Recent genomic studies have identified subtypes of uterine leiomyoma (LM) with distinctive genetic alterations. Here, we report the elucidation of the biological characteristics of the two most prevalent uterine leiomyoma subtypes, MED12-mutant (MED12-LM) and HMGA2-overexpressing (HMGA2-LM) uterine leiomyomas. Because each tumor carries only one genetic alteration, both subtypes are considered to be monoclonal. Approximately 90% of cells in HMGA2-uterine leiomyoma were smooth muscle cells (SMC) with HMGA2 overexpression. In contrast, MED12-LM consisted of similar numbers of SMC and non-SMC, which were mostly tumor-associated fibroblasts (TAF). Paradoxically, TAF carried no mutations in MED12, suggesting an interaction between SMC and TAF to coordinate their growth. The higher amount of extracellular matrix in MED12-LM than HMGA2-LM was partially due to the high concentration of collagen-producing TAF. SMC growth in a xenograft assay was driven by progesterone in both uterine leiomyoma subtypes. In contrast, TAF in MED12-LM proliferated in response to estradiol, whereas progesterone had no effect. The high concentration of estrogen-responsive TAF in MED12-LM explains the inconsistent discoveries between in vivo and in vitro studies on the mitogenic effect of estrogen and raises questions regarding the accuracy of previous studies utilizing MED12-LM cell culture. In addition, the differential effects of estradiol and progesterone on these uterine leiomyoma subtypes emphasize the importance of subtypes and genotypes in designing nonsurgical therapeutic strategies for uterine leiomyoma. Cancer Res; 77(24); 6891–901. ©2017 AACR.

Cellular mechanisms of uterine leiomyoma (LM) formation have been studied primarily utilizingin vitromodels. However, recent studies established that the cells growing in the primary cultures ofMED12-mutant LM (MED12-LM) do not carry causal mutations. To improve the accuracy of LM research, we addressed the cellular mechanisms of LM growth and regression utilizing a patient-derived xenograft (PDX) model, which faithfully replicates the patient tumorsin situ. The growth and maintenance of MED12-LMs depend on 17β-estradiol (E2) and progesterone (P4). We determined E2 and P4-activated MAPK and PI3K pathways in PDXs with upregulation of IGF1 and IGF2, suggesting that the hormone actions on MED12-LM are mediated by the IGF pathway. When hormones were removed, MED12-LM PDXs lost approximately 60% of volume within 3 days through reduction in cell size. However, in contrast to general belief, the survival of LM cells was independent of E2 and/or P4, and apoptosis was not involved in the tumor regression. Furthermore, it was postulated that abnormal collagen fibers promote the growth of LMs. However, collagen fibers of actively growing PDXs were well aligned. The disruption of collagen fibers, as found in human LM specimens, occurred only when the volume of PDXs had grown to over 20 times the volume of unstimulated PDXs, indicating disruption is the result of growth not the cause. Hence, this study revises generally accepted theories on the growth and regression of LMs.

Recent evidence suggests that neuronal Na⁺ channels (nNa_vs) contribute to catecholamine-promoted delayed afterdepolarizations (DADs) and catecholaminergic polymorphic ventricular tachycardia (CPVT). The newly identified overlap between CPVT and long QT (LQT) phenotypes has stoked interest in the cross-talk between aberrant Na⁺ and Ca²⁺ handling and its contribution to early afterdepolarizations (EADs) and DADs. Here, we used Ca²⁺ imaging and electrophysiology to investigate the role of Na⁺ and Ca²⁺ handling in DADs and EADs in wild-type and cardiac calsequestrin (CASQ2)-null mice. In experiments, repolarization was impaired using 4-aminopyridine (4AP), whereas the L-type Ca²⁺ and late Na⁺ currents were augmented using Bay K 8644 (BayK) and anemone toxin II (ATX-II), respectively. The combination of 4AP and isoproterenol prolonged action potential duration (APD) and promoted aberrant Ca²⁺ release, EADs, and DADs in wild-type cardiomyocytes. Similarly, BayK in the absence of isoproterenol induced the same effects in CASQ2-null cardiomyocytes. In vivo, it prolonged the QT interval and, upon catecholamine challenge, precipitated wide QRS polymorphic ventricular tachycardia that resembled human torsades de pointes. Treatment with ATX-II produced similar effects at both the cellular level and in vivo. Importantly, nNa_v inhibition with riluzole or 4,9-anhydro-tetrodotoxin reduced the incidence of ATX-II–, BayK-, or 4AP-induced EADs, DADs, aberrant Ca²⁺ release, and VT despite only modestly mitigating APD prolongation. These data reveal the contribution of nNa_Vs to triggered arrhythmias in murine models of LQT and CPVT-LQT overlap phenotypes. We also demonstrate the antiarrhythmic impact of nNa_V inhibition, independent of action potential and QT interval duration, and provide a basis for a mechanistically driven antiarrhythmic strategy.

Background: Monoclonal antibody (mAb) immune checkpoint inhibitor (ICI) therapies have dramatically impacted oncology this past decade. However, only about one‐third of patients respond to treatment, and biomarkers to predict responders are lacking. Recent ICI clinical pharmacology data demonstrate high baseline drug clearance (CL₀) significantly associates with shorter overall survival, independent of ICI exposure, in patients receiving ICI mAb therapies. This suggests CL₀ may predict outcomes from ICI therapy, and cachectic signalling may link elevated CL₀ and poor response. Our aim was to determine if mouse models of cancer cachexia will be useful for studying these phenomena and their underlying mechanisms.Methods: We evaluated pembrolizumab CL in the C26 and Lewis lung carcinoma mouse models of cancer cachexia. A single treatment of vehicle or pembrolizumab, at a dose of 2 or 10 mg/kg, was administered intravenously by tail vein injection. Pembrolizumab was quantified by an ELISA in serial plasma samples, and FcRn gene (Fcgrt) expression was assessed in liver using real‐time quantitative reverse transcription PCR. Non‐compartmental and mixed‐effects pharmacokinetics analyses were performed.Results: We observed higher pembrolizumab CL₀ and decreased Fcgrt expression in whole liver tissue from tumour‐bearing vs. tumour‐free mice. In multivariate analysis, presence of tumour, total murine IgG, muscle weight and Fcgrt expression were significant covariates on CL, and total murine IgG was a significant covariate on V1 and Q.Conclusions: These data demonstrate increases in catabolic clearance of monoclonal antibodies observed in humans can be replicated in cachectic mice, in which Fcgrt expression is also reduced. Notably, FcRn activity is essential for proper antigen presentation and antitumour immunity, which may permit the study of cachexia's impact on FcRn‐mediated clearance and efficacy of ICI therapies.

Immune-checkpoint inhibitor (ICI) therapy has dramatically changed the clinical landscape for several cancers, and ICI use continues to expand across many cancer types. Low baseline clearance (CL) and/or a large reduction of CL during treatment correlates with better clinical response and longer survival. Similar phenomena have also been reported with other monoclonal antibodies (mAb) in cancer and other diseases, highlighting a characteristic of mAb clinical pharmacology that is potentially shared among various mAbs and diseases. Though tempting to attribute poor outcomes to low drug exposure and arguably low target engagement due to high CL, such speculation is not supported by the relatively flat exposure–response relationship of most ICIs, where a higher dose or exposure is not likely to provide additional benefit. Instead, an elevated and/or increasing CL could be a surrogate marker of the inherent resistant phenotype that cannot be reversed by maximizing drug exposure. The mechanisms connecting ICI clearance, therapeutic efficacy, and resistance are unclear and likely to be multifactorial. Therefore, to explore the potential of ICI CL as an early marker for efficacy, this review highlights the similarities and differences of CL characteristics and CL-response relationships for all FDA-approved ICIs, and we compare and contrast these to selected non-ICI mAbs. We also discuss underlying mechanisms that potentially link mAb CL with efficacy and highlight existing knowledge gaps and future directions where more clinical and preclinical investigations are warranted to clearly understand the value of baseline and/or time-varying CL in predicting response to ICI-based therapeutics.

The neonatal Fc receptor (FcRn) is responsible for recycling of IgG antibodies and albumin throughout the body. This mechanism has been exploited for pharmaceutic delivery across an array of diseases to either enhance or diminish this function. Monoclonal antibodies and albumin-bound nanoparticles are examples of FcRn-dependent anti-cancer therapeutics. Despite its importance in drug delivery, little is known about FcRn expression in circulating immune cells. Through time-of-flight mass cytometry (CyTOF) we were able to characterize FcRn expression in peripheral blood mononuclear cell (PBMC) populations of pancreatic ductal adenocarcinoma (PDAC) patients and non-cancer donors. Furthermore, we were able to replicate these findings in an orthotopic murine model of PDAC. Altogether, we found that in both patients and mice with PDAC, FcRn was elevated in migratory and resident classical dendritic cell type 2 (cDC2) as well as monocytic and granulocytic myeloid-derived suppressor cell (MDSC) populations compared to tumor-free controls. Furthermore, PBMCs from PDAC patients had elevated monocyte, dendritic cells and MDSCs relative to non-cancer donor PBMCs. Future investigations into FcRn activity may further elucidate possible mechanisms of poor efficacy of antibody immunotherapies in patients with PDAC.