Phosphoinositide 3-kinase P110δ Research Articles

Inhibition of PI3K p110δ activity reduces IgE production in IL-4 and anti-CD40 stimulated human B cell cultures.

Phosphoinositide 3-kinase (PI3K) p110δ signalling negatively regulates the production of mouse IgE. However, there are disparities between the mouse and human IgE biology, and the role of PI3K p110δ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110δ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibodyto induce class switching to IgE and IgG1 in the presence or absence of IC87114, a small molecule inhibitor of PI3K p110δ. Using FACS, RT-PCR and ELISA we examined the effect of PI3K p110δ inhibition on IgE production and determined the mechanisms involved. Unlike in mice, we observed that PI3K p110δ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However, the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110δ inhibition. The expression levels of AID, ε and γ1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However, we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40, specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition, PI3K p110δ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion, PI3K p110δ signalling is required for the production of human IgE, which makes it a pharmacological target for the treatment of allergic disease.

The phosphoinositide-3-kinase (PI3K)-delta inhibitor seletalisib impairs monocyte-derived dendritic cells maturation, APC function, and promotes their migration to CCR7 and CXCR4 ligands.

PI3K pathway plays a crucial role in dendritic cells (DCs) functions, as it regulates different cellular processes, such as maturation and cytokines production. However, the specific role of PI3K p110δ isoform in human DCs has not been thoroughly addressed. In this study, we analyze the effects of seletalisib, a potent and specific inhibitor of PI3K p110δ, on phenotype and antigen-presenting functions of monocyte-derived DCs undergone maturation via LPS. Seletalisib treatment reduced membrane HLA-DR as well as CD83 and CD40 costimulatory molecules, whereas CD80 and CD86 expression was only partially affected. Additionally, DCs cultures showed reduced TNF-α, IL-10, and IL-12 and increased IL-23 secretion levels. This resulted ina reduced capacity of DCs to prime allogeneic T cells, with a strong decrease of Th1 differentiation. On the other hand, PI3K p110δ inhibitor seletalisib increased CXCR4 and CCR7 expression and augmented the DCs migration toward CCL19 and CXCL12 ligands. At molecular level, inhibition of PI3K p110δ isoform by seletalisib significantly down-regulated the phosphorylation of AKT and other downstream signaling molecules, such as ribosomal protein S6, 4E-BP1, and NF-κB p65. In contrast, seletalisib did not affect p38 MAP kinase phosphorylation or TLR-associated adapter molecule TIRAP in DCs. Our results indicate that PI3K p110δ can serve as an important regulatory signalfor DCs, and selective inhibition of PI3K p110δisoform by seletalisib could be used for the prevention of exaggerated and harmful immune responsesoccurring in pathologic conditions, such as autoimmune disorders.

Roles of pulmonary telocytes in airway epithelia to benefit experimental acute lung injury through production of telocyte-driven mediators and exosomes.

BackgroundTelocytes (TCs) are experimentally evidenced as an alternative of cell therapies for organ tissue injury and repair. The aims of the present studies are to explore direct roles of TCs and the roles of TC-derived exosomes in support of experimental acute lung injury (ALI) in vivo or in vitro.Materials and methodsThe roles of TCs in experimental ALI were firstly estimated. Phosphoinositide 3-kinase (PI3K) p110δ and α/δ/β isoform inhibitors were used in study dynamic alterations of bio-behaviors, and in expression of functional factors of TCs per se and TC-co-cultured airway epithelial cells during the activation with lipopolysaccharide (LPS). TC-driven exosomes were furthermore characterized for intercellular communication by which activated or non-activated TCs interacted with epithelia. ResultsOur results showed that TCs mainly prevented from lung tissue edema and hemorrhage and decreased the levels of VEGF-A and MMP9 induced by LPS. Treatment with CAL101 (PI3K p110δ inhibitor) and LY294002 (PI3Kα/δ/β inhibitor) could inhibit TC movement and differentiation and increase the number of dead TCs. The expression of Mtor, Hif1α, Vegf-a, or Mmp9 mRNA increased in TCs challenged with LPS, while Mtor, Hif1α, and Vegf-a even more increased after adding CAL101 or Mtor after adding LY. The rate of epithelial cell proliferation was higher in co-culture of human bronchial epithelial (HBE) and TCs than that in HBE alone under conditions with or without LPS challenge or when cells were treated with LPS and CAL101 or LY294002. The levels of mTOR, HIF1α, or VEGF-A significantly increased in mono-cultured or co-cultured cells, challenged with LPS as compared with those with vehicle. LPS-pretreated TC-derived exosomes upregulated the expression of AKT, p-AKT, HIF1α, and VEGF-A protein of HBE.ConclusionThe present study demonstrated that intraperitoneal administration of TCs ameliorated the severity of lung tissue edema accompanied by elevated expression of VEGF-A. TCs could nourish airway epithelial cells through nutrients produced from TCs, increasing epithelial cell proliferation, and differentiation as well as cell sensitivity to LPS challenge and PI3K p110δ and α/δ/β inhibitors, partially through exosomes released from TCs.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s10565-021-09670-5.

Dynamic Regulation of the Molecular Mechanisms of Regulatory T Cell Migration in Inflamed Skin.

The presence of regulatory T cells (Tregs) in skin is important in controlling inflammatory responses in this peripheral tissue. Uninflamed skin contains a population of relatively immotile Tregs often located in clusters around hair follicles. Inflammation induces a significant increase both in the abundance of Tregs within the dermis, and in the proportion of Tregs that are highly migratory. The molecular mechanisms underpinning Treg migration in the dermis are unclear. In this study we used multiphoton intravital microscopy to examine the role of RGD-binding integrins and signalling through phosphoinositide 3-kinase P110δ (PI3K p110δ) in intradermal Treg migration in resting and inflamed skin. We found that inflammation induced Treg migration was dependent on RGD-binding integrins in a context-dependent manner. αv integrin was important for Treg migration 24 hours after induction of inflammation, but contributed to Treg retention at 48 hours, while β1 integrin played a role in Treg retention at the later time point but not during the peak of inflammation. In contrast, inhibition of signalling through PI3K p110δ reduced Treg migration throughout the entire inflammatory response, and also in the absence of inflammation. Together these observations demonstrate that the molecular mechanisms controlling intradermal Treg migration vary markedly according to the phase of the inflammatory response.

Essential role for the p110δ isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia

AbstractThe phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activated in blast cells from patients with acute myeloid leukemia (AML) and to contribute to survival and proliferation of these cells. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110α, p110β, p110γ, and p110δ) that are responsible for Akt activation. It is not known which PI3K isoform is critical in AML. Here we show that the p110δ isoform of PI3K is consistently expressed at a high level in blast cells from AML, in contrast to the other class I isoforms, the expression of which was very variable among patients. IC87114, a p110δ-selective inhibitor, suppressed both constitutive and Flt-3–stimulated Akt activation in blasts to the same extent as Ly294002, an inhibitor of all PI3K isoforms. Moreover, IC87114 inhibited AML cell proliferation without affecting the proliferation of normal hematopoietic progenitor cells. These observations identify p110δ as a potential therapeutic target in AML.

Inhibition of PI3Kδ Enhances Poly I:C-Induced Antiviral Responses and Inhibits Replication of Human Metapneumovirus in Murine Lungs and Human Bronchial Epithelial Cells.

Viral infections of the airway can exacerbate respiratory diseases, such as asthma or chronic obstructive pulmonary disease (COPD), and accelerate disease progression. Phosphoinositide 3-kinase (PI3K)δ, a class 1A PI3K, has been studied as a potential target for achieving anti-oncogenic and anti-inflammatory effects. However, the role of PI3Kδ in antiviral responses is poorly understood. Using a synthetic double-stranded RNA poly I:C and a selective PI3Kδ inhibitor IC87114, we investigated the role of PI3Kδ signaling in poly I:C-induced expression of the T lymphocyte-inhibitory molecule programmed death 1 ligand 1 (PD-L1), inflammatory responses and antiviral interferon (IFN) responses. C57BL/6N mice were treated with IC87114 or vehicle by intratracheal (i.t.) instillation followed by i.t. administration of poly I:C. Poly I:C increased PD-L1 expression on epithelial cells, lymphocytes, macrophages, and neutrophils in the lungs and IC87114 suppressed poly I:C-induced PD-L1 expression on epithelial cells and neutrophils possibly via inhibition of the Akt/mTOR signaling pathway. IC87114 also attenuated poly I:C-induced increases in numbers of total cells, macrophages, neutrophils and lymphocytes, as well as levels of KC, IL-6 and MIP-1β in bronchoalveolar lavage fluid. Gene expression of IFNβ, IFNλ2 and IFN-stimulated genes (ISGs) were upregulated in response to poly I:C and a further increase in gene expression was observed following IC87114 treatment. In addition, IC87114 enhanced poly I:C-induced phosphorylation of IRF3. We assessed the effects of IC87114 on human primary bronchial epithelial cells (PBECs). IC87114 decreased poly I:C-induced PD-L1 expression on PBECs and secretion of IL-6 and IL-8 into culture supernatants. IC87114 further enhanced poly I:C- induced increases in the concentrations of IFNβ and IFNλ1/3 in culture supernatants as well as upregulated gene expression of ISGs in PBECs. Similar results were obtained in PBECs transfected with siRNA targeting the PIK3CD gene encoding PI3K p110δ, and stimulated with poly I:C. In human metapneumovirus (hMPV) infection of PBECs, IC87114 suppressed hMPV-induced PD-L1 expression and reduced viral replication without changing the production levels of IFNβ and IFNλ1/3 in culture supernatants. These data suggest that IC87114 may promote virus elimination and clearance through PD-L1 downregulation and enhanced antiviral IFN responses, preventing prolonged lung inflammation, which exacerbates asthma and COPD.

Roles of TGFβ1 in the expression of phosphoinositide 3-kinase isoform genes and sensitivity and response of lung telocytes to PI3K inhibitors.

The mouse lung telocyte cell line (TCSV40) recently established provides further opportunities to learn TC biology and functions. The present study aims at investigating regulatory roles of phosphoinositide 3-kinase (PI3K) isoforms in TC proliferation and movement and in TGFβ1-induced sensitivity and response of lung TCs to PI3K inhibitors. Network and molecular interactions of genes coding PI3K family or TGFβ family proteins in mouse primary TCs were defined. Mouse lung TCSV40 proliferation, apoptosis, cell cycle, and dynamical bio-behaviors were measured with or without TGFβ1 stimulation or PI3K catalytic isoform protein (PI3K/mTOR, PI3Kα/δ/β, PI3K p110δ, or pan-PI3K) inhibitions. The present study showed the difference of network characteristics and interactions of genes coding PI3K isoform proteins or TGFβ family proteins in primary lung telocytes from mouse lungs compared to those of other cells residing in the lung. TGFβ1 had diverse effects on TC proliferation with altered TC number in G2 or S phase, independent upon the administered dose of TGFβ1. PI3Kα/δ/β, PI3K/mTOR, and PI3K p110δ were involved in TC proliferation, of which PI3Kα/δ/β was more sensitive. The effects of pan-PI3K inhibitor indicate that more PI3K isoforms were stimulated by the administering of external TGFβ1 and contributed to TGFβ1-induced TC proliferation. PI3K p110δ upregulated TC proliferation and movement dynamically without TGFβ1, and downregulated TC proliferation with TGFβ1 stimulation, but not TC movement. PI3Kα/δ/β and PI3K/mTOR were more active in TGFβ1-induced S phase accumulation and had similar dynamic effects to PI3K p110δ. Gene expression of PI3K isoforms in TCs was upregulated after TGFβ1 stimulation. The expression of PIK3CA coding p110-α or PIK3CG coding p110-γ were up- or downregulated in TCs without TGFβ1, respectively, when PI3K/mTOR, PI3Kα/δ/β, PI3K p110δ, or pan-PI3K were inhibited. TGFβ1 upregulated the expression of PIK3CA and PIK3CB, while downregulated the expression of PIK3CD and PIK3CG. Our data imply that TGFβ1 plays divergent roles in the expression of PI3K isoform genes in lung TCs and can alter the sensitivity and response of lung TCs to PI3K inhibitors.

Combined Pharmacological Inhibition of Bruton's Tyrosine Kinase (BTK) and Phosphoinositide 3-Kinase (PI3K) p110δ Rescues Monocytosis, Thrombocytopenia, and Splenomegaly in a Genetic Mouse Model for JMML

Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN). It has no effective treatments and the only treatment that prolongs survival is allogeneic hematopoietic stem cell transplant (HSCT). However, even with this aggressive intervention, approximately 50% of patients relapse with JMML within five years. The most commonly mutated gene in JMML patients is PTPN11, which encodes for the non-receptor protein tyrosine phosphatase SHP2. Gain-of-function (GOF) mutations in SHP2 lead to hyperactive RAS signaling and PTPN11 is recognized as a well-established oncogene in various leukemia's. Our lab has shown that p110δ, the hematopoietic-specific catalytic subunit of phosphoinositide 3-kinase (PI3K), plays an important role downstream of SHP2-signaling. Recently, we have shown that in vivo treatment with a specific pharmacologic p110δ inhibitor significantly prolongs the overall survival and reduces the splenomegaly seen in GOF Shp2-expressing mice. Having observed the effectiveness of PI3K p110δ inhibition in correcting the mutant Shp2-induced leukemia phenotype in vitro and in prolonging survival of mice in vivo, we next explored signaling molecules with which p110δ may be interacting to promote the aberrant Shp2 signaling in myeloid cells. In recent years, a key player in B cell receptor (BCR) signaling, Bruton's tyrosine kinase (BTK), has come under intense study in the field of lymphocytic leukemia and lymphoma research. Ibrutinib, a small molecule inhibitor targeting BTK, has proven to be very effective and has received FDA-approval for the treatment of a variety of B cell malignancies. Although research on BTK up to this point has focused on B cell malignancies, BTK is also highly expressed in myeloid cells and mice lacking BTK are defective in many myeloid cell functions. Thus, it is possible that BTK signaling may also play an important role in myeloid malignancies, such as JMML. Given the collaboration of BTK and p110δ in BCR signaling, the key role of p110δ in GOF Shp2-induced leukemia, and the high expression of BTK in myeloid cells, we hypothesized that BTK and p110δ function cooperatively in GOF Shp2-expressing myeloid cells to promote MPN. To test this hypothesis, we examined the potential collaboration of GM-CSF-stimulated BTK and p110δ in GOF Shp2-expressing myeloid cells and tested the inhibitor combination in vivo. We show that BTK cooperates with p110δ to promote GOF Shp2-induced leukemia. We show a role for B cell adaptor for PI3K (BCAP) in BTK upregulation of PI3K activity. In mutant Shp2 macrophages, BCAP phosphorylation is increased specifically in the larger isoforms regulating PI3K activation, and BTK inhibition results in a dose-dependent reduction in this phosphorylation. Our findings show that the MPN caused by GOF Shp2 is due to cooperative signaling between p110δ and BTK, which forms a positive feedback loop with BCAP, thus leading to more Akt/Erk hyperphosphorylation and hyperproliferation in response to GM-CSF.Given these in vitro observations, we treated GOF Shp2 bearing mice with a combination of a PI3K p110δ-specific inhibitor and, a BTK-specific inhibitor, or single agents alone and performed hematopoietic analysis. The combination treatment scheme completely rescued monocytosis and uniquely ameliorated thrombocytopenia compared to single agent treatment of diseased mice. In addition, splenomegaly was also completely rescued in all three treatment groups compared to vehicle. Detailed flow cytometric analysis of bone marrow hematopoietic stem and progenitor cell populations revealed significant repression of LSK, HPC1 and granulocyte macrophage progenitors (GMPs) with a concomitant increase in the megakaryocyte precursors (MKPs) in drug combination treated mice vs. other groups. Consistent with these observations, a significant reduction in mature myeloid cells was noted in the spleen and a complete rescue in the production of peripheral blood platelets was observed in the mutant mice treated with a combination of the two drugs. Thus, combination therapy with PI3K p110δ- and BTK-specific inhibitors profoundly rescues disease state hallmarks of JMML, and may warrant further clinical investigation. DisclosuresNo relevant conflicts of interest to declare.

Insulin-like growth factor-1 activates different catalytic subunits p110 of PI3K in a cell-type-dependent manner to induce lipogenesis-dependent epithelial-mesenchymal transition through the regulation of ADAM10 and ADAM17.

The activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) is critical for the induction of epithelial-mesenchymal transition (EMT) by growth factors, including insulin-like growth factor 1 (IGF-1). The activation of intracellular lipogenesis provides proliferative and survival signals for cancer cells. In this study, we investigated the connection between lipogenesis-related EMT processes and IGF-1-mediated PI3K p110 isoform activation in primary (SW480 cells) and metastatic (SW620) colon carcinoma cells. We also examined the underlying signaling pathway that promotes fatty acid synthesis in IGF-1-activated colon cancer cells. IGF-1 stimulation upregulated the expression of lipogenic enzymes as well as the activation of Nardilysin (N-arginine dibasic convertase, NRD1) and its downstream targets, a disintegrin and metalloproteases 10 (ADAM10) and ADAM17. The upregulation of the Lyn/Syk-mediated PI3K p110δ isoform in SW480 cells and the Lyn-dependent PI3K p110α isoform in SW620 cells triggered fatty acid production and cell motility in IGF-1-activated colon cancer cells. Pharmacological inhibition with A66 (PI3K p110α specific inhibitor) and CAL-101 (PI3K p110δ specific inhibitor) efficiently inhibited EMT in colon cancer cells by blocking the NRD1/ADAM family protein signaling pathway. Gene silencing of NRD1 and ADAM family proteins attenuated the generation of intracellular fatty acid and the migratory activity of colon cancer cells. Our results suggest that the different isoforms of the PI3K p110 subunit could be therapeutic targets for primary and metastatic colon cancer and that regulation of the NRD1/ADAM signaling pathway controls lipogenesis-mediated EMT in IGF-1-stimulated colon cancer cells.

The phosphoinositide-3 kinase signaling is involved in neuroinflammation in hypertensive rats.

It has been demonstrated that neuroinflammation is associated with cardiovascular dysfunction. The phosphoinositide-3 kinase (PI3K) signaling in the rostral ventrolateral medulla (RVLM), a key region for sympathetic outflow, is upregulated and contributes to increased blood pressure (BP) and sympathetic outflow in hypertension. This study was designed to determine the role of the PI3K signaling in neuroinflammation in the RVLM of hypertension. The normotensive WKY rats were performed by intracisternal infusion of lipopolysaccharide (LPS) or angiotensin II (Ang II) for inducing neuroinflammation. Elisa was used to determine the level of proinflammatory cytokines. Western blot was employed to detect the protein expression of PI3K signaling pathway. Gene silencing of PI3K p110δ subunit and overexpression of angiotensin-converting enzyme 2 (ACE2) were realized by injecting related lentivirus into the RVLM. In the spontaneously hypertensive rats (SHR), the PI3K signaling in the RVLM was upregulated compared with WKY, gene silencing of PI3K in the RVLM significantly reduced BP and renal sympathetic nerve activity (RSNA), but also decreased the levels of proinflammatory cytokines. In the WKY rats, central infusion of LPS and Ang II significantly elevated BP and RSNA, but also increased the levels of proinflammatory cytokines and PI3K signaling activation in the RVLM. These changes in the Ang II-induced hypertension were effectively prevented by gene silencing of PI3K in the RVLM. Furthermore, overexpression of ACE2 in the RVLM significantly attenuated high BP and neuroinflammation, as well as decreased the activation of PI3K signaling in hypertensive rats. This study suggests that the PI3K signaling in the RVLM is involved in neuroinflammation in hypertension and plays an important role in the renin-angiotensin system-mediated changes in neuroinflammation in the RVLM.

PI3 Kinase p110 Delta Is Required for Leukemic Cell Survival and Self-Renewal in t(8;21) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) with t(8;21) comprises about 10% of adult AML cases, and is the most frequent genetic alteration in pediatric AML. In t(8;21) AML, the AML1-ETO transcription factor fusion dysregulates transcription in hematopoietic cells, interfering with apoptosis and differentiation, and leading to enhanced self-renewal. The phosphoinositide-3-kinase (PI3K) signaling pathway is frequently overactivated in AML, representing a promising target in AML. However, PI3K also plays important roles in normal hematopoiesis. Hematopoietic cells express four catalytic subunits of Class I PI3K (p110α, p110β, p110γ, and p110δ). Previous studies have demonstrated that these isoforms have redundant roles in normal hematopoietic stem cells (HSCs), but play specific roles in some genetic subtypes of AML. Therefore, inhibiting individual PI3K isoforms in AML may allow for therapeutic targeting of this pathway with lower toxicity. In fact, isoform-selective PI3K inhibitors are already in clinical development, and the p110δ-selective inhibitor idelalisib is approved by the FDA for lymphoid malignancies.To determine whether t(8;21) AML cells are dependent upon specific PI3K isoforms, we performed proliferation and apoptosis assays in Kasumi-1 cells, a t(8;21) human AML cell line, with PI3K inhibitors: pictilisib (GDC0941, a pan-PI3K inhibitor), buparlisib (BKM120, a pan-PI3K inhibitor), alpelisib (BYL719, a p110α-selective inhibitor), idelalisib (GS1101,a p110δ-selective isoform inhibitor) and KIN193 or TGX221 (p110β-selective inhibitors). We observed a decrease in proliferation and in Akt/mTOR signaling with the pan-PI3K inhibitors and alpelisib. The pan-PI3K inhibitors, alpelisib, and idelalisib also induced apoptosis in Kasumi-1 cells. In addition, we observed an increase in myeloid differentiation of Kasumi-1 cells with buparlisib or idelalisib. The splice isoform AML1-ETO9a is found in patients with t(8;21) AML, induces AML in mice when introduced into hematopoietic cells by retroviral transduction, and confers serial replating in methylcellulose (Yan et al, Nature Medicine 2006). To investigate the role of p110δ in leukemogenesis genetically, we harvested bone marrow cells from mice with a germline deletion of pik3cd, which encodes p110δ, and transduced them with a retrovirus expressing AML1-ETO9a. To study the ability of AML1-ETO9a leukemic cells to self-renew after p110δ deletion, we performed serial replating assays in methylcellulose.We found that, while AML1-ETO9a confers self-renewal activity to wild-type (WT) cells past round 8 of serial replating, p110δ-/- cells gradually lost self-renewal activity, and were unable to replate past round 4. Consistent with our genetic data, we found that the p110δ-selective inhibitor idelalisib or the pan-PI3K/mTOR inhibitor dactolisib (BEZ-235) can significantly reduce serial replating by AML1-ETO9a cells. To determine whether p110δ is also important for the initiation and progression of AML1-ETO AML in vivo, we transduced ckit-enriched bone marrow cells from WT or p110δ-/- mice with AML1-ETO9a-GFP retrovirus, and injected 200,000 GFP-sorted cells into lethally irradiated C57 Bl/6 recipient mice. During an interim analysis of this ongoing experiment, we have observed lower white cell counts in recipients of p110δ-/- cells compared with controls, suggesting a decrease in disease burden of AML1-ETO9a AML in the absence of p110δ.It has been reported that the thrombopoietin (TPO)/MPL/JAK2 signaling pathway is activated in t(8;21) AML, and is important in promoting proliferation and self-renewal of AML1-ETO leukemic cells (Pulikkan et al, Blood, 2012; Lo et al, Blood 2012). We hypothesized that p110δ may be required downstream of the TPO/MPL/JAK2 signaling pathway for leukemic self-renewal in t(8;21) AML. To determine whether TPO signaling depends upon p110δ in AML1-ETO9a cells, we treated WT and p110δ-/- cells transduced with AML1-ETO9a with TPO, and performed replating assays. We found that, although TPO treatment augmented the in vitro self-renewal of WT AML1-ETO9a cells to round 9 with increased colony numbers, p110δ-/- AML1-ETO9a cells treated with TPO failed to replate past round 5. These data suggest that p110δ is important for leukemic survival and self-renewal in AML1-ETO AML, in part by mediating signaling downstream of the TPO/MPL/JAK2 signaling axis. DisclosuresNo relevant conflicts of interest to declare.

