Gamma Isoforms Research Articles

Preclinical Characterization of PWT143, a Novel Selective and Potent Phosphatidylinositol 3-kinase Delta (PI3K delta) Inhibitor with Ex-Vivo Activity in Hematologic Malignancies.

Abstract 2907Early clinical trials with small molecule inhibitors that target kinases in the B Cell Receptor (BCR) signaling pathway have demonstrated promising activity in B cell malignancies. These kinases include PI3K delta, Bruton’s tyrosine Kinase (BTK) and spleen tyrosine kinase (SYK), with clinical validation demonstrated by the inhibitors GS-1101 (CAL-101), ibrutinib, and fostamatinib respectively. The clinical observations with GS-1101 and ibrutinib include rapid lymph node shrinkage and high lymph node response rate in refractory CLL as well clinical activity in indolent NHL and MCL, and in DLBCL for ibrutinib. In addition to B cells, PI3K delta is expressed in other hematopoietic cells such as T cells, mast cells and neutrophils, and plays a role in cellular signals transmitted by immunoreceptors such as FcεR, FcγR, and chemokine receptors. Therefore, inhibitors of PI3K delta may have utility in diverse hematological malignancies, in addition to those in B cells.PWT143 is a highly selective PI3K delta inhibitor that has been selected as a development candidate. PWT143 exhibits low nanomolar potency in cellular phosphorylation assays against PI3K delta. PWT143 exhibits cellular selectivity of 2200-, 30- and 700-fold against the alpha, beta and gamma isoforms, with no activity against approximately 500 other kinases tested, including mTOR. Low nM potency has also been demonstrated in a whole blood functional assay of basophil activity which is encouraging for translation to the clinic.The activity of PWT143 has been profiled in survival and proliferation assays in a panel of 12 human hematological malignancy cell lines, and compared to GS-1101, ibrutinib, and fostamatinib. The cell lines included DLBCL, Burkitts lymphoma, and lymphoblastic leukemias. PWT143 IC50s ranged from 30 nM to 4 μM with the majority approximating 1μM, while the comparator molecules exhibited higher IC50values: 250 nM to > 10 μM for GS-1101, 250 nM to 9.5 μM for ibrutinib, and 400 nM to > 10 μM for fostamatinib.Viability/proliferation assays were also performed in peripheral blood cells freshly isolated from patients with various hematological malignancies. In CLL samples, PWT143 displayed IC50 values < 100 nM in 3 of 4 cases, in marked contrast to GS-1101 or ibrutinib which exhibited IC50 values >10 μM for the majority of these samples tested. Potent activity was also observed for PWT143 in primary AML samples with IC50s in the 100 nM range or lower for 3 of 5 cases tested, but generally > 1 μM for GS-1101 or Ibrutinib. In previously frozen CLL and AML patient samples procured from commercial sources, PWT143 similarly exhibited several-fold lower IC50values than GS-1101 or ibrutinib. These data suggest increased sensitivity of CLL and AML patient samples to PWT143.The lack of activity of GS-1101 and ibrutinib at low micromolar concentrations in the primary cell assays is consistent with the published mechanism of action. Rather than a direct inhibition of tumor cell viability, the major axis of clinical activity is inhibition of stromal-tumor interactions mediated by BCR “inside-out” signaling which normally maintains tumor cells in the lymph node. Accordingly, the clinical activity of GS-1101, ibrutinib and fostamatinib is associated with marked lymphocytosis due to release of tumor cells from the lymph nodes into peripheral blood, observed in the initial weeks of treatment and often persists for many months.The direct inhibition of viability by PWT143, as well as the established stromal-mediated mechanism of action of PI3K delta inhibitors, may translate to increased clinical activity for PWT143. PWT143 is a potent and selective PI3K delta inhibitor, and preclinical data indicate that it is an attractive candidate for clinical development. Disclosures:O'Farrell:Pathway Therapeutics: Employment, own equity as a Pathway employee Other. Ventura:Pathway Therapeutics: Employment, own equity as a Pathway employee Other. Tai:Pathway Therapeutics: Consultancy. Tyner:Pathway Therapeutics: Research Funding. Loriaux:Pathway Therapeutics: Research Funding. Mahadevan:Pathway Therapeutics: Research Funding. Morales:Pathway Therapeutics: Research Funding. Brown:Pathway Therapeutics: Consultancy. Matthews:Pathway Therapeutics: Employment, Own equity as a Pathway employee Other.

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.

Plasma Protein Characteristics of Long-Term Hemodialysis Survivors

Hemodialysis (HD) patients are under recurrent circulatory stress, and hemodialysis has a high mortality rate. The characteristics of plasma proteomes in patients surviving long-term HD remain obscure, as well as the potential biomarkers in predicting prognoses. This study reports the proteome analyses of patient plasma from non-diabetic long-term HD (LHD, dialysis vintage 14.9±4.1 years, n = 6) and the age/sex/uremic etiology-comparable short-term HD (SHD, dialysis vintage 5.3±2.9 years, n = 6) using 2-DE and mass spectrometry. In addition, a 4-year longitudinal follow-up of 60 non-diabetic HD patients was subsequently conducted to analyze the baseline plasma proteins by ELISA in predicting prognosis. Compared to the SHD, the LHD survivors had increased plasma vitamin D binding proteins (DBP) and decreased clusterin, apolipoprotein A-IV, haptoglobin, hemopexin, complement factors B and H, and altered isoforms of α1-antitrypsin and fibrinogen gamma. During the 45.7±15 months for follow-up of the 60 HD patient cases, 16 patients died. Kaplan-Meier analysis demonstrated that HD patients with the lowest tertile of the baseline plasma DBP level have a significantly higher mortality rate. Multivariate Cox regression analysis further indicated that DBP is an independent predictor of mortality. In summary, the altered plasma proteins in LHD implicated accelerated atherosclerosis, defective antioxidative activity, increased inflammation/infection, and organ dysfunction. Furthermore, lower baseline plasma DBP in HD patients is related to mortality. The results suggest that the proteomic approach could help discover the potential biomarker in HD prognoses.

Early induction of a brown-like phenotype by rosiglitazone in the epicardial adipose tissue of fatty Zucker rats

The epicardial adipose tissue (EAT) is "hypertrophied" in the obese. Thiazolidinediones are anti-diabetic, hypolipidemic drugs and are selective agonists for the gamma isoform of peroxisome proliferator-activated receptor (PPARγ). We evaluated the short-term effects of the prototype rosiglitazone (RSG, 5mgkg(-1)day(-1) for 4 days)on the expression of the genes and proteins (by real-time PCR and Western blot) involved in fatty acid (FA) metabolism in EAT of the obesefatty Zucker rat and compared the levels of expression with those in retroperitoneal adipose tissue (RAT). The glyceroneogenic flux leading to fatty acid re-esterification was assessed by the incorporation of 14C from [1-14C]-pyruvate into neutral lipids. RSG upregulated the mRNA for phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase kinase 4, glycerol kinase, adipocyte lipid binding protein, adipose tissue triglyceride lipase and lipoprotein lipase in both RAT and EAT with a resulting increase in glyceroneogenesis that, however, was more pronounced in EAT than in RAT. Under RSG, fatty acid output was decreased in both tissues but unexpectedly less so in EAT than in RAT. RSG also induced the expression of thekey genes for fatty acid oxidation [carnitinepalmitoyl transferase-1, medium chain acyl dehydrogenase and very long chain acyl dehydrogenase (VLCAD)]in EAT and RAT with a resulting significant rise of the expression of VLCAD protein.In addition, the expression of the genes encoding proteins involved in mitochondrial processing and density PPARγ coactivator 1 alpha (PGC-1α), NADH dehydrogenase 1 and cytochrome oxidase (COX4) were increased by RSG treatment only in EAT, with a resulting significant up-regulation of PGC1-α and COX4 protein. This was accompanied by a rise in the expression of PR domain containing 16 and uncoupling protein 1, two brown adipose tissue-specific proteins. In conclusion, this study reveals that PPAR-γ agonist could induce a rapid browning of the EAT that probably contributes to the increase in lipid turnover.

Abstract 3741: Inhibition of PI3Kα kinase by a selective small molecule inhibitor suppresses B-cell proliferation and leukemic cell growth

Abstract Background: Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellular lipid kinases that phosphorylate the 3 position hydroxyl group of the inositol ring of phosphotidylinositol. The PI3K pathway is frequently activated in human cancers and thus represents an attractive target for small molecule inhibitors. Pan-PI3K inhibitors currently in development have been associated with adverse side-effects such as insulin resistance, thus necessitating the need to develop isoform specific inhibitors of PI3K. Herein, we describe the biological and pharmacokinetic properties of RP5264, a small molecule PI3Kα inhibitors with scope to be further developed as a clinical candidate for hematological malignancies mediated by B-cells. Methods: Activity of RP5264 on individual PI3K isoforms was determined by a Homogenous Time Resolved Fluorescence assay (Millipore, Billerica, MA) with modifications. Cell based selectivity assays against β, α, or ≤ isoforms was assessed by testing the effect of the compound on PDGF, LPA, or c5a induced Akt phosphorylation in NIH-3T3 or RAW cells. Similarly, inhibition of cellular PI3Kα activity was determined in an IgM induced human B-cell proliferation as well as LPS-induced CD19 assays. Ability to arrest cell growth and induce apoptosis was also tested. Viability assays was conducted to determine the growth inhibitory effect of the compounds in leukemic cells. Pharmacokinetic behaviour of compounds in plasma after single dose oral administration was determined in female Balb/c mice. Results: RP5264 inhibited PI3Kα activity in enzyme and cell based assays with IC50 and EC50 values of 22.2 &amp; 24.3 nM respectively. The compound displayed a high degree of selectivity over the alpha (&amp;gt;1000 fold), beta (&amp;gt;30-50 fold), and gamma (&amp;gt;15-50 fold) isoforms. Additionally, the compound caused a half-maximal inhibition of human whole blood CD19 cell proliferation between 100-300 nM. Treatment of PBMC with RP5264 resulted initially in a G2/M arrest followed by subsequent increase in the number of Sub G0 cells. Viability assays demonstrated that the compound caused a significant inhibition in growth as well as Akt phosphorylation of immortalized and primary leukemic cells. Further, the compound exhibited good oral absorption with favourable pharmacokinetic properties in rodents. Conclusions: Results demonstrate the PI3K delta selective nature of RP5264 along with an ability to suppress proliferation and Akt phosphorylation in cancer cells. In vitro selectivity and potency data indicate the therapeutics potential of the compounds in hematological cancers without the deleterious effects commonly associated with the Pan PI3K inhibitors. RP5264 is poised to enter clinical development in 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3741. doi:1538-7445.AM2012-3741

Tyrosine hydroxylase and Lewy body molecules immunoreactivity in the SNC neurons of an AS/AGU mutantrat

The AS/AGU rat has a recessive single point mutation in the gene coding for the gamma isoform of protein kinase C (PKC-γ) resulting in a failure to release dopamine in the striatum and impaired movement including a staggering gait, difficulty in initiating movement and a slight whole body tremor. This study examined the levels tyrosine hydroxylase, ubiquitin and parkin in individual SNC cell bodies, there was no evidence of a reduction in tyrosine hydroxylase levels although levels of ubiquitin and parkin were elevated in the cytoplasm. The findings support the hypothesis that the initial bar to dopamine availability in the striatum is reduced release, with substantia nigra cell death being a later phenomenon.

Frontal affinity chromatography with MS detection of the ligand binding domain of PPARγ receptor: Ligand affinity screening and stereoselective ligand–macromolecule interaction

In this study we report the development of new chromatographic tools for binding studies based on the gamma isoform ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARγ) belonging to the nuclear receptor superfamily of ligand-activated transcription factors. PPARγ subtype plays important roles in the functions of adipocytes, muscles, and macrophages with a direct impact on type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In order to set up a suitable immobilization chemistry, the LBD of PPARγ receptor was first covalently immobilized onto the surface of aminopropyl silica particles to create a PPARγ-Silica column for zonal elution experiments and then onto the surface of open tubular (OT) capillaries to create PPARγ-OT capillaries following different immobilization conditions. The capillaries were used in frontal affinity chromatography coupled to mass spectrometry (FAC–MS) experiments to determine the relative binding affinities of a series of chiral fibrates. The relative affinity orders obtained for these derivatives were consistent with the EC50 values reported in literature. The optimized PPARγ-OT capillary was validated by determining the Kd values of two selected compounds. Known the role of stereoselectivity in the binding of chiral fibrates, for the first time a detailed study was carried out by analysing two enantioselective couples on the LBD-PPARγ capillary by FAC and a characteristic two-stairs frontal profile was derived as the result of the two saturation events. All the obtained data indicate that the immobilized form of PPARγ-LBD retained the ability to specifically bind ligands.

Abstract B62: Role of classical protein kinase C (PKC) in intestinal cancer

Abstract PKCs represent a family of serine/threonine kinases consisting of a least 10 isoforms, divided into three subclasses based on their Ca2+ and DAG dependency. The classical PKC (cPKC) isoforms alpha, beta-I, beta-II, and gamma are calcium and diacylglycerol (DAG) dependent whereas novel PKCs (delta, epsilon, eta, and theta) are calcium independent. In contrast the atypical PKC zeta and lambda/iota do neither need calcium ions nor DAG for their activation. All PKCs are thought to be involved in cell growth and differentiation. In particular, studies focusing on expression profiles of PKCs during tumor formation and progression imply a functional link. For example, have we been able to show that PKC-alpha and -beta are differently regulated in the APCMin mouse model, which represents a well-established model for intestinal cancer. Based on these data, we further have identified PKC-alpha as to act like a tumor suppressor in this context. Given the fact that PKCs are activated by PMA/TPA and therefore defined as tumor promotors, this was a very surprising observation. Material and Methods: This project makes use of mouse intestinal epithelial cell lines that we established from various genetic backgrounds. Using standard Western blot protocols, we first analyzed the abundance and activation status of different signal cascades upon epidermal growth factor (EGF) stimulation. Results: Earlier studies by us (Oster and Leitges, 2006) have indentified an alteration of the EGF receptor signaling in the APCMin model due to PKC-alpha deficiency. To understand the underlying mechanism, we analyzed EGF receptor signaling in mouse intestinal epithelial cell lines. Thus far it became obvious that PKC-alpha deficiency causes a prolonged Erk1/2 and Akt activity whereas JNK activation was only detectable in the alpha deficiency. Conclusion: In sum, we have shown that EGF receptor signaling capacity is functionally linked to PKC-alpha activity. Literature: Oster H, Leitges M. Protein kinase C alpha but not PKC zeta suppresses intestinal tumor formation in APCmin mice. Cancer Res. 2006;66:6955–6963. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B62.

Antiviral protection mechanisms mediated by ginbuna crucian carp interferon gamma isoforms 1 and 2 through two distinct interferon gamma-receptors

Fish genomes possess three type II interferon (IFN) genes, ifnγ1, ifnγ2 and ifnγ-related (ifnγrel). The IFNγ-dependent STAT signalling pathway found in humans and mice had not been characterized in fish previously. To identify the antiviral functions and signalling pathways of the type II IFN system in fish, we purified the ifnγ1, ifnγ2 and ifnγrel proteins of ginbuna crucian carp expressed in bacteria and found them to elicit high antiviral activities against crucian carp hematopoietic necrosis virus. We also cloned two distinct ifnγ receptor alpha chain (ifngr1) isoforms, 1 and 2, and stably expressed them in HeLa cells by transfecting the cells with ifngr1-1 or ifngr1-2 cDNA. When receptor transfectants were treated with the ligands in a one-ligand-one-receptor manner (ifnγ1 and ifngr1-2 or ifnγ2 and ifngr1-1), the stat1 protein was phosphorylated at both serine-727 and tyrosine-701 residues. Gel shift mobility analysis and reporter assay clearly showed that the specific ligand-receptor interaction resulted in the binding of the stat1 protein to the GAS element and enhanced transcription. Therefore, the actions of ifnγ1 and ifnγ2 were found to be mediated by a specific receptor for each signalling pathway via a stat1-dependent mechanism.

Peroxisome Proliferator-Activated Receptors: Role of Isoform Gamma in the Antineoplastic Effect of Iodine in Mammary Cancer

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors. Three subtypes--PPAR alpha, PPAR beta, and PPAR gamma--have been identified and are differentially expressed in tissues. Originally, they were described as molecular regulators of lipid metabolism; recently, it has been shown that they are also involved in regulating the cell cycle and apoptosis in both normal and tumoral cells. In fact, some synthetic PPAR ligands are used to treat dyslipidemia, metabolic diseases, and type 2 diabetes. Here, we review the role of PPAR gamma (PPARγ) in tumor initiation and progression, emphasizing the relationship between this isoform and the cellular and molecular mechanisms involved in the antineoplastic effect of iodine on mammary cancer.

