P110δ Expression Research Articles

Paradoxical dominant negative activity of an immunodeficiency-associated activating PIK3R1 variant.

PIK3R1 encodes three regulatory subunits of class IA phosphoinositide 3-kinase (PI3K), each associating with any of three catalytic subunits, namely p110α, p110β or p110δ. Constitutional PIK3R1 mutations cause diseases with a genotype-phenotype relationship not yet fully explained: heterozygous loss-of-function mutations cause SHORT syndrome, featuring insulin resistance and short stature attributed to reduced p110α function, while heterozygous activating mutations cause immunodeficiency, attributed to p110δ activation and known as APDS2. Surprisingly, APDS2 patients do not show features of p110α hyperactivation, but do commonly have SHORT syndrome-like features, suggesting p110α hypofunction. We sought to investigate this. In dermal fibroblasts from an APDS2 patient, we found no increased PI3K signalling, with p110δ expression markedly reduced. In preadipocytes, the APDS2 variant was potently dominant negative, associating with Irs1 and Irs2 but failing to heterodimerise with p110α. This attenuation of p110α signalling by a p110δ-activating PIK3R1 variant potentially explains co-incidence of gain-of-function and loss-of-function PIK3R1 phenotypes.

Leverage of nuclease-deficient CasX for preventing pathological angiogenesis

Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2%± 6.5%) invitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1%± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression.

Genome Editing of Pik3cd Impedes Abnormal Retinal Angiogenesis.

Abnormal angiogenesis is associated with myriad human diseases, including proliferative diabetic retinopathy (PDR). Signaling transduction through phosphoinositide 3-kinases (PI3Ks) plays a critical role in angiogenesis. Herein, we showed that p110δ, the catalytic subunit of PI3Kδ, was highly expressed in pathological retinal vascular endothelial cells (ECs) in a mouse model of oxygen-induced retinopathy (OIR) and in fibrovascular membranes from patients with PDR. To explore novel intervention with PI3Kδ expression, we developed a recombinant dual adeno-associated viral (rAAV) system for delivering CRISPR/Cas9 in which Streptococcus pyogenes (Sp) Cas9 expression was driven by an endothelial specific promoter of the intercellular adhesion molecule 2 (pICAM2) to edit genomic Pik3cd, the gene encoding p110δ. We then demonstrated that infection of cultured mouse vascular ECs with the dual rAAV1s of rAAV1-pICAM2-SpCas9 and rAAV1-SpGuide targeting genomic Pik3cd resulted in 80% DNA insertion/deletion in the locus of genomic Pik3cd and 70% depletion of p110δ expression. Furthermore, we showed that in the mouse model of OIR editing retinal Pik3cd with the dual rAAV1s resulted in not only a significant decrease in p110δ expression, and Akt activation, but also a dramatic reduction in pathological retinal angiogenesis. These findings reveal that Pik3cd editing is a novel approach to treating abnormal retinal angiogenesis.

PI3 kinase isoform p110δ is more important than p110α in KIT signaling in hematopoietic cells

PI3 kinases are important for KIT signaling and KIT mutants mediated cell transformation. In order to know the difference of PI3 kinase isoforms p110α and p110δ in the signaling of wild-type KIT and the often occurred KIT mutation D816V in hematopoietic malignancy mastocytosis, the predominant PI3 kinase isoform p110δ in hematopoietic tissues was knocked out in hematopoietic cells. We found that loss of p110δ expression dramatically inhibits PI3 kinase activation mediated by both wild-type KIT and KIT/D816V. By over expression of p110α in p110δ knock out cells, wild-type KIT mediated PI3 kinase activation was not changed while over expression of p110δ increased PI3 kinase activation. Similarly, in KIT/D816V expressing cells without p110δ expression, over expression of p110δ but not p110α restored PI3 kinase activation. In agreement with the signaling results, cell proliferation, cell survival and cell cycle assay further showed that over expression of p110δ but not p110α in p110δ knock out cells increases both wild-type KIT and KIT/ D816V mediated cell survival and proliferation. These results suggested that p110δ plays a more important role than p110α in KIT signaling and KIT mutant mediated cell transformation in hematopoietic cells.

PI 3‐kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3‐kinase (PI3K) and its product phosphatidylinositol (3,4,5)‐trisphosphate (PIP3). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion.

Echinochrome A Promotes Ex Vivo Expansion of Peripheral Blood-Derived CD34+ Cells, Potentially through Downregulation of ROS Production and Activation of the Src-Lyn-p110δ Pathway.

Intracellular reactive oxygen species (ROS) play an important role in the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). HSPCs are difficult to be expanded ex vivo while maintaining their stemness when they are exposed to oxidative damage after being released from the bone marrow. There have been efforts to overcome this limitation by using various cytokine cocktails and antioxidants. In this study, we investigated the effects of echinochrome A (Ech A)-a well-established and non-toxic antioxidant-on the ex vivo expansion of HSPCs by analyzing a CD34+ cell population and their biological functions. We observed that Ech A-induced suppression of ROS generation and p38-MAPK/JNK phosphorylation causes increased expansion of CD34+ cells. Moreover, p38-MAPK/JNK inhibitors SB203580 and SP600125 promoted ex vivo expansion of CD34+ cells. We also demonstrated that the activation of Lyn kinase and p110δ is a novel mechanism for Ech A to enhance ex vivo expansion of CD34+ cells. Ech A upregulated phospho-Src, phospho-Lyn, and p110δ expression. Furthermore, the Ech A-induced ex vivo expansion of CD34+ cells was inhibited by pretreatment with the Src family inhibitor PP1 and p110δ inhibitor CAL-101; PP1 blocked p110δ upregulation and PI3K/Akt activation, whereas CAL-101 and PI3K/Akt pathway inhibitor LY294002 did not block Src/Lyn activation. These results suggest that Ech A initially induces Src/Lyn activation, upregulates p110δ expression, and finally activates the PI3K/Akt pathway. CD34+ cells expanded in the presence of Ech A produced equal or more hematopoietic colony-forming cells than unexpanded CD34+ cells. In conclusion, Ech A promotes the ex vivo expansion of CD34+ cells through Src/Lyn-mediated p110δ expression, suppression of ROS generation, and p38-MAPK/JNK activation. Hence, Ech A is a potential candidate modality for the ex vivo, and possibly in vivo, expansion of CD34+ cells.

PB1647 EXPRESSION OF PHOSPHORYLATED AKT AND CLASS I PI3K ISOFORMS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA

Background : Acute lymphoblastic leukemia (ALL) is the most common neoplastic disease in children and represents 30% of all childhood malignancies. Although more than 80% of ALL cases are long term survivors, there is still a percentage of resistant or relapsed disease. The identification of genes and molecular pathways that are involved is crucial for targeted therapy design. The intracellular signaling pathway PI3K/AKT gets induced by growth factors and plays an important role in triggering many biological processes of the cell such as cell growth, metabolism, proliferation, survival, metabolism of insulin, protein synthesis and apoptosis. Recently it has been found that changes in the expression and mutations in many molecules of that pathway are involved in tumor genesis, which makes them an attractive target for cancer therapy. Aims: The study of the expression of the P110β, P110δ and P-Akt proteins in leukemic blasts of bone marrow from children with ALL at diagnosis and lymphomononuclear cells from children in remission, compared to the expression in lymphomononuclear cells from bone marrow of children with solid tumors without marrow involvement. Methods: Bone marrow mononuclear cells from 31 children with ALL at diagnosis, 25 children with ALL in remission and 20 children with solid tumors without bone marrow involvement was collected. Proteins were extracted and were analyzed with Western blot. The detection of P-Akt, P110β and P110δ was done with Thermo Scientific™ Pierce™ ECL Western Blotting Substrate,.The analysis of the results was performed with Image Lab and the statistical analysis with IBM SPSS Statistics version 25 (Kruskal-Wallis test and Mann-Whitney test). Results: The expression level of P-Akt in ALL diagnosis was significantly lower compared with ALL in remission (0.66 ± 0.21 vs 1.97 ± 0.41, p = 0.004) as well as with the solid tumors without bone marrow involvement (0.66 ± 0.21 vs 1.03 ± 0.25, p = 0.019). Additionally, the expression of P110δ estimated significantly lower in ALL diagnosis compared with ALL remission (0.92 ± 0.37 vs 1.52 ± 0.41, p = 0.012). On the other hand, there was no statistically significant difference of P110β expression values between ALL diagnosis, remission and solid tumors. Summary/Conclusion: P-Akt and P110δ expression was estimated decreased in ALL diagnosis although P110β expression was not found to be different between ALL diagnosis, ALL remission and solid tumors. Further studies are needed in order to clarify the relation of these proteins’ expression with other established prognostic factors and their role to the leukemia course, outcome and prognosis.

Phosphoinositide 3-kinase-delta could be a biomarker for eosinophilic nasal polyps

Nasal polyps (NP) cause diverse clinical symptoms of chronic rhinosinusitis (CRS). Chronic inflammation of sinonasal mucosa is known to be crucial in NP formation. We aimed to define the implications of phosphoinositide 3-kinase (PI3K)-δ in nasal inflammation associated with NP by analyzing NP tissue obtained from CRS patients. Results showed that expression of p110δ, a regulatory subunit of PI3K-δ, in NP tissue was increased compared to control tissue. Increased p110δ expression was closely correlated with more severe CRS features. Interestingly, p110δ expression was increased in eosinophilic NP, which are closely related to more complicated clinical courses of the disease. Furthermore, CRS patients possessing NP with higher p110δ expression displayed more eosinophils in NP tissue and blood, higher levels of IL-5 in NP tissue, and more severe features of the disease. Therefore, PI3K-δ may contribute to the formation of NP, especially eosinophilic NP associated with more severe clinical presentations and radiological features.

Phosphatidylinositol 3-kinase p110δ expression in Merkel cell carcinoma.

The prognosis of stage III/IV Merkel cell carcinoma (MCC) is very poor. The Phosphatidylinositol 3-kinase p110δ specific inhibitor idelalisib has recently been reported to induce complete clinical remission in a stage IV MCC patient. Here we assessed the expression of p110δ in primary MCC and MCC cell lines including its functionality.Immunofluorescence microscopy revealed a specific cytoplasmic p110δ expression in 71.4% of the tested MCCs and in all tested MCC cell lines. Compared to the B cell leukemia cell line REH all MCC cell lines, except MKL-1, revealed a lower response towards the treatment with idelalisib. MKL-1 showed a 10-fold higher IC50 compared to REH which was accompanied by a significant decrease of Akt phosphorylation. However, treating the MCC cells with the specific PI3K p110α subunit inhibitor BYL719 led to a more effective decrease of the cell viability compared to idelalisib: WaGa cells 30-fold, PeTa cells 15-fold and all other MCC cell lines 3-fold.Although PI3K p110δ is expressed in the majority of MCCs and cell lines its inhibition by idelalisib alone does not suffice to effectively affect MCC cells viability.

A p110δ-specific inhibitor combined with bortezomib blocks drug resistance properties of EBV-related B cell origin cancer cells via regulation of NF-κB.

Epstein-Barr virus (EBV) infection is closely related to carcinogenesis of various cancers, and is also associated with the development of drug resistance in cancer stem cells. However, in EBV-positive cancer cells, the mechanistic details of the downstream signaling and the connection of PI3K with the NF-κB pathway for development of drug resistance remain controversial. Diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM) cells infected by EBV display drug resistance-related proteins (MDR1, MRP1 and MRP2) and stem cell markers (OCT4 and SOX2). EBV-infected HT (HT/EBV) and H929 (H929/EBV) cells activated p110δ expression, but downregulated the expression of p110α and p110β. A combination of CAL-101, a p110δ-specific inhibitor, with bortezomib treatment of HT/EBV cells synergistically suppressed proliferation, reduced levels of drug resistance-related proteins, activated caspase cleavage and recovered expression of p110α/p110β. Additionally, co-treatment with CAL-101 and bortezomib attenuated the expression of OCT4 and SOX2 via inhibition of activated NF-κB. Co-treatment with CAL-101 and bortezomib also attenuated drug resistance and NF-κB activity of EBV-infected H929 cells. Our results provide supportive evidence for the clinical application of CAL-101 and bortezomib to treat EBV-infected hematologic cancer.

Class IA Phosphatidylinositol 3-Kinase Isoform p110α Mediates Vascular Remodeling.

Neointima formation after vascular injury remains a significant problem in clinical cardiology, and current preventive strategies are suboptimal. Phosphatidylinositol 3'-kinase is a central downstream mediator of growth factor signaling, but the role of phosphatidylinositol 3'-kinase isoforms in vascular remodeling remains elusive. We sought to systematically characterize the precise role of catalytic class IA phosphatidylinositol 3'-kinase isoforms (p110α, p110β, p110δ), which signal downstream of receptor tyrosine kinases, for vascular remodeling in vivo. Western blot analyses revealed that all 3 isoforms are abundantly expressed in smooth muscle cells. To analyze their significance for receptor tyrosine kinases-dependent cellular responses, we used targeted gene knockdown and isoform-specific small molecule inhibitors of p110α (PIK-75), p110β (TGX-221), and p110δ (IC-87114), respectively. We identified p110α to be crucial for receptor tyrosine kinases signaling, thus affecting proliferation, migration, and survival of rat, murine, and human smooth muscle cells, whereas p110β and p110δ activities were dispensable. Surprisingly, p110δ exerted noncatalytic functions in smooth muscle cell proliferation, but had no effect on migration. Based on these results, we generated a mouse model of smooth muscle cell-specific p110α deficiency (sm-p110α(-/-)). Targeted deletion of p110α in sm-p110α(-/-) mice blunted growth factor-induced cellular responses and abolished neointima formation after balloon injury of the carotid artery in mice. In contrast, p110δ deficiency did not affect vascular remodeling in vivo. Receptor tyrosine kinases-induced phosphatidylinositol 3'-kinase signaling via the p110α isoform plays a central role for vascular remodeling in vivo. Thus, p110α represents a selective target for the prevention of neointima formation after vascular injury, whereas p110β and p110δ expression and activity do not play a significant role.

Abstract LB-7: Design and development of a novel series of orally active, selective PI3Kp110β/δ inhibitors for the treatment of cancer

Abstract The phosphatidylinositide 3-kinases (PI3Ks) have emerged as attractive therapeutic targets for the treatment of cancer. The majority of PI3K inhibitors currently in clinical development primarily target either all four class I enzymes (p110α, β, δ and γ) with limited selectivity, or are pan-class I/mTOR dual inhibitors. Over recent years, a switch of focus to more selective PI3K inhibitors for the treatment of specific tumor types has occurred. Of particular note, p110b has been reported to be the principal isoform in driving PTEN-deficient tumorigenesis; additionally, high levels of p110δ expression in solid tumor cells have been shown to suppress PTEN activity, generating cellular sensitivity to p110δ inhibition through PTEN activation. Moreover, PI3K pathway activation in certain PTEN-null solid tumors has been shown to be mediated by both the p110β and p110δ catalytic subunits, suggesting the potential of a dual-selective p110β/δ inhibitor in this setting. During the past 12 months, the discovery of activating mutant forms of both p110β and p110δ has been reported. The strategy outlined in this presentation centres on the design and development of a new class of orally-active small molecules that selectively inhibit p110β and p110δ, and which display high degrees of specificity over protein kinases and other lipid kinases. Moreover, a spectrum of p110β/δ selectivity has been achieved in this series, which has facilitated interrogation of preferred isoform inhibition profiles required to confer tumor growth inhibition; additionally, by tuning selectivity over the other class I isoforms, elimination of the mechanism-related toxicities associated with the pan-class I inhibitors currently in the clinic can be realized, making such agents potentially more conducive to combination therapy. In vitro and in vivo therapy data for advanced lead compounds will be disclosed. Citation Format: Stephen J. Shuttleworth. Design and development of a novel series of orally active, selective PI3Kp110β/δ inhibitors for the treatment of cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-7. doi:10.1158/1538-7445.AM2014-LB-7

Innate PI3K p110δ Regulates Th1/Th17 Development and Microbiota-Dependent Colitis

The p110δ subunit of class IA PI3K modulates signaling in innate immune cells. We previously demonstrated that mice harboring a kinase-dead p110δ subunit (p110δ(KD)) develop spontaneous colitis. Macrophages contributed to the Th1/Th17 cytokine bias in p110δ(KD) mice through increased IL-12 and IL-23 expression. In this study, we show that the enteric microbiota is required for colitis development in germfree p110δ(KD) mice. Colonic tissue and macrophages from p110δ(KD) mice produce significantly less IL-10 compared with wild-type mice. p110δ(KD) APCs cocultured with naive CD4+ Ag-specific T cells also produce significantly less IL-10 and induce more IFN-γ- and IL-17A-producing CD4+ T cells compared with wild-type APCs. Illustrating the importance of APC-T cell interactions in colitis pathogenesis in vivo, Rag1(-/-)/p110δ(KD) mice develop mild colonic inflammation and produced more colonic IL-12p40 compared with Rag1(-/-) mice. However, CD4+ CD45RB(high/low) T cell Rag1(-/-)/p110δ(KD) recipient mice develop severe colitis with increased percentages of IFN-γ- and IL-17A-producing lamina propria CD3+D4+ T cells compared with Rag1(-/-) recipient mice. Intestinal tissue samples from patients with Crohn's disease reveal significantly lower expression of PIK3CD compared with intestinal samples from non-inflammatory bowel disease control subjects (p < 0.05). PIK3CD expression inversely correlates with the ratio of IL12B:IL10 expression. In conclusion, the PI3K subunit p110δ controls homeostatic APC-T cell interactions by altering the balance between IL-10 and IL-12/23. Defects in p110δ expression and/or function may underlie the pathogenesis of human inflammatory bowel disease and lead to new therapeutic strategies.

Isoform-selective induction of human p110δ PI3K expression by TNFα: identification of a new and inducible PIK3CD promoter

PI3Ks (phosphoinositide 3-kinases) are signalling molecules and drug targets with important biological functions, yet the regulation of PI3K gene expression is poorly understood. Key PI3Ks are the class IA PI3Ks that consist of a catalytic subunit (p110α, p110β and p110δ) in complex with a p85 regulatory subunit. Whereas p110α and p110β are ubiquitously expressed, high levels of p110δ are mainly found in white blood cells, with most non-leucocytes expressing low levels of p110δ. In the present paper we report that TNFα (tumour necrosis factor α) stimulation induces p110δ expression in human ECs (endothelial cells) and synovial fibroblasts, but not in leucocytes, through transcription start sites located in a novel promoter region in the p110δ gene (PIK3CD). This promoter is used in all cell types, including solid tumour cell lines that express p110δ, and is activated by TNFα in ECs and synovial fibroblasts. We further present a detailed biochemical and bioinformatic characterization of p110δ gene regulation, demonstrating that PIK3CD has distinct promoters, some of which can be dynamically activated by pro-inflammatory mediators. This is the first molecular identification of a PI3K promoter under the control of acute extracellular stimulation.

High levels of p110δ PI3K expression in solid tumor cells suppress PTEN activity, generating cellular sensitivity to p110δ inhibitors through PTEN activation

Class IA PI3K isoforms have divergent, nonredundant cell biological roles. In untransformed cells and tissues, p110α and p110β are ubiquitously expressed, whereas p110δ expression is highly enriched in leukocytes. High levels of p110δ expression have been documented in some solid tumor cell lines, but the functional role is unknown. This study aimed to elucidate the link between elevated expression of p110δ PI3K and cancer. We report that in breast and prostate cancer cells that contain leukocyte levels of p110δ, p110δ activity dampens the activity of the PTEN tumor suppressor. Indeed, inactivation of p110δ in these cells led to PTEN activation, suppression of Akt phosphorylation, and inhibition of cell proliferation, with inhibition of PTEN activity being able to counterbalance p110δ inactivation. Likewise, forced overexpression of p110δ in cells with low p110δ expression reduced PTEN activity, resulting in increased Akt phosphorylation. Our data indicate that the oncogenic potential of p110δ PI3K overexpression might at least partially act through PTEN inactivation, and that p110δ-selective PI3K inhibitors can have a dual antitumor mechanism, namely by directly inhibiting p110δ signaling and by a broader inhibition of class I PI3K activity through PTEN activation. These data may have important implications in the intervention of breast cancer.

Altered Macrophage Function Contributes to Colitis in Mice Defective in the Phosphoinositide-3 Kinase Subunit p110δ

Innate immune responses are crucial for host defense against pathogens but need to be tightly regulated to prevent chronic inflammation. Initial characterization of mice with a targeted inactivating mutation in the p110δ subunit of phosphoinositide 3-kinase (PI3K p110δ(D910A/D910A)) revealed defects in B- and T-cell signaling and chronic colitis. Here, we further characterize features of inflammatory bowel diseases in these mice and investigate underlying innate immune defects. Colons and macrophages from PI3K p110δ(D910A/D910A) mice were evaluated for colonic inflammation and innate immune dysfunction. Colonic p110δ messenger RNA expression was examined in interleukin (IL)-10(-/-) and wild-type germ-free mice during transition to a conventional microbiota. To assess polygenic impact on development of colitis, p110δ(D910A/D910A) mice were backcrossed to IL-10(-/-) mice. A mild spontaneous colitis was shown in PI3K p110δ(D910A/D910A) mice at 8 weeks, with inflammation increasing with age. An inflammatory mucosal and systemic cytokine profile was characterized by expression of IL-12/23. In PI3K p110δ(D910A/D910A) macrophages, augmented toll-like receptor signaling and defective bactericidal activity were observed. Consistent with an important homeostatic role for PI3K p110δ, wild-type mice raised in a germ-free environment markedly up-regulated colonic PI3K p110δ expression with the introduction of the enteric microbiota; however, colitis-prone IL-10(-/-) mice did not. Moreover, PI3K p110δ(D910A/D910A) mice crossed to IL-10(-/-) mice developed severe colitis at an early age. This study describes a novel model of experimental colitis that highlights the importance of PI3K p110δ in maintaining mucosal homeostasis and could provide insight into the pathogenesis of human inflammatory bowel disease.

Regulation of p110delta PI 3-kinase gene expression.

BackgroundDespite an intense interest in the biological functions of the phosphoinositide 3-kinase (PI3K) signalling enzymes, little is known about the regulation of PI3K gene expression. This also applies to the leukocyte-enriched p110δ catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy.Principal FindingsWe show that p110δ expression is mainly regulated at the transcriptional level. In fibroblasts, lymphocytes and myeloid cells, p110δ gene transcription appears to be constitutive and not subject to acute stimulation. 5′RACE experiments revealed that p110δ mRNA transcripts contain distinct upstream untranslated exons (named exon -1, -2a, -2b, -2c and -2d), which are located up to 81 kb upstream of the translational start codon in exon 1. The levels of all the different p110δ transcripts are higher in leukocytes compared to non-leukocytes, with the p110δ transcript containing exon -2a most abundantly expressed. We have identified a highly conserved transcription factor (TF) binding cluster in the p110δ gene which has enhanced promoter activity in leukocytes compared to non-leukocytes. In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a.ConclusionThis study identifies a conserved PIK3CD promoter region that may account for the predominant leukocyte expression of p110δ.

