Selective PI3Kδ Inhibitor Research Articles

Discovery of Novel PI3-Kinase δ Specific Inhibitors for the Treatment of Rheumatoid Arthritis: Taming CYP3A4 Time-Dependent Inhibition

PI3Kδ is a lipid kinase and a member of a larger family of enzymes, PI3K class IA(α, β, δ) and IB (γ), which catalyze the phosphorylation of PIP2 to PIP3. PI3Kδ is mainly expressed in leukocytes, where it plays a critical, nonredundant role in B cell receptor mediated signaling and provides an attractive opportunity to treat diseases where B cell activity is essential, e.g., rheumatoid arthritis. We report the discovery of novel, potent, and selective PI3Kδ inhibitors and describe a structural hypothesis for isoform (α, β, γ) selectivity gained from interactions in the affinity pocket. The critical component of our initial pharmacophore for isoform selectivity was strongly associated with CYP3A4 time-dependent inhibition (TDI). We describe a variety of strategies and methods for monitoring and attenuating TDI. Ultimately, a structure-based design approach was employed to identify a suitable structural replacement for further optimization.

PI3 Kinase δ Is a Key Regulator of Synoviocyte Function in Rheumatoid Arthritis

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.

Targeting phosphoinositide 3-kinase δ for allergic asthma

Chronic inflammation in the lung has long been linked to the pathogenesis of asthma. Central to this airway inflammation is a T-cell response to allergens, with Th2 cytokines driving the differentiation, survival and function of the major inflammatory cells involved in the allergic cascade. PI3Kδ (phosphoinositide 3-kinase δ) is a lipid kinase, expressed predominantly in leucocytes, where it plays a critical role in immune receptor signalling. A selective PI3Kδ inhibitor is predicted to block T-cell activation in the lung, reducing the production of pro-inflammatory Th2 cytokines. PI3Kδ is also involved in B-cell and mast cell activation. Therefore the inhibition of PI3Kδ should dampen down the inflammatory cascade involved in the asthmatic response through a wide breadth of pharmacology. Current anti-inflammatory therapies, which are based on corticosteroids, are effective in controlling inflammation in mild asthmatics, but moderate/severe asthmatic patients remain poorly controlled, experiencing recurrent exacerbations. Corticosteroids have no effect on mast cell degranulation and do not act directly on B-cells, so, overall, a PI3Kδ inhibitor has the potential to deliver improvements in onset of action, efficacy and reduced exacerbations in moderate/severe asthmatics. Additionally, PI3Kδ inhibition is expected to block effects of Th17 cells, which are increasingly implicated in steroid-insensitive asthma.

Phosphatidylinositol-3 Kinase Delta (PI3Kδ) Inhibitor AMG 319 Is a Potent, Selective and Orally Bioavailable Small Molecule Inhibitor That Suppresses PI3K-Mediated Signaling and Viability in Neoplastic B Cells

Abstract 4964Immune receptors such as the B cell receptor (BCR) require key signaling intermediate phosphatidylinositol-3 kinase delta (PI3Kδ) for normal immune cell survival, development and function. PI3Kδ is a class IA lipid kinase, is expressed primarily within the hematopoietic system and is composed of a catalytic subunit p110δ and a regulatory subunit p85. Recently, deregulated BCR-PI3Kδ signaling has been reported to play a role in B-cell malignancies such as chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) by mediating abnormal B-cell growth and survival. Indeed, the constitutive phosphorylation of downstream signaling intermediate AKT is associated with poor prognosis in several B cell malignancies. Here, we have investigated the potential of a novel small molecule inhibitor of PI3Kδ, AMG 319, to suppress PI3K signaling in human B cell lines and assessed the subsequent effects on viability as a single agent and in combination with chemotherapeutic drugs in preclinical models.Small molecule AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent preclinical pharmacokinetic (PK) properties. AMG 319 was found to potently inhibit PI3Kδ in enzyme assays (IC50 <10 nM). AMG 319 also potently suppressed the phosphorylation of AKT (pAKTS473) in primary murine splenocytes (IC50<5 nM) after BCR cross linking and demonstrated a less than 10 fold shift in human whole blood B cells using a similar BCR pAKT assay in vitro. In a cell based selectivity screen, AMG 319 was selective for PI3Kδ against other PI3K class I isoforms (200 to >5000 fold). Furthermore, AMG 319 was considered inactive at 10 μM on non-PI3K class I kinases in a broader kinome screen of 402 kinases. In preclinical PK studies, AMG 319 had low systemic clearance, T1/2 range of 2–4 hr, oral bioavailability of >45% and unbound fractions in plasma of 5–19%.Here, we have investigated the potential for AMG 319 to inhibit constitutive PI3K mediated signaling and effects on human B cell line viability. In a broad screen of >20 cell lines derived from B cell malignancies, the majority of lines were found to express PI3Kδ protein, all cells lines expressed the PI3Kα and β isoforms and variable levels of constitutive pAKTS473 were detected. AMG 319 was found to potently suppress constitutive pAKTS473 in the cell lines with IC50 in the low single to double digit nM range. Cellular viability was inhibited by AMG 319 though lines were variably sensitive to drug (range low double digit nM to μM IC50). As cell lines were variably sensitive to AMG 319 as a single agent, we examined if AMG 319 could enhance the efficacy of chemotherapeutic agents in vitro and in vivo. These studies focused on a DLBCL cell line HT which was relatively insensitive to AMG 319 as a single agent (IC50 ∼10 μM) in viability assays even though pAKTS473 was potently suppressed (IC50 ∼ 0.030 μM). Treatment with AMG 319 was found to synergize with the effects of vincristine to reduce cell viability in vitro using a 72 hr viability assay. Next we examined whether the enhanced cytotoxicity using these drugs in combination could be observed in vivo. Using the human B-cell lymphoma HT xenograft model, we found that AMG 319 in combination with vincristine enhanced tumor growth inhibition above that observed with either agent alone. Taken together, these findings suggest that the inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancies.In conclusion, AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent PK properties. AMG 319 inhibited constitutive pAKTS473, reduced the viability of B cell lines and synergized with vincristine in vitro and in vivo. The safety, PK and preliminary efficacy of AMG 319 are currently being investigated in a Phase I trial in patients with relapsed or refractory lymphoid malignancies. Disclosures:Sinclair:Amgen: Employment, Stock and Options. Metz:Amgen, Inc: Employment, Stock and Options. Cushing:Amgen, Inc: Employment, Stock and Options. Liu:Amgen, Inc: Employment, Stock and Options. Brake:Amgen, Inc: Employment, Stock and Options. Starnes:Amgen, Inc: Employment, Stock and Options. Means:Amgen, Inc: Employment, Stock and Options. Henne:Amgen, Inc: Employment, Stock and Options. Archibeque:Amgen: Employment, Stock and Options. Mattson:Amgen, Inc: Employment, Stock and Options. Drew:Amgen, Inc: Employment, Stock and Options. Busse:Amgen, Inc: Employment, Stock and Options. Wang:Amgen, Inc: Employment, Stock and Options. Al-Assaad:Amgen, Inc: Employment, Stock and Options. Molineux:Amgen: Employment, Stock and Options.

NPM-ALK Regulated GSK3β Phosphorylation Results in Mcl-1 and CDC25A Accumulation and Enhanced Oncogenic Potential

Abstract 586Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. ALCLs are frequently characterized by translocations involving the anaplastic lymphoma kinase (ALK) gene. In 70–80% of these cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of ALK with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK tyrosine kinase. NPM-ALK is an oncogenic tyrosine kinase which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules in ALCLs have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3β, as a potential signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the phosphorylation of GSK3β at serine-9 (pS9-GSK3β) in a time and dose-dependent manner. Expression of NPM-ALK in 293T cells led to increased phosphorylation of GSK3β at serine-9 compared to kinase-defective K210R mutant NPM-ALK but did not affect total GSK3β levels. Inducible knockdown of ALK in SU-DHL-1 cells also led to decreased phosphorylation of GSK3β at serine-9 without affecting total GSK3β levels. Selective inhibition of PI3Kδ by CAL-101 resulted in decreased phosphorylation of GSK3β at serine-9 in a dose dependent manner without affecting total GSK3β or NPM-ALK activity. Ablation of ALK kinase activity resulted in proteasomal degradation of GSK3β substrates Mcl-1 and CDC25A. This degradation was recovered upon chemical inhibition of GSK3β (GSK3 IX). To address the role of GSK3β in ALCL proliferation, WST-1 assay revealed that ALK inhibition resulted in a decrease in cell viability which was rescued by GSK3β inhibition. ALK inhibition resulted in significant cellular apoptosis as detected by propidium iodide and Annexin V staining followed by flow cytometry. This apoptotic response was rescued by GSK3β inhibition. The effect of GSK3β on cellular oncogenic potential was addressed by colony formation assay using ALCL-derived cell lines. While ALK inhibition resulted in decreased colony numbers, simultaneous ALK and GSK3β inhibition recovered colony numbers compared to ALK inhibition alone. Additionally, stable GSK3β knockdown conferred resistance to growth inhibitory effect of ALK inhibition as determined by colony formation assay. Furthermore, pS9-GSK3β and its known substrate, CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies and showed a significant correlation (p<0.001). Conversely, ALK-ALCL tissue biopsies showed no significant correlation of pS9-GSK3β and CDC25A expression (p<0.2). Our results demonstrate that NPM-ALK signals through PI3K/AKT to phosphorylate GSK3β at serine-9. This phosphorylation inhibits GSK3β kinase activity which results in the accumulation of CDC25A and Mcl-1 thus providing growth advantage and protection from apoptosis. These findings provide support for the role of GSK3β as a novel mediator of NPM-ALK oncogenesis. Disclosures:No relevant conflicts of interest to declare.

The Allosteric AKT Inhibitor MK-2206 Demonstrates Potent Antiproliferative Activity in Lymphoma Cells and Synergizes with the HDAC Inhibitor Vorinostat,

Abstract 3729The serine/threonine kinase Akt plays a critical signaling role downstream of phosphatidylinositol-3-kinase (PI3K) and is important in promoting cell survival and inhibiting apoptosis. Indeed, Akt activation and overexpression is often associated with resistance to chemotherapy or radiotherapy. Previous studies demonstrated the potential therapeutic value of targeting the PI3K pathway in lymphoma, as both the selective PI3Kδ inhibitor CAL-101, and everolimus and temsirolimus, which target PI3K and mTOR, produce clinical responses in a variety of lymphomas. We evaluated the effect of the novel allosteric Akt inhibitor, MK-2206, in a panel of lymphoma cell lines and primary lymphoma cells. We found that Akt, and activated pAkt, are highly expressed in lymphoma cells. After 72 hours of incubation, the Akt inhibitor MK-2206 demonstrated antiproliferative activity in a variety of lymphoma cell lines, with an IC50 ranging between 0.1 and 5μM. There was no correlation between pre-treatment levels of pAKT, PI3K isoforms, or PTEN protein expression and sensitivity to MK-2206. Within the diffuse large B cell lymphoma cell lines, those of GCB cell of origin were more sensitive to MK-2206, compared with the ABC-derived cell lines. Resistant cell lines tended to had weak or absent expression of p-GSK3 and p-4EBPI. Mechanistically, MK-2206 treatment decreased the level of p-Akt (Ser473), and p-Akt (Thr308), irrespective of drug sensitivity. Furthermore, MK-2206 decreased the phosphorylation level of Akt downstream targets, including p-GSK3 beta and p-PRAS40, upregulated p27. and dephosphorylated p70S6K. Moreover, MK-2206 treatment decreased HIF-1 alpha and VEGF expression. Depending on the cell of origin, the antiproliferative effect resulted from cycle arrest at the G0/G1 phase, autophagy, orapoptosis. MK-2206 showed synergistic effect in combination with the HDAC inhibitor, Vorinostat. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, the combination of MK-2206 and Vorinostat effectively altered p53 and p27 levels, which were associated with increased PARP cleavage and induction of apoptosis. Our data demonstrate that AKT is a promising target for the treatment of lymphoma, and provide a rationale for an ongoing trial, evaluating MK-2206 for the treatment of patients with relapsed lymphoma. Disclosures:No relevant conflicts of interest to declare.

Phosphatidylinsoitol-3 Kinase Delta (PI3Kδ) Inhibitor AMG 319 Combined with Vincristine Enhances G2/M Arrest and Apoptotic Death in Neoplastic B Cells

Abstract 4963Phosphatidylinositol-3 kinase delta (PI3Kδ) is a class IA lipid kinase expressed primarily in leukocytes and is a key signaling molecule for immune receptors such as the B cell receptor (BCR). Dysregulated BCR-PI3Kδ signaling has been reported to play a role in B cell malignancy by mediating abnormal growth and survival. Recently we have identified that the potent and selective PI3Kδ inhibitor AMG 319 in combination with vincristine synergistically reduced cell viability in vitro and enhanced xenograft tumor regression in vivo when DLBCL cell line HT, which was relatively insensitive to AMG 319 in viability assays, was used as a model system. These findings suggest that inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancy.To investigate the mechanism by which AMG 319 synergizes with vincristine in HT cells, we evaluated cell cycle and apoptosis after treatment with single agents or the combination. Vincristine alone at high concentration (4 nM) induced extensive G2/M arrest (51% versus 10% in vehicle control) at 24 hours while little or no G2/M arrest was detected at lower concentrations (0.8 and 0.16 nM). AMG 319 alone, at 0.33 μM, 3.3 μM and 10 μM did not induce G2/M arrest in HT cells. However, when AMG 319 was combined with a low concentration of vincristine at 0.8 nM, increased G2/M arrest (49%) was detected after 24 hours compared to each agent alone (21% in vincristine at 0.8 nM and 9% in AMG 319 at 10 μM) or vehicle control group (16%). This effect was AMG 319 concentration dependent (25%, 34% and 49% in vincristine at 0.8 nM combined with AMG 319 at 0.33, 3.3 and 10 μM, respectively). Interestingly, the enhanced G2/M cell cycle arrest with the drug combination was not sustained at 48 hours as cells underwent apoptosis as demonstrated by an increase in the SubG1 population and caspase-3 activity as well as a decrease in cell viability. In further support of these findings, a substantial increase in Annexin V (+) apoptotic cells (up to 68%) was observed in an AMG 319 concentration dependent manner at 48 and 72 hours in combination with vincristine as compared to that observed with the single agents (17% in vincristine at 0.8 nM; between 11%-23% in AMG 319 at 0.33, 3.3 and 10 μM) or vehicle control (around 10%).We then examined phosphorylation of Histone H3 at Ser10 and phosphorylation of cell cycle check point kinases Chk2 at Ser68 and Chk1 at Ser317, which are known to be associated with G2/M arrest. The phosphorylation of Histone H3 and Chk2 was increased between 3 to 18 fold in the drug combination group compared with either agent alone or the vehicle control group at 24 hours. In contrast, phosphorylation of Chk1 at Ser317 was unchanged. Lastly, modulation of pAkt (Ser473 and Ser308) was assessed and vincristine alone did not inhibit pAkt at all three concentrations (0.16 nM, 0.8 nM and 4 nM) up to 24 hours or change the maximum levels of pAkt observed. As expected, AMG 319 alone (in concentrations ranging from 3.3 nM to 3.3 μM) significantly inhibited pAkt (> 85% of inhibition in AMG 319 at 3.3 μM) at both 6 and 24 hour time points in a concentration-dependent fashion. The combination of vincristine with AMG 319 did not lead to further inhibition of pAkt.Taken together, these data suggest that the up-regulation of Chk2 (Ser68) is one of the underlying molecular events associated with the synergistic effects of AMG 319-vincristine combination on G2/M arrest, leading to cell growth inhibition and apoptotic death in HT cells. Therefore, inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancy by inducing cell cycle arrest and apoptosis. Disclosures:Liu:Amgen: Employment. Sun:Amgen: Employment. Pistillo:Amgen: Employment. Payton:Amgen: Employment. Wang:Amgen: Employment. Archibeque:Amgen: Employment. Molineux:Amgen: Employment. Sinclair:Amgen: Employment.

The phosphoinositide 3′-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia

AbstractIn lymphocytes, the phosphoinositide 3′-kinase (PI3K) isoform p110δ (PI3Kδ) transmits signals from surface receptors, including the B-cell receptor (BCR). CAL-101, a selective inhibitor of PI3Kδ, displays clinical activity in CLL, causing rapid lymph node shrinkage and a transient lymphocytosis. Inhibition of pro-survival pathways, the presumed mechanism of CAL-101, does not explain this characteristic pattern of activity. Therefore, we tested CAL-101 in assays that model CLL-microenvironment interactions in vitro. We found that CAL-101 inhibits CLL cell chemotaxis toward CXCL12 and CXCL13 and migration beneath stromal cells (pseudoemperipolesis). CAL-101 also down-regulates secretion of chemokines in stromal cocultures and after BCR triggering. CAL-101 reduces survival signals derived from the BCR or from nurse-like cells, and inhibits BCR- and chemokine-receptor–induced AKT and MAP kinase (ERK) activation. In stromal cocultures, CAL-101 sensitizes CLL cells toward bendamustine, fludarabine, and dexamethasone. These results are corroborated by clinical data showing marked reductions in circulating CCL3, CCL4, and CXCL13 levels, and a surge in lymphocytosis during CAL-101 treatment. Thus, CAL-101 displays a dual mechanism of action, directly decreasing cell survival while reducing interactions that retain CLL cells in protective tissue microenvironments. These data provide an explanation for the clinical activity of CAL-101, and a roadmap for future therapeutic development.

Phase I study of CAL-101, an isoform-selective inhibitor of phosphatidylinositol 3‐kinase P110d, in patients with previously treated chronic lymphocytic leukemia.

6631 Background: The class I phosphatidylinositol 3-kinases (PI3Ks) regulate cellular functions relevant to oncogenesis. Expression of the PI3K p110δ isoform (PI3Kδ) is restricted to cells of hematopoietic origin. CAL-101 is an isoform-selective inhibitor of PI3Kδ that induces apoptosis of chronic lymphocytic leukemia (CLL) cells. Methods: This phase I study evaluated CAL-101 in patients with previously treated hematologic cancers. CAL-101 was administered orally 1 or 2 times per day (QD or BID) continuously in 28-day cycles, until disease progression or unacceptable toxicity. Efficacy analyses were based on standard criteria. Results: At data cutoff, the study had enrolled 54 patients with CLL. Patient characteristics included: median [range] age of 62 [37‐82] years; 82% males; 72% with refractory disease, 81% with bulky disease, 36% with del(17p), and a median [range] of prior therapies of 5 [2‐15]. CAL-101 dose levels were 50 mg, 100 mg, 150 mg, 200 mg, and 350 mg BID; and 300 mg QD. The median [range] of CAL‐101 treatment cycles was 8 [1-19]. Grade ≥3 adverse events included pneumonia (24%), neutropenia (24%), thrombocytopenia (7%), neutropenic fever (7%), anemia (6%) and ALT/AST increase (6%). CAL‐101 reduced lymphadenopathy by ≥50% in 80% of patients. A transient increase of >50% from baseline in peripheral absolute lymphocyte counts occurred in 58% of patients. The overall intention-to-treat response rate by IWCLL 2008 criteria was 26%. Medians for duration of response and progression-free survival had not been reached (>11 months), with 46% of patients continuing on treatment. CAL-101 reduced constitutive overexpression of phospho‐AKT in patient CLL cells and normalized plasma concentrations of CCL3, CCL4, and CXCL13. PK analyses showed minimal increases in plasma Cmax and AUC at doses >150 mg BID. Accrual is ongoing and updated data will be presented. Conclusions: CAL-101, an oral PI3Kδ-selective inhibitor, shows acceptable safety and promising clinical activity in heavily pretreated patients with CLL. Dose‐response assessments support the 150 mg BID dose for future single-agent and combination chemo/immunotherapy studies.