Dynamic remodeling of the actin cytoskeleton by FMNL1γ is required for structural maintenance of the Golgi complex

Formin-like 1 (FMNL1) is a member of the formin family of actin nucleators, and is one of the few formins for which in vitro activities have been well characterized. However, the functional roles of this mammalian formin remain ill-defined. In particular, it is unclear how the unique in vitro biochemical properties of FMNL1 relate to its regulation of cellular processes. Here, we demonstrate that FMNL1 depletion caused a dramatic increase in cellular F-actin content, which resulted in Golgi complex fragmentation. Moreover, increased F-actin and maintenance of Golgi structure were distinctly regulated by the gamma isoform of FMNL1, which required binding to actin. Importantly, in addition to Golgi fragmentation, increased F-actin content in the absence of FMNL1 also led to cation-independent mannose 6-phosphate receptor dispersal, lysosomal enlargement and missorting of cathepsin D. Taken together, our data support a model in which FMNL1 regulates cellular F-actin levels required to maintain structural integrity of the Golgi complex and lysosomes.

Progesterone Prevents Nerve Injury-Induced Allodynia and Spinal NMDA Receptor Upregulation in Rats

Peripheral nerve injury-evoked neuropathic pain still remains a therapeutic challenge. Recent studies support the notion that progesterone, a neuroactive steroid, may offer a promising perspective in pain modulation. Evaluate the effect of progesterone administration on the development of neuropathic pain-associated allodynia and on the spinal expression of N-Methyl-D-Aspartate Receptor subunit 1 (NR1), its phosphorylated form (pNR1), and the gamma isoform of protein kinase C (PKCγ), all key players in the process of central sensitization, in animals subjected to a sciatic nerve constriction. Male Sprague-Dawley rats were subjected to a sciatic nerve single ligature constriction and treated with daily subcutaneous injections of progesterone (16 mg/kg) or vehicle. The development of hindpaw mechanical and thermal allodynia was assessed using the von Frey and Choi tests, respectively. Twenty two days after injury, the number of neuronal profiles exhibiting NR1, pNR1, or PKCγ immunoreactivity was determined in the dorsal horn of the lumbar spinal cord. Injured animals receiving progesterone did not develop mechanical allodynia and showed a significantly lower number of painful responses to cold stimulation. In correlation with the observed attenuation of pain behaviors, progesterone administration significantly reduced the number of NR1, pNR1, and PKCγ immunoreactive neuronal profiles. Our results show that progesterone prevents allodynia in a rat model of sciatic nerve constriction and reinforce its role as a potential treatment for neuropathic pain.

Identification and prioritization ofNUAK1andPPP1CCas positional candidate loci for skeletal muscle strength phenotypes

Muscle strength is an important determinant in elite sports performance as well as in the activities of daily living. Muscle metabolism also plays a role in the genesis, and therefore prevention, of common pathological conditions and chronic diseases. Even though heritability estimates between 31 and 78% suggest a significant genetic component in muscle strength, only a limited number of genes influencing muscle strength have been identified. This study aimed to identify and prioritize positional candidate genes within a skeletal muscle strength quantitative trait locus on chromosome 12q22-23 for follow-up. A two-staged gene-centered fine-mapping approach using 122 single nucleotide polymorphisms (SNPs) in stage 1 identified a family-based association (n=500) between several tagSNPs located in the ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 (ATP2A2; rs3026468), the NUAK family, SNF1-like kinase, 1 (NUAK1; rs10861553 and rs3741886), and the protein phosphatase 1, catalytic subunit, gamma isoform (PPP1CC; rs1050587 and rs7901769) genes and knee torque production (P values up to 0.00092). In stage 2, family-based association tests on additional putatively functional SNPs (e.g., exonic SNPs, SNPs in transcription factor binding sites or in conserved regions) in an enlarged sample (n=536; 464 individuals overlap with stage 1) did not identify additional associations with muscle strength characteristics. Further in-depth analyses will be necessary to elucidate the exact role of ATP2A2, PPP1CC, and NUAK1 in muscle strength and to find out which functional polymorphisms are at the base of the interindividual strength differences.

