Malignant DFFB isoform switching promotes leukemia survival in relapse pediatric T-cell acute lymphoblastic leukemia.

With modern treatment most children with acute lymphoblastic leukemia (ALL) survive without relapse. However, for children who relapse the prognosis is still poor, especially in children with T-cell phenotype (T-ALL) and remains the major cause of death. The exact mechanism of relapse is currently not known. While contribution of RNA processing alteration has been linked to other hematological malignancies, its contribution in pediatric T-ALL may provide new insights. Almost all human genes express more than one alternative splice isoform. Thus, gene modulation producing a diverse repertoire of the transcriptome and proteome have become a significant molecular marker of cancer and a potential therapeutic vulnerability. To study this, we performed RNA-sequencing analysis on patient-derived samples followed by splice isoform-specific PCR. We uncovered a distinct RNA splice isoform expression pattern characteristic for relapse samples compared to the leukemia samples from the time of diagnosis. We also identified deregulated splicing and apoptosis pathways specific for relapse T-ALL. Moreover, patients with T-ALL displayed pro-survival splice isoform switching favoring pro-survival isoforms compared to normal healthy stem cells. Cumulatively, pro-survival isoform switching and DFFB isoform regulation of SOX2 and MYCN may play a role in T-ALL proliferation and survival, thus serving as a potential therapeutic option.

The immune cell atlas of human neuroblastoma.

Understanding the complete immune cell composition of human neuroblastoma (NB) is crucial for the development of immunotherapeutics. Here, we perform single-cell RNA sequencing (scRNA-seq) on 19 human NB samples coupled with multiplex immunohistochemistry, survival analysis, and comparison with normal fetal adrenal gland data. We provide a comprehensive immune cell landscape and characterize cell-state changes from normal tissue to NB. Our analysis reveals 27 immune cell subtypes, including distinct subpopulations of myeloid, NK, B, and T cells. Several different cell types demonstrate a survival benefit. In contrast to adult cancers and previous NB studies, we show an increase in inflammatory monocyte cell state when contrasting normal and tumor tissue, while no differences in cytotoxicity and exhaustion score for T cells, nor in Treg activity, are observed. Our receptor-ligand interaction analysis reveals a highly complex interactive network of the NB microenvironment from which we highlight several interactions that we suggest for future therapeutic studies.

Inactivation of Pmel alters melanosome shape but has only a subtle effect on visible pigmentation.

PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel⁻/⁻). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel⁻/⁻ melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation.

Central nociceptin/orphanin FQ system elevates food consumption by both increasing energy intake and reducing aversive responsiveness.

Nociceptin/orphanin FQ (N/OFQ), the nociceptin opioid peptide (NOP) receptor ligand, increases feeding when injected centrally. Initial data suggest that N/OFQ blocks the development of a conditioned taste aversion (CTA). The current project further characterized the involvement of N/OFQ in the regulation of hunger vs. aversive responses in rats by employing behavioral, immunohistochemical, and real-time PCR methodology. We determined that the same low dose of the NOP antagonist [Nphe(1)]N/OFQ(1-13)NH(2) delivered via the lateral ventricle diminishes both N/OFQ- and deprivation-induced feeding. This anorexigenic effect did not stem from aversive consequences, as the antagonist did not cause the development of a CTA. When [Nphe(1)]N/OFQ(1-13)NH(2) was administered with LiCl, it moderately delayed extinction of the LiCl-induced CTA. Injection of LiCl + antagonist compared with LiCl alone generated an increase in c-Fos immunoreactivity in the central nucleus of the amygdala. The antagonist alone elevated Fos immunoreactivity in the paraventricular nucleus of the hypothalamus, nucleus of the solitary tract, and central nucleus of the amygdala. Hypothalamic NOP mRNA levels were decreased during energy intake restriction induced by aversion, as well as in non-CTA rats food-restricted to match CTA-reduced consumption. Brain stem NOP was upregulated only in aversion. Prepro-N/OFQ mRNA showed a trend toward upregulation in restricted rats (P = 0.068). We conclude that the N/OFQ system promotes feeding by affecting the need to replenish lacking calories and by reducing aversive responsiveness. It may belong to mechanisms that shift a balance between the drive to ingest energy and avoidance of potentially tainted food.

Distribution of [3H]-corticosterone in urine, feces and blood of male Sprague-Dawley rats after tail vein and jugular vein injections.

The present study aimed to investigate the time-course and distribution of [(3)H]-corticosterone in urine, feces and blood of male Sprague-Dawley rats after intravenous administration of a low dose (1 microCi), and to investigate whether different intravenous routes of administration may affect the dynamics of excreted [(3)H]-corticosterone in the feces. One microCi [(3)H]-corticosterone was injected intravenously either through the tail vein in manually restrained rats or through a jugular vein catheter three days after surgical implantation. Urine and feces were collected at different time points over 78 h from the rats injected in the tail vein, and blood and feces were collected over 48 h from rats injected in the jugular vein. In the blood, radioactivity peaked immediately and decreased rapidly within 90 minutes. The radioactivity was excreted in urine within six h and in feces after at least 12 h. Sixty percent of the radioactivity was detected in the urine and 40% in feces during the study period of 78 h. The detected amount of radioactivity in feces was higher and displayed a more pronounced peak 12 h after injection when the substance was administered through a jugular vein catheter compared to tail vein injection. The data obtained in the present study may serve as an important benchmark when choosing time points for fecal collection for quantification of corticosterone or corticosterone metabolites as a non-invasive measure of preceding HPA-axis activation.

Lrig1 is an endogenous inhibitor of Ret receptor tyrosine kinase activation, downstream signaling, and biological responses to GDNF.

Glial cell line-derived neurotrophic factor (GDNF)/Ret signaling has potent trophic effects on ventral midbrain dopaminergic, motor, sensory, and sympathetic neurons. The molecular mechanisms that restrict Ret receptor tyrosine kinase activation are not well understood. Here, we show that Lrig1, a transmembrane protein containing leucine-rich repeats and Ig-like domains in its extracellular region, acts in a negative feedback loop to regulate the activity of Ret receptor tyrosine kinase. In particular, we demonstrate that Lrig1 is capable of physically interacting with Ret and that Lrig1/Ret association inhibits GDNF binding, recruitment of Ret to lipid rafts, receptor autophosphorylation, and mitogen-activated protein kinase (MAPK) activation in response to GDNF. In neuronal cells, Lrig1 overexpression also inhibits GDNF/Ret-induced neurite outgrowth in a cell-autonomous manner. Downregulation of Lrig1 using small interference RNA knock-down experiments potentiates both neuronal differentiation and MAPK activation in response to GDNF. Together, these results provide an insight into Lrig1 function and establish a new physiological mechanism to restrict signaling and biological responses induced by GDNF and Ret in neuronal cells.