing HDR-mediated variants and accelerates the production of disease models.

Advances in induced pluripotent stem cell (iPSC) technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM) models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs) are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA) analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias.