RET-mediated autophagy suppression as targetable co-dependence in acute myeloid leukemia.

Many cases of AML are associated with mutational activation of receptor tyrosine kinases (RTKs) such as FLT3. However, RTK inhibitors have limited clinical efficacy as single agents, indicating that AML is driven by concomitant activation of different signaling molecules. We used a functional genomic approach to identify RET, encoding an RTK, as an essential gene in multiple subtypes of AML, and observed that AML cells show activation of RET signaling via ARTN/GFRA3 and NRTN/GFRA2 ligand/co-receptor complexes. Interrogation of downstream pathways identified mTORC1-mediated suppression of autophagy and subsequent stabilization of leukemogenic drivers such as mutant FLT3 as important RET effectors. Accordingly, genetic or pharmacologic RET inhibition impaired the growth of FLT3-dependent AML cell lines and was accompanied by upregulation of autophagy and FLT3 depletion. RET dependence was also evident in mouse models of AML and primary AML patient samples, and transcriptome and immunohistochemistry analyses identified elevated RET mRNA levels and co-expression of RET and FLT3 proteins in a substantial proportion of AML patients. Our results indicate that RET-mTORC1 signaling promotes AML through autophagy suppression, suggesting that targeting RET or, more broadly, depletion of leukemogenic drivers via autophagy induction provides a therapeutic opportunity in a relevant subset of AML patients.

JUN is a key transcriptional regulator of the unfolded protein response in acute myeloid leukemia.

The transcription factor JUN is frequently overexpressed in multiple genetic subtypes of acute myeloid leukemia (AML); however, the functional role of JUN in AML is not well defined. Here we report that short hairpin RNA (shRNA)-mediated inhibition of JUN decreases AML cell survival and propagation in vivo. By performing RNA sequencing analysis, we discovered that JUN inhibition reduces the transcriptional output of the unfolded protein response (UPR), an intracellular signaling transduction network activated by endoplasmic reticulum (ER) stress. Specifically, we found that JUN is activated by MEK signaling in response to ER stress, and that JUN binds to the promoters of several key UPR effectors, such as XBP1 and ATF4, to activate their transcription and allow AML cells to properly negotiate ER stress. In addition, we observed that shRNA-mediated inhibition of XBP1 or ATF4 induces AML cell apoptosis and significantly extends disease latency in vivo tying the reduced survival mediated by JUN inhibition to the loss of pro-survival UPR signaling. These data uncover a previously unrecognized role of JUN as a regulator of the UPR as well as provide key new insights into the how ER stress responses contribute to AML and identify JUN and the UPR as promising therapeutic targets in this disease.

PKCε as a novel promoter of skeletal muscle differentiation and regeneration.

INTRODUCTION: Satellite cells are muscle resident stem cells and are responsible for muscle regeneration. In this study we investigate the involvement of PKCε during muscle stem cell differentiation in vitro and in vivo. Here, we describe the identification of a previously unrecognized role for the PKCε-HMGA1 signaling axis in myoblast differentiation and regeneration processes. METHODS: PKCε expression was modulated in the C2C12 cell line and primary murine satellite cells in vitro, as well as in an in vivo model of muscle regeneration. Immunohistochemistry and immunofluorescence, RT-PCR and shRNA silencing techniques were used to determine the role of PKCε and HMGA1 in myogenic differentiation. RESULTS: PKCε expression increases and subsequently re-localizes to the nucleus during skeletal muscle cell differentiation. In the nucleus, PKCε blocks Hmga1 expression to promote Myogenin and Mrf4 accumulation and myoblast formation. Following in vivo muscle injury, PKCε accumulates in regenerating, centrally-nucleated myofibers. Pharmacological inhibition of PKCε impairs the expression of two crucial markers of muscle differentiation, namely MyoD and Myogenin, during injury induced muscle regeneration. CONCLUSION: This work identifies the PKCε-HMGA1 signaling axis as a positive regulator of skeletal muscle differentiation.

The p53 family and programmed cell death.

The p53 tumor suppressor continues to hold distinction as the most frequently mutated gene in human cancer. The ability of p53 to induce programmed cell death, or apoptosis, of cells exposed to environmental or oncogenic stress constitutes a major pathway whereby p53 exerts its tumor suppressor function. In the past decade, we have discovered that p53 is not alone in its mission to destroy damaged or aberrantly proliferating cells: it has two homologs, p63 and p73, that in various cellular contexts and stresses contribute to this process. In this review, the mechanisms whereby p53, and in some cases p63 and p73, induce apoptosis are discussed. Other reviews have focused more extensively on the contribution of individual p53-regulated genes to apoptosis induction by this protein, whereas in this review, we focus more on those factors that mediate the decision between growth arrest and apoptosis by p53, p63 and p73, and on the post-translational modifications and protein-protein interactions that influence this decision.

Medullary lateral tegmental field: an important synaptic relay in the baroreceptor reflex pathway of the cat.

