Humanin is an endogenous activator of chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) serves as quality control during stress conditions through selective degradation of cytosolic proteins in lysosomes. Humanin (HN) is a mitochondria-associated peptide that offers cytoprotective, cardioprotective, and neuroprotective effects in vivo and in vitro. In this study, we demonstrate that HN directly activates CMA by increasing substrate binding and translocation into lysosomes. The potent HN analogue HNG protects from stressor-induced cell death in fibroblasts, cardiomyoblasts, neuronal cells, and primary cardiomyocytes. The protective effects are lost in CMA-deficient cells, suggesting that they are mediated through the activation of CMA. We identified that a fraction of endogenous HN is present at the cytosolic side of the lysosomal membrane, where it interacts with heat shock protein 90 (HSP90) and stabilizes binding of this chaperone to CMA substrates as they bind to the membrane. Inhibition of HSP90 blocks the effect of HNG on substrate translocation and abolishes the cytoprotective effects. Our study provides a novel mechanism by which HN exerts its cardioprotective and neuroprotective effects.

The lncRNA CASC15 regulates SOX4 expression in RUNX1-rearranged acute leukemia.

BACKGROUND: Long non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia. RESULTS: CASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter. CONCLUSIONS: Our findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action.

BALR-6 regulates cell growth and cell survival in B-lymphoblastic leukemia.

BACKGROUND: A new class of non-coding RNAs, known as long non-coding RNAs (lncRNAs), has been recently described. These lncRNAs are implicated to play pivotal roles in various molecular processes, including development and oncogenesis. Gene expression profiling of human B-ALL samples showed differential lncRNA expression in samples with particular cytogenetic abnormalities. One of the most promising lncRNAs identified, designated B-ALL associated long RNA-6 (BALR-6), had the highest expression in patient samples carrying the MLL rearrangement, and is the focus of this study. RESULTS: Here, we performed a series of experiments to define the function of BALR-6, including several novel splice forms that we identified. Functionally, siRNA-mediated knockdown of BALR-6 in human B-ALL cell lines caused reduced cell proliferation and increased cell death. Conversely, overexpression of BALR-6 isoforms in both human and mouse cell lines caused increased proliferation and decreased apoptosis. Overexpression of BALR-6 in murine bone marrow transplantation experiments caused a significant increase in early hematopoietic progenitor populations, suggesting that its dysregulation may cause developmental changes. Notably, the knockdown of BALR-6 resulted in global dysregulation of gene expression. The gene set was enriched for leukemia-associated genes, as well as for the transcriptome regulated by Specificity Protein 1 (SP1). We confirmed changes in the expression of SP1, as well as its known interactor and downstream target CREB1. Luciferase reporter assays demonstrated an enhancement of SP1-mediated transcription in the presence of BALR-6. These data provide a putative mechanism for regulation by BALR-6 in B-ALL. CONCLUSIONS: Our findings support a role for the novel lncRNA BALR-6 in promoting cell survival in B-ALL. Furthermore, this lncRNA influences gene expression in B-ALL in a manner consistent with a function in transcriptional regulation. Specifically, our findings suggest that BALR-6 expression regulates the transcriptome downstream of SP1, and that this may underlie the function of BALR-6 in B-ALL.

Chemical modulation of chaperone-mediated autophagy by retinoic acid derivatives.

Chaperone-mediated autophagy (CMA) contributes to cellular quality control and the cellular response to stress through the selective degradation of cytosolic proteins in lysosomes. A decrease in CMA activity occurs in aging and in age-related disorders (for example, neurodegenerative diseases and diabetes). Although prevention of this age-dependent decline through genetic manipulation in mice has proven beneficial, chemical modulation of CMA is not currently possible, owing in part to the lack of information on the signaling mechanisms that modulate this pathway. In this work, we report that signaling through retinoic acid receptor α (RARα) inhibits CMA and apply structure-based chemical design to develop synthetic derivatives of all-trans-retinoic acid to specifically neutralize this inhibitory effect. We demonstrate that chemical enhancement of CMA protects cells from oxidative stress and from proteotoxicity, supporting a potential therapeutic opportunity when reduced CMA contributes to cellular dysfunction and disease.

Design, synthesis and biological evaluation of 2H-benzo[b][1,4] oxazine derivatives as hypoxia targeted compounds for cancer therapeutics.

A small library of 2H-benzo[b][1,4] oxazine derivative was synthesized and their biological activity was tested on HepG2 cells under normoxic and hypoxic conditions. From preliminary screening, we found compound 10 and 11 specifically inhibit hypoxic cancer cell growth IC(50) 87+/-1.8microM and IC(50) 10+/-3.7microM while sparing 'normoxic' cells IC(50) >600M and >1mM (not applicable), respectively. We tested the effect of 10 on MTT, clonogenic and hypoxia induced genes. The MTT correlates with clonogenic assays and most importantly compound 10 down regulates hypoxia induces genes (HIF-1alpha, P21 and VEGF) appropriately. We are in the process to explore the molecular mechanism of action of oxazine derivative compounds on hypoxia tumor cells.

Synthesis of novel ketoconazole derivatives as inhibitors of the human Pregnane X Receptor (PXR; NR1I2; also termed SXR, PAR).

PXR, pregnane X receptor, in its activated state, is a validated target for controlling certain drug-drug interactions in humans. In this context, there is a paucity of inhibitors directed toward activated PXR. Using prior observations with ketoconazole as a PXR inhibitor, the target compound 3 was synthesized from (s)-glycidol with overall 56% yield. (+)-Glycidol was reacted with 4-bromophenol and potassium carbonate in DMF to yield the ring opened compound 6. This was then heated to reflux in benzene along with 2', 4'-difluoroacetophenone and catalytic amount of para-toluene sulfonic acid to yield 8. The resultant acetal 8 was then functionalized using Palladium chemistry to yield the target compound 3. The activity of the compound was compared with ketoconazole and UCL2158H. However, in contrast with ketoconazole (IC(50) approximately 0.020 microM; approximately 100% inhibition), 3 has negligible effects on inhibition of microsomal CYP450 (maximum approximately 20% inhibition) at concentrations >40 microM. In vitro, micromolar concentration of ketoconazole is toxic to passaged human cell lines, while 3 does not exhibit cytotoxicity up to concentrations approximately 100 microM (viability >85%). This is the first demonstration of a chemical analog of a PXR inhibitor that retains activity against activated PXR. Furthermore, in contrast with ketoconazole, 3 is less toxic in human cell lines and has negligible CYP450 activity.

High yield selective acylation of polyamines: proton as protecting group.

An advance in the selective acylation of polyamines having identical or similar amine functions is reported. While nucleophilicity differences between the various amine functions are slight, the corresponding conjugate acids exhibit pKa values over a significant range. We have used proton as polyamine protecting group: the monoamine resulting from single deprotonation of a polyammonium compound has allowed for high yields of selective acylation.