Regulation of Energy Stores and Feeding by Neuronal and Peripheral CREB Activity in Drosophila

The cAMP-responsive transcription factor CREB functions in adipose tissue and liver to regulate glycogen and lipid metabolism in mammals. While Drosophila has a homolog of mammalian CREB, dCREB2, its role in energy metabolism is not fully understood. Using tissue-specific expression of a dominant-negative form of CREB (DN-CREB), we have examined the effect of blocking CREB activity in neurons and in the fat body, the primary energy storage depot with functions of adipose tissue and the liver in flies, on energy balance, stress resistance and feeding behavior. We found that disruption of CREB function in neurons reduced glycogen and lipid stores and increased sensitivity to starvation. Expression of DN-CREB in the fat body also reduced glycogen levels, while it did not affect starvation sensitivity, presumably due to increased lipid levels in these flies. Interestingly, blocking CREB activity in the fat body increased food intake. These flies did not show a significant change in overall body size, suggesting that disruption of CREB activity in the fat body caused an obese-like phenotype. Using a transgenic CRE-luciferase reporter, we further demonstrated that disruption of the adipokinetic hormone receptor, which is functionally related to mammalian glucagon and β-adrenergic signaling, in the fat body reduced CRE-mediated transcription in flies. This study demonstrates that CREB activity in either neuronal or peripheral tissues regulates energy balance in Drosophila, and that the key signaling pathway regulating CREB activity in peripheral tissue is evolutionarily conserved

Brain Region–Specific Decrease in the Activity and Expression of Protein Kinase A in the Frontal Cortex of Regressive Autism

Autism is a severe neurodevelopmental disorder that is characterized by impaired language, communication, and social skills. In regressive autism, affected children first show signs of normal social and language development but eventually lose these skills and develop autistic behavior. Protein kinases are essential in G-protein-coupled, receptor-mediated signal transduction and are involved in neuronal functions, gene expression, memory, and cell differentiation. We studied the activity and expression of protein kinase A (PKA), a cyclic AMP–dependent protein kinase, in postmortem brain tissue samples from the frontal, temporal, parietal, and occipital cortices, and the cerebellum of individuals with regressive autism; autistic subjects without a clinical history of regression; and age-matched developmentally normal control subjects. The activity of PKA and the expression of PKA (C-α), a catalytic subunit of PKA, were significantly decreased in the frontal cortex of individuals with regressive autism compared to control subjects and individuals with non-regressive autism. Such changes were not observed in the cerebellum, or the cortices from the temporal, parietal, and occipital regions of the brain in subjects with regressive autism. In addition, there was no significant difference in PKA activity or expression of PKA (C-α) between non-regressive autism and control groups. These results suggest that regression in autism may be associated, in part, with decreased PKA-mediated phosphorylation of proteins and abnormalities in cellular signaling

Role of BCR in Down-Modulation of BCR-ABL Oncogenicity in CML

Abstract Bcr-Abl acquires its transforming ability through its up-regulated Abl tyrosine kinase activity. Bcr is a phosphoprotein with a novel serine/threonine kinase activity encoded by its first exon. Over-expression of BCR in K562 cells produces a phosphoserine form of Bcr and interferes with the oncogenic effects of BCR-ABL in mice (Lin et al., Oncogene 2001). We have recently shown the inhibitory effects of Bcr on Bcr-Abl, in a nude mouse solid tumor model. Expression of BCR/GFP in TonB210 cells used for injection delayed tumor formation and tumors were 50% smaller compared to the TonB210/GFP control. In contrast two point mutants in the BCR kinase domain (Y360F and S354A), not only blocked Bcr’s inhibitory effects but enhanced the oncogenic effects of BCRABL (Perazzona et.al. Oncogene, 2008). Similar Bcr effects were observed in a mouse leukemia model. We are investigating the mechanism of the interaction between Bcr and Bcr-Abl proteins. Using TonB210 cells, in which BCR-ABL expression is controlled by a tetracycline-inducible promoter and Bcr is stably transduced by lentivirus infection, we observed that increasing levels of Bcr-Abl expression increased the levels of the Bcr protein. Treatment of TonB210 cells with imatinib mesylate decreased the levels of Bcr- Abl and surprisingly the Bcr protein as well, indicating that the tyrosine kinase function of Bcr-Abl is required to up-regulate Bcr protein expression. In addition, withdrawal of doxycycline also reduced Bcr-Abl and Bcr protein levels, confirming that Bcr-Abl is required for increased expression of the Bcr protein. In order to examine the levels of Bcr in cells lacking Bcr-Abl, we transduced BCR/GFP with lentivirus infection into BaF3 and 32D cells. Surprisingly, these cell lines expressed extremely low levels of Bcr, despite 90% expression of the GFP marker. Expression of Bcr was restored by overnight treatment with the proteasome inhibitor calpain inhibitor I. Forced expression of Bcr-Abl in BCR- transduced cells restored high expression of Bcr protein, confirming that Bcr- Abl is required for preventing degradation of the Bcr protein. Together these findings indicate that Bcr-Abl up-regulated Bcr expression by interfering with proteasomemediated degradation of the Bcr protein. Additional studies indicated that Bcr increases expression of the myeloid membrane surface marker Mac-1 in Bcr-Abl TonB210 cells, which originated from the mouse pro-B cell BaF3. We propose that Bcr may play a role in generating the myeloid phenotype caused by Bcr-Abl in CML patients and may be an important player in the chronic phase of CML by down-modulating Bcr-Abl.</jats:p