CAL-101, a Selective Inhibitor of the p110δ Isoform of Phosphatidylinositol 3-Kinase, Effectively Induces Apoptosis in Primary Chronic Lymphocytic Leukemia Cells Providing a Novel Therapeutic Strategy for the Treatment of This Disease

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a pivotal role in cell proliferation and survival that underlies the biology of many cancers including CLL. Of the eight distinct mammalian isoforms of PI3K, it is the class IA PI3Ks (p110α, p110β and p110δ) that are responsible for Akt activation and cellular transformation. The p110α and p110β isoforms both have a ubiquitous tissue distribution in adults, whereas p110δ expression is restricted to cells of hematopoietic origin. Recent studies have established a dominant role of p110δ isoform in B-cell responses. Deletion or inactivation of p110δ ablates B-cell antigen receptor (BCR)-induced phosphorylation of Akt and impairs cell cycle progression. Furthermore, CD40-ligand (CD40L) dependent survival is compromised in the absence of p110δ activity. Considering the role of p110δ-mediated BCR and CD40L-CD40 signaling to the enhanced survival in normal B cells, we hypothesized that inhibition of this kinase will induce cytotoxicity in B-CLL cells. We first examined the protein expression of p110δ in primary tumor CD19+ B cells from CLL patients and show that 24/24 CLL consistently overexpressed p110δ. The expression levels of p110α and p110β however, varied more widely, and were often undetectable. Treatment of primary tumor cells with CAL-101, a novel selective p110δ inhibitor, at concentrations of 0.1–10μM resulted in significant cell killing (linear mixed model; p=0.0004). As an example, 5μM CAL-101 resulted in a median of 59.6% viable cells (n=18 CLL patient samples). CAL-101 induced cytotoxicity was accompanied by PARP and caspase 3 cleavage. Previous published studies have demonstrated that CD40L-CD40 signaling promotes activation of CLL cells (as measured by up-regulation of CD40 and CD86) and also protection from spontaneous apoptosis ex vivo. Treatment of CLL cells in the presence of CAL-101 diminished the activation markers CD40 and CD86 induced by CD40L. In addition, an increase in CLL cell viability induced by CD40L was reversed by CAL-101 treatment. Contrasting with this, diminishment of apoptosis with IL-4 was not observed. Given the common finding of innate and cellular immune effects induced by therapies utilized in CLL, we next assessed the effect of CAL-101 on normal NK cells and T cells. Treatment of NK cells and T cells in vitro from healthy volunteers had no effect on cell viability. The lack of cytotoxic effect on normal NK cells and T cells was also assessed in vivo from a completed phase I trial of healthy volunteers that serves as a forerunner to the phase 1 clinical trial in CLL and related lymphoproliferative diseases currently ongoing. Here, treatment of normal human volunteers with CAL-101 for seven days achieved peak plasma concentrations up to 5μM without changes in general hematology or subpopulations of NK cells and T cells. Overall, our results identify the p110δ isoform as a potential therapeutic target in CLL where selective cytotoxicity is observed as compared to normal immune effector cells and the very important CD40-CD40L survival pathway is disrupted. Together, these in vitro and in vivo data provide sound validation for the ongoing Phase 1 clinical trial for the treatment of patients with CLL and related lymphoid malignancies.

