Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases.

Lung metastases are the primary cause of death for osteosarcoma (OS) patients. We recently validated interleukin-11 receptor α (IL-11Rα) as a molecular target for the inhibition of OS lung metastases. Since there is no clinically approved antibody against this receptor, we sought to identify downstream targets that mediate the effects of IL-11Rα signaling. We used shRNA to deplete IL-11Rα from OS cells; as a complementary approach, we added IL-11 exogenously to OS cells. The resulting changes in gene expression identified EZH2 as a downstream candidate. This was confirmed by knockdown of IL-11Rα in OS cells, which led to increased expression of genes repressed by histone methyltransferase EZH2, including members of the WNT pathway, a known target pathway of EZH2. Exogenous IL-11 increased the global levels of histone H3 lysine 27 trimethylation, evidence of EZH2 activation. Treatment with the EZH2 inhibitor GSK126 significantly reduced in vitro proliferation and increased cell-cycle arrest and apoptosis, which were partially mediated through the WNT pathway. In vivo, treatment of an orthotopic nude mouse model of OS with GSK126 inhibited lung metastatic growth and prolonged survival. In addition, significantly shorter recurrence-free survival was seen in OS patients with high levels of EZH2 in their primary tumors (P < .05). This suggests that IL-11Rα promotes OS lung metastasis via activation of EZH2. Thus, blocking EZH2 activity may be an effective strategy for inhibiting OS lung metastasis and improving prognosis.

Co-clinical Trial of Novel Bispecific Anti-HER2 Antibody Zanidatamab in Patient-Derived Xenografts.

Zanidatamab is a bispecific human epidermal growth factor receptor 2 (HER2)-targeted antibody that has demonstrated antitumor activity in a broad range of HER2-amplified/expressing solid tumors. We determined the antitumor activity of zanidatamab in patient-derived xenograft (PDX) models developed from pretreatment or postprogression biopsies on the first-in-human zanidatamab phase I study (NCT02892123). Of 36 tumors implanted, 19 PDX models were established (52.7% take rate) from 17 patients. Established PDXs represented a broad range of HER2-expressing cancers, and in vivo testing demonstrated an association between antitumor activity in PDXs and matched patients in 7 of 8 co-clinical models tested. We also identified amplification of MET as a potential mechanism of acquired resistance to zanidatamab and demonstrated that MET inhibitors have single-agent activity and can enhance zanidatamab activity in vitro and in vivo. These findings provide evidence that PDXs can be developed from pretreatment biopsies in clinical trials and may provide insight into mechanisms of resistance. SIGNIFICANCE: We demonstrate that PDXs can be developed from pretreatment and postprogression biopsies in clinical trials and may represent a powerful preclinical tool. We identified amplification of MET as a potential mechanism of acquired resistance to the HER2 inhibitor zanidatamab and MET inhibitors alone and in combination as a therapeutic strategy. This article is featured in Selected Articles from This Issue, p. 695.

Effects of Extruded Compared with Pelleted Diets on Laboratory Mice Housed in Individually Ventilated Cages and the Cage Environment.

The physical form of the diet fed to laboratory animals should be evaluated to reduce experimental variations and confoundingfactors. This 14-d study evaluated the effects of diet form (pelleted or extruded) on intracage ammonia concentrations,feed disappearance, body weight, cage weight, and the degree of cage soilage and whether these effects were influenced bystrain or stock or sex. Mice (C57BL/6, ICR, and nude; age, 4 wk) were randomly assigned to 4 treatment groups representingpelleted and extruded diets from each of 2 vendors (pelleted diet groups, P1 and P2; extruded diet groups, E1 and E2).Intracage ammonia concentrations depended on strain or stock, diet, and day and were higher in cages housing nude micethat consumed P1. Diet type did not affect the weight of mice at the end of the study. Feed disappearance was dependent ondiet type and mouse strain or stock and was greatest in the cages of mice that consumed P1. In addition, the greatest feeddisappearance was seen with ICR mice, whereas the least was seen with C57BL/6 mice. Cages housing male nude mice hadgreater cage soilage than those housing female nude mice. The degree of cage soilage was influenced by diet type and dayalso. These results show that diet form and mouse strain or stock significantly affect intracage ammonia concentrations, feeddisappearance, cage weight, and the degree of cage soilage.