K562-R As a Model to Study Jak2 in a Non Bcr-Abl Addicted Cell Line

Abstract Abstract 4412 Chronic myeloid leukemia (CML) is a hematological disease caused by the fusion protein Bcr-Abl tyrosine kinase. Development of the tyrosine kinase inhibitor Imatinib Mesylate (IM) has significantly improved the long-term survival of early stage CML patients. However, occurrence of drug resistance, permanence of residual disease and recurrence of active leukemia if IM is discontinued, remain problems awaiting solution. Therefore, new therapeutic strategies aimed at targeting alternative signaling pathways or CML progenitor cells that survive IM treatment are needed. We have previously shown that Janus kinase 2 (Jak2) is activated in Bcr-Abl+ cells. We have demonstrated that reduction of Jak2 activity by the Jak2-specific inhibitor TG101209 (TG) or by genetic knock down (Jak2 shRNA and siRNA) in Bcr-Abl+ cell lines, IM-resistant cells and CML blast crisis cell lines resulted in reduced levels of phosphorylation of Tyr177 and of total Bcr-Abl protein. Jak2 inhibition results in diminished activation of the Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5 (Samanta et al., Leukemia 2011). During these studies we observed that K562 cells and IM-resistant cell line K562-R had different susceptibility to the effect of TG, with K562-R showing increase sensitivity to lower concentrations of TG resulting in faster destabilization of the Bcr-Abl protein. Based on these observations, we hypothesized that increased sensitivity of the K562-R cells was due to the different state of activation of Jak2. In addition, based on recent studies by (Dawson et al., Nat 2009) and by (Rinaldi et al., Blood 2010) we also hypothesized that different levels of Jak2 activation may influence the localization of Jak2 in the cell. We used cell fractionation and western blotting analysis to show that in K562-R cells, active Jak2 is mostly localized in the nucleus with a minor pool found in the cytoplasm. In K562 cells, active Jak2 is equally distributed in both cytoplasmic and nuclear compartment. In addition, immuno-fluorescence confocal analysis of total Jak2 distribution in K562 shows a very organized localization of Jak2 at one pole of the cells but this organization is lost in K562-R and total Jak2 appears uniformly distributed within the cell. K562-R cells were isolated as a Bcr-Abl independent IM resistant cell line that expressed high levels of activated Lyn kinase (Donato et al., Blood 2003). We used K562-R as a model to study the role of Jak2 in a non Bcr-Abl addicted cell line. Since we have previously published that Jak2 up-regulates Lyn kinase activity (Samanta et al., Oncogene 2009), we propose that the higher activation of Jak2 in K562-R is the main driver of oncogenicity and IM resistance and that this cell line may be used to model the role of Jak2 in a cell that is not Bcr-Abl “addicted.” Disclosures: No relevant conflicts of interest to declare. </jats:sec

Jak2 Regulates Bcr-Abl In CD34+ Cells From Imatinib Mesylate-Resistant CML Patients.

Abstract Abstract 1220 Despite the success of imatinib mesylate (IM), complete molecular remission is difficult to achieve in IM-treated CML patients. After continuing treatment with IM, a population of leukemic stem/progenitor cells remains, which are insensitive to IM. Our previous studies have shown that Jak2 regulates Bcr-Abl signaling and maintains levels of Bcr-Abl in CML cell lines, fresh cells from blast crisis patients, and mouse hematopoietic cells transformed by wild-type and imatinib-resistant mutants of BCR-ABL (Samanta et al. ASH 2009 meeting, submitted). We hypothesize that Jak2 plays similar role in Bcr-Abl+ CD34+ stem/progenitor cells, and that inhibition of Jak2 will induce apoptosis by inhibiting critical signaling pathways. In our current studies we have examined Bcr-Abl CD34+ progenitor cells from CML blast crisis patients. These cells are resistant to IM but quite sensitive to cell death induction by the selective Jak2 inhibitor TG101209 at 5–10 μM concentrations for 48 h. Similar results were obtained with cells from IM-resistant accelerated phase and IM-resistant chronic phase patients. Western blotting of CD34+ cells from an IM-resistant blast crisis CML patient established that a four h treatment with TG101209 at 10 μM severely reduced levels of Bcr-Abl protein and levels of phosphotyrosine 177 within Bcr-Abl. To begin to assess whether Jak2 effects on Bcr-Abl signaling also occur in early stage Bcr-Abl induced disease, we assayed CD34+ cord blood cells transformed by Bcr-Abl in short-term culture (Chu S et al. Cancer Res 2007; 67: 7045–7053). The results showed that a six h treatment with TG101209 (10 μM) reduced levels of: Jak2 protein; activated Jak2 (lower levels of pTyr 1007/8); Tyr 177 within Bcr-Abl; and the Bcr-Abl protein itself. These results support our earlier findings that Jak2 regulates Bcr-Abl signaling in CML cell lines (Samanta AK, et al. Cancer Res 66 (13):6468-72, 7/2006; Samanta AK, et al., Oncogene 28 (14):1669-81, 4/2009; Tao W., et al., Oncogene 27(22):3194-200, 5/2008). Furthermore, these results suggest that Jak2 plays a critical role in progenitor CML cells from early stage and late stage patients with CML disease. Disclosures: Bhatia: Novartis: Consultancy. </jats:sec