Cloning and characterization of barley caryopsis FCA

The RNA binding protein, flowering control locus A, (FCA) regulates flowering in rice and Arabidopsis. FCA interacts with FY to auto-regulate its own transcripts as well as to control flowering by downregulating flowering locus C (FLC). We report the cloning and characterization of the gamma (γ) isoform of FCA from barley (Hordeum vulgare cv. McLeod). The deduced protein contained two RNA recognition motifs (RRMs), a glycine-rich region at the N-terminal end, a polyglutamine region immediately downstream of a WW domain. Barley FCA had greater protein sequence homology to wheat and rice FCA than to its Arabidopsis homolog. In developing barley embryos, FCA transcripts could be detected from 2 days after pollination (DAP) up to 40 DAP. FCA transcript levels in mature barley embryo were more abundant in non-germinated than in germinated seeds, with the levels declining as germination progressed. ABA inhibition of germination inhibited the decline of barley embryo FCA. Transient co-expression of FCA or a truncated FCA (lacking RRM) with maize VP1 promoter or wheat Em gene promoter in barley aleurone protoplasts led to increased VP1 and Em gene promoter activity. Barley FCA or truncated FCA localized in the nucleus suggested its role in gene regulation.

Differential expression of 14-3-3 protein isoforms in developing rat hippocampus, cortex, rostral migratory stream, olfactory bulb, and white matter

We investigated the differential immunoexpression of 14-3-3 proteins according to their 7 isoforms during the postnatal development of rat brains, primarily in the hippocampus, cortex, rostral migratory stream (RMS), olfactory bulb, and white matter. Wistar rats at different developmental stages, on postnatal days 2 (P2), P7, P14, P21 and P100 were obtained, and were incubated with each type of anti-14-3-3 isoform antibody. 14-3-3 common (COM)-like immunoreactivity (IR) which represents an epitope shared among the 7 isoforms was initially expressed in the olfactory bulb on P2. This IR was partially expressed in the dentate granule cells and hippocampal pyramidal neurons from P7, and increased during development. These chronological changes were similar to those obtained with beta, gamma, and eta isoforms. Epsilon isoform-like IR was initially identified in the cell body of cortical neurons and glia-like cells on P2. After P7, the IR was more intense in the neuropil of the cortex. This epsilon isoform-like IR was markedly accentuated in the stratum lucidum of the hippocampus after P7, where hippocampal mossy fibers terminate, functioning as a giant synapse. This suggests that epsilon isoforms may be associated with synaptogenesis of the hippocampal mossy fibers. Sigma isoform-like IR was observed in the nuclei of external plexiform layer cells of the olfactory bulb from P2 to P21, in the nuclei of the hippocampal pyramidal and dentate granule cells after P7 and in the nuclei of RMS cells after P7. Zeta and tau isoform-like IRs were mainly identified in the white matter and in oligodendroglial cells from P7 to P21. Different immunolocalizations of the 7 isoforms suggest that 14-3-3 protein isoforms are individually associated with neuronal development and synaptogenesis during postnatal formation of the rat brain.

Abstract 4489: Preclinical profile of novel, potent, and selective PI3K delta inhibitors

Abstract Background: Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellular lipid kinases that phosphorylate the 3 position hydroxyl group of the inositol ring of phosphotidylinositol. The PI3K pathway is frequently activated in human cancers and thus represents an attractive target for small molecule inhibitors. Pan-PI3K inhibitors currently in development have been associated with adverse side-effects such as insulin resistance, thus necessitating the need to develop isoform specific inhibitors of PI3K. Herein, we describe the biological and pharmacokinetic properties of representative molecules from a series of novel and small molecule PI3KΔ inhibitors with scope to be further developed as clinical candidates for hematological malignancies such as acute myeloid leukemia and Non-Hodkin's lymphoma. Methods: Activity of test compounds on individual PI3K isoforms was determined by a Homogenous Time Resolved Fluorescence assay (Millipore, Billerica, MA) with modifications. Cell based selectivity assays against α, β, or γ isoforms was assessed by testing the effect of the compounds on PDGF, LPA, or c5a induced Akt phosphorylation in NIH-3T3 or RAW cells. Similarly, inhibition of cellular PI3KΔ activity was determined in a IgM induced human B-cell proliferation assay. Cell viability assay was conducted to determine the growth inhibitory effect of the compounds in cell lines representative of human acute and chronic leukaemia and lymphoma. Pharmacokinetic behaviour of compounds in plasma after single dose oral administration was determined in female Balb/c mice. Results: Among the compounds evaluated, RP5175 and RP5182 inhibited PI3KΔ activity in enzyme and cell based assays with IC50 values of 9.6 &amp; 37.3 nM and EC50 values of 7.6 &amp; 18.9 nM respectively. Besides, the compounds displayed a high degree of selectivity over the alpha (&amp;gt;500 fold), beta (&amp;gt;400 fold), and gamma (&amp;gt;15-40 fold) isoforms. Viability assays demonstrated that the compounds caused a significant inhibition in growth of THP-1, TOLEDO, and HL-60 cells. Pharmacokinetic studies in female Balb/c mice indicated good oral absorption with favourable peak plasma concentrations. Conclusions: Results demonstrate the PI3K delta selective nature of RP5175 and 5182 along with an ability to suppress proliferation of cancer cells. In vitro selectivity and potency data indicate the therapeutics potential of the compounds in hematological cancers without the deleterious effects commonly associated with the Pan PI3K inhibitors. The compounds are currently being tested for in vitro and in vivo efficacy across various cancer cell lines and xenograft models besides selectivity against other kinases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4489. doi:10.1158/1538-7445.AM2011-4489