Compromised regulation of the collecting duct ENaC activity in mice lacking AT1a receptor.

ENaC-mediated sodium reabsorption in the collecting duct (CD) is a critical determinant of urinary sodium excretion. Existing evidence suggest direct stimulatory actions of Angiotensin II (Ang II) on ENaC in the CD, independently of the aldosterone-mineralocorticoid receptor (MR) signaling. Deletion of the major renal AT1 receptor isoform, AT1a R, decreases blood pressure and reduces ENaC abundance despite elevated aldosterone levels. The mechanism of this insufficient compensation is not known. Here, we used patch clamp electrophysiology in freshly isolated split-opened CDs to investigate how AT1a R dysfunction compromises functional ENaC activity and its regulation by dietary salt intake. Ang II had no effect on ENaC activity in CDs from AT1a R -/- mice suggesting no complementary contribution of AT2 receptors. We next found that AT1a R deficient mice had lower ENaC activity when fed with low (<0.01% Na+ ) and regular (0.32% Na+ ) but not with high (∼2% Na+ ) salt diet, when compared to the respective values obtained in Wild type (WT) animals. Inhibition of AT1 R with losartan in wild-type animals reproduces the effects of genetic ablation of AT1a R on ENaC activity arguing against contribution of developmental factors. Interestingly, manipulation with aldosterone-MR signaling via deoxycosterone acetate (DOCA) and spironolactone had much reduced influence on ENaC activity upon AT1a R deletion. Consistently, AT1a R -/- mice have a markedly diminished MR abundance in cytosol. Overall, we conclude that AT1a R deficiency elicits a complex inhibitory effect on ENaC activity by attenuating ENaC Po and precluding adequate compensation via aldosterone cascade due to decreased MR availability.

Collecting duct prorenin receptor knockout reduces renal function, increases sodium excretion, and mitigates renal responses in ANG II-induced hypertensive mice.

Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na+ reabsorption via activation of epithelial Na+ channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANG II-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na+ handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP by using a mouse model with specific deletion of PRR in the CD (CDPRR-KO). At basal conditions, CDPRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na+ excretion and lower ANG II levels in urine. After 14 days of ANG II infusion (400 ng·kg-1·min-1), the increases in systolic BP and diastolic BP were mitigated in CDPRR-KO mice. CDPRR-KO mice had lower abundance of cleaved αENaC and γENaC, as well as lower ANG II and renin content in urine compared with wild-type mice. In isolated CD from CDPRR-KO mice, patch-clamp studies demonstrated that ANG II-dependent stimulation of ENaC activity was reduced because of fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANG II infusion by enhancing renin activity, increasing ANG II, and activating ENaC in the distal nephron segments.

The renal TRPV4 channel is essential for adaptation to increased dietary potassium.

To maintain potassium homeostasis, kidneys exert flow-dependent potassium secretion to facilitate kaliuresis in response to elevated dietary potassium intake. This process involves stimulation of calcium-activated large conductance maxi-K (BK) channels in the distal nephron, namely the connecting tubule and the collecting duct. Recent evidence suggests that the TRPV4 channel is a critical determinant of flow-dependent intracellular calcium elevations in these segments of the renal tubule. Here, we demonstrate that elevated dietary potassium intake (five percent potassium) increases renal TRPV4 mRNA and protein levels in an aldosterone-dependent manner and causes redistribution of the channel to the apical plasma membrane in native collecting duct cells. This, in turn, leads to augmented TRPV4-mediated flow-dependent calcium ion responses in freshly isolated split-opened collecting ducts from mice fed the high potassium diet. Genetic TRPV4 ablation greatly diminished BK channel activity in collecting duct cells pointing to a reduced capacity to excrete potassium. Consistently, elevated potassium intake induced hyperkalemia in TRPV4 knockout mice due to deficient renal potassium excretion. Thus, regulation of TRPV4 activity in the distal nephron by dietary potassium is an indispensable component of whole body potassium balance.

Effects of Environmental Enrichment on the Fertility and Fecundity of Zebrafish (Danio rerio).