Abstract 3555: Anti-tumor activity of CAL-101, a potent selective inhibitor of the p110Δ isoform of PI3K, in models of human glioblastoma

Abstract Phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation play a pivotal role in cell proliferation and survival, transducing signals from several cell surface receptors through PI3K isoforms (designated p110α, β, Δ, and γ). Our previous research has elucidated a significant role of the PI3K p110Δ (PI3KΔ) isoform in hematologic malignancies. Aberrant upregulation of the PI3K/Akt pathway is a frequently observed defect in human malignancies, including glioblastoma, but the role of the PI3KΔ isoform, has not been systematically evaluated. Selective inhibition of PI3KΔ with the orally bioavailable investigational drug, CAL-101, offers the potential for targeting PI3KΔ as a therapeutic approach to glioblastoma while avoiding undesirable side effects associated with pan-inhibitors that broadly block PI3K isoforms in multiple cell types. CAL-101 potently inhibits PI3KΔ (with an EC50 of 65 nM in a whole-blood assay) and shows &amp;gt;200-fold selectivity over other Class I PI3K isoforms and no activity against Class II and III PI3K family members or other PI3K-related proteins, including mTOR and DNA-PK. We examined expression levels of PI3KΔ and constitutive pathway activation in a panel of glioma cell lines (LN18, LN229, SF-539, U87MG, U138MG, and U251). PI3KΔ was consistently expressed and was functionally active, inducing high basal levels of phosphorylated-Akt (pAkt) in all tested cell lines. Treatment with CAL-101 decreased p-Akt levels in all cell lines and also reduced phosphorylation of the downstream pathway target, S6. Furthermore, PI3KΔ inhibition with CAL-101 resulted in an accumulation of cells in G1 and a decrease in the S phase population. Consistent with the effects of CAL-101 on G1 cell-cycle arrest, immunoblotting showed a decrease in cyclin D1 levels. In mice bearing U87MG xenografts, in vivo PI3KΔ inhibition resulted in significant anti-tumor effects, delaying time to tumor progression (tumor volume of &amp;gt;1000 mm3) by 30 days (p&amp;lt;0.001). Taken together, these data suggest that PI3KΔ overexpression and heightened functional activity contribute to glioblastoma proliferation, and that selective targeting of PI3KΔ may offer a novel therapeutic approach for patients with central nervous system tumors. 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 3555. doi:10.1158/1538-7445.AM2011-3555

PI3Kδ drives the pathogenesis of experimental autoimmune encephalomyelitis by inhibiting effector T cell apoptosis and promoting Th17 differentiation

The Class IA phosphoinositide 3-kinase delta (PI3Kδ) has been implicated in multiple signaling pathways involved in leukocyte activation and hence is an attractive target in many human autoimmune diseases, including multiple sclerosis (MS). Here, using mice expressing a catalytically inactive form of the PI3Kδ subunit p110δ, we show that signaling through PI3Kδ is required for full and sustained pathology of experimental autoimmune encephalomyelitis (EAE), a Th17-driven model of MS. In p110δ-inactivated mice, T cell activation and function during EAE was markedly reduced and fewer T cells were observed in the central nervous system (CNS). The decrease in T cell activation is unlikely to be due to defects in dendritic cell (DC) function, as p110δ-inactivated DCs migrate and present antigen normally. However, significant increases in the proportion of T cells undergoing apoptosis at early stages of EAE were evident in the absence of PI3Kδ activity. Furthermore, a profound defect in Th17 cellular responses during EAE was apparent in the absence of PI3Kδ activity while Th1 responses were less affected. A highly selective PI3Kδ inhibitor, IC87114, also had greater inhibitory effects on Th17 cell generation in vitro than it did on Th1 cell generation. Thus, PI3Kδ plays an important role in Th17 responses in EAE, suggesting that small molecule inhibitors of PI3Kδ may be useful therapeutics for treatment of MS and other autoimmune diseases.