This study was designed to test the hypothesis that the medullary lateral tegmental field (LTF) is an important synaptic relay in the baroreceptor reflex pathway controlling sympathetic nerve discharge (SND) of urethan-anesthetized cats. We determined the effects of blockade of excitatory amino acid-mediated neurotransmission in the LTF on three indexes of baroreceptor reflex function: cardiac-related power in SND, strength of linear correlation (coherence value) of SND to the arterial pulse (AP), and inhibition of SND during increased arterial pressure produced by abrupt obstruction of the abdominal aorta. Bilateral microinjection of D-(-)-2-amino-5-phosphonopentanoic acid, an N-methyl-D-aspartate (NMDA) receptor antagonist, abolished cardiac-related power and coherence of SND to the AP, and it prevented inhibition of SND during aortic obstruction. These data support the view that NMDA receptor-mediated neurotransmission in the LTF is critical for baroreceptor reflex control of SND. Bilateral microinjection of 1,2, 3,4-tetrahydro-6-nitro-2,3-dioxobenzo-[f]-quinoxaline-7-sulfonamid e, a non-NMDA receptor antagonist, decreased cardiac-related power and total power in the 0- to 6-Hz band of SND; however, the AP-SND coherence value remained high, and inhibition of SND during aortic obstruction was preserved. These data imply that non-NMDA receptor-mediated neurotransmission in the LTF is involved in setting the level of excitatory drive to sympathetic nerves.

The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

Photoaging effects on spectral transmittance of plastic filters.

We examined the effects of ultraviolet (UV) radiation in combination with high levels of infrared (IR) radiation on the spectral transmittance of plastic filters. The biological action spectrum for damage to the human eye and skin changes dramatically in the 300-400 nm wavelength range. Cut-off filters used in this region to shape the spectrum of exposure sources are thus critical to the design of experiments which use broadband light sources. The changes in transmittance of three types of plastic filters were observed over an exposure period of 1000 h. One set of three filters was exposed mainly to UV radiation, while the other set was exposed to UV radiation plus IR radiation. Filters exposed to both UV and IR radiation showed spectral changes in their transmittance, while the filters exposed to UV only showed no measurable changes.

Recovery of absolute threshold with UVA-induced retinal damage.

A within-trial psychophysical procedure tracked the initial loss and subsequent recovery of visual thresholds in albino rats exposed to ultraviolet light at 350 nanometers and 0.4 milliwatts per square centimeter. Absolute thresholds increased up to 5 log units immediately following the 15 hour ultraviolet exposure, with a daily recovery of 1-2 log to asymptotic thresholds over a 7-day post-exposure period. The corresponding retinal damage on Day 1 included extensive vesiculation of the photoreceptor outer segments, vacuolation of the inner segments, and pyknosis of cell nuclei. The total number of photoreceptor nuclei and outer segments was unchanged relative to control eyes through post-exposure Day 3. Both nuclei and outer segment counts then consistently decreased 15-20 percent between Days 3-7. The two-stage loss of photoreceptors but daily recovery of absolute thresholds again suggests a significant dissociation of retinal structure and psychophysical function in light-induced ocular pathology.

Changes in absolute threshold with light-induced retinal damage.

Albino rats were trained on a mixed signalled reinforcement procedure designed to yield repeated absolute visual thresholds within each session. After threshold stability animals were exposed 12 hours per night to 1000 lux of light froma cool-white fluorescent source. Log threshold rose as an approximate linear function of exposure time to a maximum of 2.0 log units above baseline after 36 cumulative hours of exposure. Light and electron microscopic analysis of the irradiated retinas revealed vesiculated photoreceptor outer segments, with varying degrees of vacuolation of the inner segments and pyknosis of photoreceptor nuclei depending on exposure time. The various combinations of retinal pathology suggests that damage to photoreceptor outer segments and inner segments interact to jointly affect psychophysical thresholds in light-induced retinal damage.

Control of wheel running by near-ultraviolet light.

Wheel running by mice was compared during incandescent and incandescent plus near-ultraviolet (UV) illumination in three experiments. During the daily 12-hour light period, running rates decreased with supplemental UV compared to previous incandescent only baselines. The decrements were systematically replicated in a second experiment using alternating UV exposure and recovery phases programmed across blocks of sessions. Again, running rates consistently decreased with supplemental UV irradiation of phases two and four compared to preceding baselines of phases one and three. In comparison, running rates were inconsistently affected during the 12-hour dark period in both experiments. A third experiment compared the relative decremental effects of UV, blue and red illumination. Baseline running rates monotonically decreased as an inverse function of wavelength for all subjects. The data suggest that the spectral composition of ambient illumination may affect behavior.

Damage to the monkey retina by broad-spectrum fluorescent light.

Adult rhesus and pigtail monkeys were exposed to a uniform field of light from daylight fluorescent lamps to determine the initial site and characteristics of structural retinal damage induced by continuous exposure and the threshold intensity required to produce the effects. Electron microscopic examination revealed that the initial site of damage is the photoreceptor outer segments. Damage remained restricted to the outer segments at intensities as high as 24,700 lux and with 12 hr exposure periods repeated for up to 4 days. Rods were swollen at the distal tip and showed disc membrane separation. Come outer segments were affected at the proximal end with vesiculation and membrane rearrangement. The threshold intensity for morphological changes to comes in the macula for a single 12 hr exposure was between 195 and 361 mu W/cm2 at the retina (400 to 700 mm; uncorrected for ocular transmittance), whereas the threshold for changes in rods was higher, at between 361 and 615 mu W/cm2. These levels correspond to between 5900 and 10,800 lux (550 to 1000 ft-cd) for comes and 10,800 to 19,400 lux (1000 to 1800 ft-cd) for rods of monkeys with fully dilated pupils. The paramacular areas of the retina were less sensitive to damage than macular areas of the same animal. No paramacular changes were observed in animals exposed to 10,800 lux or less. The patched (control) eyes of each monkey remained structurally normal in both the macula and paramacula at all exposure levels. These results demonstrate the sensitivity of the adult primate retina to damage by relatively moderate levels of light.