Activation of PLEKHG2 RhoGEF by selective G beta gamma isoforms released from multiple classes of G proteins

PLEKHG2 is a novel Gβγ‐activated Rho guanine nucleotide exchange factor (GEF) which catalyzes the activation of the RhoGTPase family of proteins. Overexpression of PLEKHG2 has been associated with enhanced cell transformation. Additionally, PLEKHG2 may mediate lysophosphatidic acid (LPA)‐induced cell spreading. However; the mechanism of PLEKHG2 regulation has yet to be elucidated. In this study we aim to identify how LPA stimulation of G proteins activates PLEKHG2. Using a luciferase‐based reporter assay, we found that PLEKHG2 activation via LPA is selectively mediated by Gβγ subunits released from G proteins, as activation is completely abolished by scavengers of Gβγ, GαT or the C‐terminal tail of G protein‐coupled receptor kinase 2. Interestingly, PLEKHG2 activation is only partially inhibited by Pertussis toxin, the Gαq minigene, or the Gα13 minigene that selectively uncouple the Gi/o, Gq/11, and G12/13 classes of G proteins. The combination of these three inhibitors has additive effect on reducing activation, indicating that activation of PLEKHG2 involves Gβγ subunits released from multiple classes of G proteins. By comparing the activation of PLEKHG2 by different Gβ isoforms, we further provided evidence that activation of PLEKHG2 is Gβ isoform specific. Taken together, our data indicate that PLEKHG2 is a RhoGEF activated by selective Gβγ isoforms released from multiple classes of G proteins.

Identification of the target proteins of rosiglitazone in 3T3-L1 adipocytes through proteomic analysis of cytosolic and secreted proteins.

Rosiglitazone, one of the thiazolidinedione (TZD), is an oral antidiabetic drug that activates a gamma isoform of peroxisome proliferator-activated receptor (PPARγ). To identify target proteins induced by rosiglitazone in adipocytes, we first performed simultaneous in-depth proteomic profiling of cytosolic proteins and secreted proteins (secretome) from 3T3-L1 adipocytes using a label-free quantification method with nano-UPLC MS/MS. In total, we identified 646 proteins from 3T3-L1 adipocytes, of which 172 and 162 proteins were upregulated and downregulated >1.5-fold, respectively, in rosiglitazone-treated cells, as compared to controls. Some differentially expressed proteins in particular, including fatty acid translocase (FAT)/CD36, fatty acid binding protein, lipoprotein lipase, acetyl CoA acyltransferase, carnitine O-palmitoyltransferase 2, sterol carrier protein, adiponectin, and phosphoenolpyruvate carboxykinase could explain the current action mechanism of TZDs. Furthermore, this study is the first to report on two potential target proteins of rosiglitazone, such as adenomatosis polyposis coli 2 (APC2), and eukaryotic translation initiation factor 5A-1 (eIF5A) related to apoptosis and cell division. Our data clearly suggest that in-depth proteomic approaches using cytosolic and secreted proteins are important and necessary for identification of drug targets at the protein level.