CAL-101, a Specific Inhibitor of the p110δ Isoform of Phosphatidylinositide 3-Kinase Induces Cytotoxicity in Multiple Myeloma (MM)

The phosphatidylinositide 3-kinase (PI3K)/Akt pathway mediates cell proliferation, cell cycle regulation, apoptosis, and autophagy. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110α, p110β, p110δ, and p110γ) that are responsible for Akt activation. Recently, p110δ expression has been shown in colon and bladder carcinoma, glioblastoma, and acute myeloid leukemia cells. In this study, we demonstrate expression and examined biologic sequelae of p110δ signaling in MM using a novel specific inhibitor CAL-101. Overexpression of p110δ protein was observed in 24/24 primary tumor cells from MM patients. High level of p110δ expression was detected in two (INA-6 and LB) of the 11 MM cell lines examined. Treatment of INA-6 and LB cells with CAL-101 (1.0 μM for 1.5, 3, 6 hr) significantly inhibited phosphorylation of Akt at serine 473 and threonine 308, suggesting that p110δ is the isoform responsible for PI3K signaling in these cells. Furthermore, CAL-101 induced cytotoxicity in INA-6 cells in a dose-dependent fashion, with an IC50 of 0.625 μM-1.25 μM at 48 h. In contrast, minimal cytotoxicity was observed in p110δ-negative cell lines. Importantly, CAL-101 (0.625 μM) triggered cytotoxicity against tumor cells from MM patients without affecting the survival of normal peripheral blood mononuclear cells at concentrations as high as 20 μM. The specificity of CAL-101 towards p110δ was confirmed by the observation that p110δ small interfering RNA knock-down, but not control, caused inhibition of cell growth and cytotoxicity that was consistent with CAL-101 treatment, further confirming the key role of p110δ in MM cells. Interleukin 6 (IL-6) and insulin-like growth factor I (IGF-I) and cell-cell contact with stromal cells have been shown to induce proliferative and anti-apoptotic responses in MM cells that abrogate cytotoxic effects of conventional therapies. Importantly, neither either IL-6 and IGF-1 treatment nor co-culture of MM cells with bone marrow stromal cells abrogated CAL-101-induced MM cell growth inhibition. Moreover, CAL-101 inhibited adherence of MM cells to bone marrow stromal cells. Since the PI3K/Akt pathway is involved in autophagy, we also examined whether CAL-101 induced autophagic cell death in MM cells. CAL-101 (6 hr treatment) significantly increased LC3-II expression, assessed by western blot and immunofluorescent analyses; conversely, autophagy inhibitor 3-MA (100 μM) blocked LC3-II expression induced by CAL-101. Acridine orange staining and electron microscopic analysis to assess the relative number of autolysosomes further confirmed induction of autophagy by CAL-101. Finally, combined CAL-101 with bortezomib showed synergistic cytotoxicity against MM cells (combination index = 0.64). In conclusion, our studies showed that p110δ is a novel therapeutic target in MM and provide the basis for clinical evaluation of CAL-101 in ongoing Phase 1/2 trial in hematologic malignancies including MM.

P110δ is required for innate immunity to transplantable lymphomas

NK cell (natural killer cells) are lymphocytes of innate immunity that kill tumour cells and respond to infections, without prior stimulation. A balance of activating and inhibitory signals regulates NK cell cytotoxicity, but the molecular mechanisms are not fully understood. General inhibitors of PI3K (phosphoinositide 3-kinase) suppress cytotoxicity in human and mouse NK cells. However, which isoforms and how they regulate NK cell activation is unknown, and no data have been published on mice carrying PI3K mutations. p110delta expression is restricted to leucocytes, where it plays central roles in lymphocyte development and signalling. We have used mice carrying a catalytically inactive mutant form of p110delta in order to determine its role in NK cell biology. We show here that p110delta is not required to kill tumour cells, but unexpectedly p110delta mutant mice failed to fully reject transplanted lymphomas. Our results show for the first time a critical role for p110delta in NK cell biology in vivo.