A Novel Method of Preparing (S)-5-Fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)propyl]-3H-quinazolin-4-one

Idelalisib ((S)-5-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin -4-one, 9) is the first-in-class, highly selective inhibitor of phosphoinositide 3-kinase p110δ; it has been clinic...

Targeting PI3Kδ and PI3Kγ signalling disrupts human AML survival and bone marrow stromal cell mediated protection.

Phosphoinositide-3-kinase (PI3K) is an enzyme group, known to regulate key survival pathways in acute myeloid leukaemia (AML). It generates phosphatidylinositol-3,4,5-triphosphate, which provides a membrane docking site for protein kinaseB activation. PI3K catalytic p110 subunits are divided into 4 isoforms; α,β,δ and γ. The PI3Kδ isoform is always expressed in AML cells, whereas the frequency of PI3Kγ expression is highly variable. The functions of these individual catalytic enzymes have not been fully resolved in AML, therefore using the PI3K p110δ and p110γ-targeted inhibitor IPI-145 (duvelisib) and specific p110δ and p110γ shRNA, we analysed the role of these two p110 subunits in human AML blast survival. The results show that PI3Kδ and PI3Kγ inhibition with IPI-145 has anti-proliferative activity in primary AML cells by inhibiting the activity of AKT and MAPK. Pre-treatment of AML cells with IPI-145 inhibits both adhesion and migration of AML blasts to bone marrow stromal cells. Using shRNA targeted to the individual isoforms we demonstrated that p110δ-knockdown had a more significant anti-proliferative effect on AML cells, whereas targeting p110γ-knockdown significantly inhibited AML migration. The results demonstrate that targeting both PI3Kδ and PI3Kγ to inhibit AML-BMSC interactions provides a biologic rationale for the pre-clinical evaluation of IPI-145 in AML.

Human Cytomegalovirus Induces an Atypical Activation of Akt To Stimulate the Survival of Short-Lived Monocytes.

Human cytomegalovirus (HCMV) is a pervasive herpesvirus responsible for significant morbidity and mortality among immunodeficient/naive hosts. Following a primary HCMV infection, circulating blood monocytes mediate the systemic spread of the virus. Extending the short 48-h life span of monocytes is critical to the viral dissemination process, as these blood-borne cells are nonpermissive for virus replication until they are fully differentiated into macrophages. Here, we show that HCMV glycoprotein gB binding to cellular epidermal growth factor receptor (EGFR) during HCMV entry initiated a rapid (within 15 min) activation of the apoptosis suppressor Akt, which was maintained through 72 h. The virus-induced activation of Akt was more robust than that with the normal myeloid growth factor macrophage colony-stimulating factor (M-CSF) and was essential for infected monocytes to bypass the 48-h viability checkpoint. Activation of phosphoinositide 3-kinase (PI3K) following EGFR engagement by HCMV mediated the phosphorylation of Akt. Moreover, HCMV entry drove a switch away from the PI3K p110δ isoform, which was required for the viability of uninfected monocytes, to the p110β isoform in order to facilitate the Akt-dependent prosurvival state within infected cells. Simultaneously, in contrast to M-CSF, HCMV promoted a rapid increase in SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) expression, leading to signaling through a noncanonical Akt activation pathway. To ensure maximum Akt activity, HCMV also induced an early phosphorylation-dependent inactivation of the negative regulator phosphatase and tensin homolog. Overall, our data indicate that HCMV hijacks the upstream Akt signaling network to induce a nontraditional activation of Akt and subsequently a prosurvival decision at the 48-h cell fate checkpoint, a vital step for HCMV's dissemination and persistence strategy. HCMV is found throughout the world with a prevalence of 55 to 100% within the human population. HCMV infection is generally asymptomatic in immunocompetent or naive individuals but is a significant cause of morbidity and mortality among the immunocompromised. Widespread organ inflammation is associated with symptomatic infections, which is a direct consequence of the viral dissemination strategy. Inflammatory peripheral blood monocytes facilitate the spread of HCMV. However, HCMV must subvert the naturally short life span of monocytes. In this work, we demonstrate that HCMV induces the activation of Akt, an antiapoptotic protein, in a manner distinct from that of normal myeloid growth factors. Moreover, we decipher how HCMV dysregulates the upstream Akt signaling network during viral entry to promote an Akt-dependent prosurvival state following infection. Delineation of the virus-specific mechanisms that regulate cellular prosurvival pathways in order to drive the survival of HCMV-infected monocytes is important to identifying new anti-HCMV therapeutic targets.

Genetic Inhibition of PI3K p110delta Antagonizes Survival Signals and Induces Immune Activation in Chronic Lymphocytic Leukemia (CLL)

Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) p110δ pathway. In addition, immune tolerance, a critical mechanism for cancer immune invasion, remains a major barrier for effective anti-cancer therapy. PI3K p110δ also plays critical roles in T cell development and function. Studies using genetic p110δ kinase-dead mice have showed that p110δ inhibition can reverse the tumor induced immune tolerance, impairs the expansion of regulatory T cells (Treg) thereby enhancing the cytotoxic CD8+ T cells to induce tumor regression in various solid tumor models.Methods: Here we use a PI3K p110δ kinase-dead (δD910A/D910A) mouse model and a well-characterized Eμ-TCL1 mouse CLL model to delineate the role of p110δ signaling in CLL leukemia pathogenesis and microenvironmental immune tolerance. The δD910A/D910A has a point mutation on the p110δ kinase domain, which results in the complete inactivation of p110δ activity without affecting the expression of p110δ or the other isoforms. TCL1 mice exhibit B-cell leukemia similar to that in humans beginning around 4-6 months. Adoptive transfer experiments are performed by tail-vein injection of 5e6 splenic lymphocytes from a leukemic Eμ-TCL1 mouse. Leukemia progression is measure by flow cytometry for the CD5+ CD19+ % lymphocytes in the peripheral blood. Whole blood cytotoxicity assay was performed by incubating whole blood from recipient mice with cryopreserved cells from the original donor to assess the their cytotoxic effects towards the donor cells.Results: δD910A/D910A mice were crossed with TCL1 mice to examine the effect of systemic p110δ inactivation in CLL. We show that global inactivation of PI3K p110δ significantly prevented spontaneous leukemia development. Both δWT /TCL1 and δWT/D910A /TCL1 mice developed CLL like disorder starting 4 months whereas δD910A/D910A /TCL1 mice did not show any sign of disease up to 14 months. This indicates that p110δ is a critical kinase for CLL disease initiation and expansion.We then performed adoptive transfer of TCL1 leukemic cells into p110δ mice to examine the role of p110δ in the microenvironment. We show that inhibiting p110δ in the microenvironment impairs leukemia engraftment. On day 31 after engraftment, 7 out of 10 mice from the δWT group developed leukemia while no mice from δD910A/D910A group had detectable leukemia. On day 43, δD910A/D910A group started to have leukemia incidence (2 out of 7) when δWT group continued to progress. δWT/D910A group showed intermediate leukemia incidence and disease development, which were different from the phenotype we saw in δWT/D910A /TCL1. This suggests that p110δ plays a different role outside of the leukemia compartment.To identify potential effector cells, we performed adoptive transfer of ovalbumin (OVA) -expressing TCL1 leukemia cells into p110δ mice. The TCR (OT1) specific SIINFEKL peptide of OVA is used to induce the expansion of OVA antigen specific CD8+ T cells. Interestingly, upon engraftment, δWT and δWT/D910A mice showed similar levels (4% and 3.5%, respectively) of OVA specific CD8+ T cells whereas the δD910A/D910A mice only have 2%. This appears to be contradicting to the less leukemia development we observed previously. However, in vitro whole blood killing assay showed that whole blood from δD910A/D910A mice exhibited higher cytotoxicity to TCL1 leukemia cells despite their impaired development of the antigen specific anti-tumor CD8+ T cells.Conclusions: This study demonstrates that blocking p110δ in CLL not only abrogates survival signals in leukemic cells but also has the potential to reverse immune suppression in the micoenvironment. Studies are ongoing to pinpoint the effector cell subsets. The finding from this study will suggest that idelalisib, the PI3K p110δ inhibitor recently approved by FDA for the treatment of CLL, or other inhibitors of this pathway can potentially be used to treat a wide range of cancers by unleashing host anti-tumor immunity. DisclosuresByrd:Acerta Pharma BV: Research Funding.

Abstract B44: Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton's tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. The PI3K p110δ inhibitor, idelalisib, was granted a breakthrough therapy designation by the FDA in relapsed CLL and the BTK inhibitor, ibrutinib, has received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies. Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, IPI-145, which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of IPI-145 in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM IPI-145 for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to IPI-145, displaying about 20% decrease in viability at concentrations greater than 0.5μM, however the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM IPI-145 for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, IPI-145 is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, IPI-145. This study provides solid rationale for ongoing clinical development of IPI-145 in CLL patients. Additionally, as IPI-145 can continue to inhibit the PI3K/AKT pathway in the setting of a C481S BTK mutation, it provides justification for clinical studies of this agent in this subset of patients. Citation Format: Amy J. Johnson, Shuai Dong, Daphne Guinn, Jason A. Dubovsky, Yiming Zhong, Amy Lehman, Jeff Kutok, Jennifer A. Woyach, John C. Byrd. Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B44.

P110Delta Inhibits Monocyte Infiltration by Thioglycollate-Induced Periotoneal Inflammation but Not HCD-Induced Inflammation and Atherosclerosis in APOE KO Mice.

We have previously reported that phosphoinositide 3-kinase p110δ knockout (p110δ KO) diminished the adhesion of leukocytes to capillary venules and suppressed the peritoneal infiltration of leukocytes, both functions that play important roles in atherosclerosis. Therefore, we hypothesized that p110δ deficiency might be protective against atherosclerosis. Apolipoprotein E knockout (ApoE KO) mice were crossed with p110δ KO mice to generate homozygous double knockout mice (ApoE/p110δ DKO). The present study showed that ApoE/p110δ DKO mice fed with a high cholesterol diet (HCD) demonstrated less peritoneal infiltration of leukocytes and monocytes compared with ApoE KO mice after intraperitoneal injection of thioglycollate, an inducer of acute peritoneal inflammation. Unexpectedly, atherosclerosis in the aortic root and in the entire aorta was similar between the ApoE/p110δ DKO and ApoE KO groups. No difference in Mac-3 expression, indicative of macrophage infiltration, was found between the two groups. Further analysis showed that ApoE KO mice chronically fed with HCD had increased levels of total cholesterol, low-density lipoprotein in the blood and counts and percentages of circulating monocytes compared with ApoE KO mice fed with a normal diet. Consistently, the deficiency of p110δ affected neither the counts nor the percentages of monocytes nor the lipid profiles in the blood. The results suggested that p110δ plays an important role in acute but not in chronic inflammation, the latter being included in the early characteristics of atherosclerosis, which might explain the finding that p110δ deficiency fails to inhibit early atherosclerosis.

The Phosphoinositide 3-Kinase p110α Isoform Regulates Leukemia Inhibitory Factor Receptor Expression via c-Myc and miR-125b to Promote Cell Proliferation in Medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and represents the main cause of cancer-related death in this age group. The phosphoinositide 3-kinase (PI3K) pathway has been shown to play an important role in the regulation of medulloblastoma cell survival and proliferation, but the molecular mechanisms and downstream effectors underlying PI3K signaling still remain elusive. The impact of RNA interference (RNAi)-mediated silencing of PI3K isoforms p110α and p110δ on global gene expression was investigated by DNA microarray analysis in medulloblastoma cell lines. A subset of genes with selectively altered expression upon p110α silencing in comparison to silencing of the closely related p110δ isoform was revealed. Among these genes, the leukemia inhibitory factor receptor α (LIFR α) was validated as a novel p110α target in medulloblastoma. A network involving c-Myc and miR-125b was shown to be involved in the control of LIFRα expression downstream of p110α. Targeting the LIFRα by RNAi, or by using neutralizing reagents impaired medulloblastoma cell proliferation in vitro and induced a tumor volume reduction in vivo. An analysis of primary tumors revealed that LIFRα and p110α expression were elevated in the sonic hedgehog (SHH) subgroup of medulloblastoma, indicating its clinical relevance. Together, these data reveal a novel molecular signaling network, in which PI3K isoform p110α controls the expression of LIFRα via c-Myc and miR-125b to promote MB cell proliferation.

Phosphoinositide 3-kinase p110δ promotes lumen formation through the enhancement of apico-basal polarity and basal membrane organization.

Signalling triggered by adhesion to the extracellular matrix plays a key role in the spatial orientation of epithelial polarity and formation of lumens in glandular tissues. Phosphoinositide 3-kinase signalling in particular is known to influence the polarization process during epithelial cell morphogenesis. Here, using Madin-Darby canine kidney epithelial cells grown in 3D culture, we show that the p110δ isoform of phosphoinositide 3-kinase co-localizes with focal adhesion proteins at the basal surface of polarized cells. Pharmacological, siRNA- or kinase-dead-mediated inhibition of p110δ impair the early stages of lumen formation, resulting in inverted polarized cysts, with no laminin or type IV collagen assembly at cell/extracellular matrix contacts. p110δ also regulates the organization of focal adhesions and membrane localization of dystroglycan. Thus, we uncover a previously unrecognized role for p110δ in epithelial cells in the orientation of the apico-basal axis and lumen formation.

Pharmacologic Inhibition with Duvelisib (IPI-145) and Genetic Inhibition of PI3K p110δ Antagonizes Intrinsic and Extrinsic Survival Signals in Chronic Lymphocytic Leukemia (CLL)

Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen, and bone marrow. CLL cells display up-regulated B-cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton’s tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. Both the PI3K p110δ inhibitor, idelalisib, and the BTK inhibitor, ibrutinib, have received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies.Methods/Results: Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, duvelisib (IPI-145), which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of duvelisib in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM duvelisib for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to duvelisib, displaying about 20% decrease in viability at concentrations greater than 0.5μM; however, the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM duvelisib for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, duvelisib is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in the leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose-dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, duvelisib. DisclosuresDubovsky:Principia Inc.: Research Funding. Kutok:Infinity Pharmaceuticals, Inc.: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.