Delta inulin: a novel, immunologically active, stable packing structure comprising -D-[2 -&gt; 1] poly(fructo-furanosyl) -D-glucose polymers

We report a novel isoform of β-D-[2 → 1] poly(fructo-furanosyl) α-D-glucose termed delta inulin (DI), comparing it with previously described alpha (AI), beta (BI) and gamma (GI) isoforms. In vitro, DI is the most immunologically active weight/weight in human complement activation and in binding to monocytes and regulating their chemokine production and cell surface protein expression. In vivo, this translates into potent immune adjuvant activity, enhancing humoral and cellular responses against co-administered antigens. As a biocompatible polysaccharide particle, DI is safe and well tolerated by subcutaneous or intramuscular injection. Physico-chemically, DI forms as an insoluble precipitate from an aqueous solution of suitable AI, BI or GI held at 37-48°C, whereas the precipitate from the same solution at lower temperatures has the properties of AI or GI. DI can also be produced by heat conversion of GI suspensions at 56°C, whereas GI is converted from AI at 45°C. DI is distinguished from GI by its higher temperature of solution in dilute aqueous suspension and by its lower solubility in dimethyl sulfoxide, both consistent with greater hydrogen bonding in DI's polymer packing structure. DI suspensions can be dissolved by heat, re-precipitated by cooling as AI and finally re-converted back to DI by repeated heat treatment. Thus, DI, like the previously described inulin isoforms, reflects the formation of a distinct polymer aggregate packing structure via reversible noncovalent bonding. DI forms the basis for a potent new human vaccine adjuvant and further swells the growing family of carbohydrate structures with immunological activity.

Autophagy Is a Key Myeloma Survival Pathway That Can Be Manipulated Therapeutically to Enhance Apoptosis

AbstractAbstract 4083In normal cells, autophagy is up-regulated under conditions of stress ensuring cell survival, suggesting that if it is inhibited cell death will ensue. In some cancer settings however it has been shown to enhance cell death. Therefore if autophagy is to be a target for anti-cancer therapy, it is important to determine whether to promote or inhibit the process. Cellular entry to autophagy can be manipulated via the PI3K/AKT/mTOR pathway, a pathway known to be important for myeloma cell growth and survival. In this study we investigated the effects of PI-103, a dual Class I PI3K and mTOR inhibitor, which activates autophagy, and used it as a tool to investigate the interaction of autophagy with other myeloma therapies. As the PI3K/AKT/mTOR pathway has been suggested to be a central pathway controlling entry into autophagy, we first determined the basal expression of key members of the pathway in a panel of myeloma cell lines. PI3K alpha, beta delta and gamma isoforms were expressed to varying degrees and constitutive activation of the pathway, (pAKT or pMTOR), was seen in the majority of cell lines. As predicted, treatment with PI-103 induced autophagy in myeloma cells as demonstrated by an increase in cellular inclusions staining positively with acridine orange, cleavage of the autophagosome marker, LC3, and a decrease in p62. PI-103 was shown to inhibit proliferation of all the myeloma cell lines and patient cells tested to varying degrees, although 100% growth inhibition was not seen. Bone marrow stromal cells were unaffected. The main mode of action of PI-103 was autophagy activation, and in keeping with this the extent of cell death measured by Annexin V/PI binding and trypan blue exclusion, was minimal. Cell cycle analysis demonstrated an increase in G0-G1 phase. The unfolded protein response (UPR) is important in myeloma cells and UPR activation and autophagy have been reported to be interlinked. Following exposure to PI-103, splicing of XBP1 mRNA to its active form, XBP1s, was seen, CHOP and ATF4 mRNA levels were also increased, consistent with activation of at least two branches of the UPR in response to PI3K/mTOR inhibition and autophagy induction. As myeloma cells activate autophagy as a pro-survival pathway following PI3-kinase inhibition, we were interested to understand the effect of blocking autophagy in this context. When PI-103 was combined with the autophagy inhibitor, Bafilomycin greatly enhanced apoptosis was seen. This increased apoptosis was seen in cells constitutively expressing p-AKT, with a complete loss of both phospho- and total levels of AKT and mTOR, an increase in the cleaved forms of caspase 3 and Bcl2, and massive activation of the IRE1 and PERK branches of the UPR. This effect was not seen in cells lacking p-AKT, a phenomenon described as ‘context-dependent oncogene addiction’ suggesting that measurement of p-AKT may be a useful predictive marker for response to joint PI3K/autophagy inhibition. Importantly the pro-apoptotic effects of the combination are not overcome by the presence of bone marrow cytokines, a more representative model of the physiological situation in vivo. In conclusion our data highlights the interplay between known myeloma growth and survival pathways and autophagy and suggests that combining PI3K inhibitors, with agents that target autophagy, may be beneficial for the treatment of myeloma, particularly in >50% of patients that express p-AKT. Disclosures:No relevant conflicts of interest to declare.