Zebrafish (Danio rerio) are a popular vertebrate model in biomedical research, but information describing the effects of environmental enrichment on fertility and fecundity of zebrafish is sparse. In the current study, 18 breeding pairs were placed in divided 1.5-L breeding tanks containing 1 of 3 enrichment conditions: plastic grass (n = 6), plastic leaves (n = 6), or no enrichment (n = 6, control). The pairs were allowed to spawn for 3 h the next day, after which eggs were counted and breeding pairs were returned to holding tanks for use in subsequent sessions. Spawning sessions were repeated at 7-d intervals until the completion of 9 trials, with pairs rotating to a different condition at each interval. Total egg count (mean ± SEM) after 3 h was greater for zebrafish spawning in the grass environment (48.0 ± 7.7 eggs) than in the leaf or control environments (29.4 ± 5.3 and 20.4 ± 3.7 eggs, respectively). An interaction emerged between enrichment type and the age of the spawning pair on the number of fry at 6 d postfertilization (dpf). Initially, more fry were obtained from 110- and 160-dpf pairs with the grass enrichment, but from 173- and 180-dpf pairs there were more obtained with leaf enrichment than grass. A separate experiment showed that enrichment type did not have an effect on fry survivability. Overall, our data indicates that, under certain conditions, zebrafish fertility and fecundity are greater in a breeding tank containing environmental enrichment than in a bare tank.

Fish Lice (Argulus japonicus) in Goldfish (Carassius auratus).

Fish lice (Argulus spp; family Argulidae) are branchiuran crustaceans that parasitize both marine and freshwater fishes. Argulus spp can be a major threat to fish health, because heavy infestations can cause significant morbidity and mortality. In addition, fish lice are known to be the vehicle for other fish diseases. During rounds at our facility, Argulus japonicus was collected from the caudal and anal fins of 3 goldfish (Carassius auratus). These goldfish were asymptomatic, and no additional cases were noted after manual removal of the lice. As soon as any Argulus organisms are identified, management and treatment are recommended because infections can escalate rapidly. Currently, there are no FDA-approved drugs for the control and treatment of this parasite, but several chemicals including organophosphates and diflubenzuron have been used with success. The screening and quarantine of incoming fish is the best way to avoid a facility-wide Argulus infestation.

Chronic angiotensin II infusion drives extensive aldosterone-independent epithelial Na+ channel activation.

The inability of mineralocorticoid receptor (MR) blockade to reduce hypertension associated with high angiotensin (Ang) II suggests direct actions of Ang II to regulate tubular sodium reabsorption via the epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron. We used freshly isolated aldosterone-sensitive distal nephron from mice to delineate the synergism and primacy between aldosterone and Ang II in controlling functional ENaC activity. Inhibition of MR specifically prevented the increased number of functionally active ENaC, but not ENaC open probability elicited by a low sodium diet. In contrast, we found no functional role of glucocorticoid receptors in the regulation of ENaC activity by dietary salt intake. Simultaneous inhibition of MR and Ang II type 1 receptors ameliorated the enhanced ENaC activity caused by low dietary salt intake and produced significantly greater natriuresis than either inhibitor alone. Chronic systemic Ang II infusion induced more than 2 times greater increase in ENaC activity than observed during dietary sodium restriction. Importantly, ENaC activity remained greatly above control levels during maximal MR inhibition. We conclude that during variations in dietary salt intake both aldosterone and Ang II contribute complementarily to the regulation of ENaC activity in the aldosterone-sensitive distal nephron. In contrast, in the setting of Ang II-dependent hypertension, ENaC activity is upregulated well above the physiological range and is not effectively suppressed by inhibition of the aldosterone-MR axis. This provides a mechanistic explanation for the resistance to MR inhibition that occurs in hypertensive subjects having elevated intrarenal Ang II levels.

Bilateral neuroepithelial choristomas of the optic disc in a cynomolgus monkey (Macaca fascicularis): a case report.

A 6-year-old intact male cynomolgus monkey of Chinese origin was received at the Sierra Biomedical Facility. While physical examination revealed good body condition with no abnormalities, routine ophthalmic examination revealed bilateral proliferative optic neuropathy involving the dorsal aspect of the optic disc. No changes were noted in the appearance of the lesions over 8 months, and fluoroescein angiography revealed no abnormalities other than obstruction of the view of the retinal vessels by the lesions. Histopathologic studies revealed characteristics consistent with a diagnosis of bilateral neuroepithelial choristoma.