Phosphatidylinositol 3‐kinase‐δ up‐regulates L‐type Ca2+ currents and increases vascular contractility in a mouse model of type 1 diabetes

Vasculopathies represent the main cause of morbidity and mortality in diabetes. Vascular malfunctioning in diabetes is associated with abnormal vasoconstriction and Ca(2+) handling by smooth muscle cells (SMC). Phosphatidylinositol 3-kinases (PI3K) are key mediators of insulin action and have been shown to modulate the function of voltage-dependent L-type Ca(2+) channels (Ca(V) 1.2). In the present work, we investigated the involvement of PI3K signalling in regulating Ca(2+) current through Ca(V) 1.2 (I(Ca,L) ) and vascular dysfunction in a mouse model of type I diabetes. Changes in isometric tension were recorded on myograph. Ca(2+) currents in freshly dissociated mice aortic SMCs were measured using the whole-cell patch-clamp technique. Antisense techniques were used to knock-down the PI3Kδ isoform. KEY RESULTS Contractile responses to phenylephrine and KCl were strongly enhanced in diabetic aorta independent of a functional endothelium. The magnitude of phenylephrine-induced I(Ca,L) was also greatly augmented. PI3Kδ expression, but not PI3Kα, PI3Kβ, PI3Kγ, was increased in diabetic aortas and treatment of vessels with a selective PI3Kδ inhibitor normalized I(Ca,L) and contractile response of diabetic vessels. Moreover, knock-down of PI3Kδin vivo decreased PI3Kδ expression and normalized I(Ca,L) and contractile response of diabetic vessels ex vivo. Phosphatidylinositol 3-kinase δ was essential to the increased vascular contractile response in our model of type I diabetes. PI3Kδ signalling was up-regulated and most likely accounted for the increased I(Ca,L,) leading to increased vascular contractility. Blockade of PI3Kδ may represent a novel therapeutic approach to treat vascular dysfunction in diabetic patients.

Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals

AbstractTargeted therapy with imatinib in chronic myeloid leukemia (CML) prompted a new treatment paradigm. Unlike CML, chronic lymphocytic leukemia (CLL) lacks an aberrant fusion protein kinase but instead displays increased phosphatidylinositol 3-kinase (PI3K) activity. To date, PI3K inhibitor development has been limited because of the requirement of this pathway for many essential cellular functions. Identification of the hematopoietic-selective isoform PI3K-δ unlocks a new therapeutic potential for B-cell malignancies. Herein, we demonstrate that PI3K has increased enzymatic activity and that PI3K-δ is expressed in CLL cells. A PI3K-δ selective inhibitor CAL-101 promoted apoptosis in primary CLL cells ex vivo in a dose- and time-dependent fashion that was independent of common prognostic markers. CAL-101–mediated cytotoxicity was caspase dependent and was not diminished by coculture on stromal cells. In addition, CAL-101 abrogated protection from spontaneous apoptosis induced by B cell–activating factors CD40L, TNF-α, and fibronectin. In contrast to malignant cells, CAL-101 does not promote apoptosis in normal T cells or natural killer cells, nor does it diminish antibody-dependent cellular cytotoxicity. However, CAL-101 did decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Collectively, these studies provide rationale for the clinical development of CAL-101 as a first-in-class targeted therapy for CLL and related B-cell lymphoproliferative disorders.

Inhibition of phosphoinositide 3 kinase delta attenuates allergic airway inflammation in mice

Phosphoinositide 3‐kinase p110δ (PI3Kδ) plays a pivotal role in the recruitment and activation of inflammatory cells. The role of PI3Kδ in a mouse model of cockroach antigen (CRA)‐induced acute allergic airway inflammation was investigated using IC87114, a selective PI3Kδ inhibitor. BALB/c mice exposed to CRA developed airway eosinophilia associated with increased expression of IL‐2, IL‐17, IFNγ, IL‐4 and IL‐5 in lung tissue as well as of pro‐inflammatory chemokines eotaxin‐1 and MCP‐1 in bronchoalveolar lavage fluid (BALF) and lung tissue. Further, these mice developed airway hyperresponsiveness (AHR) to inhaled methacholine. Oral administration of IC87114 significantly attenuated CRA‐induced influx of inflammatory cells in the BALF (eosinophils and macrophages) and lung tissue and suppressed CRA‐induced AHR. Moreover, levels of IL‐2, IL‐17, IFNγ, IL‐4, and IL‐5 in lung tissue and of eotaxin‐1 and MCP‐1 in BALF as well as lung tissue were substantially reduced. Treatment of human eosinophils with IC87114 significantly inhibited eosinophil rolling on human umbilical vein endothelial cells as well as VCAM‐1 in vitro. Overall, these findings implicate that inhibition of the PI3Kδ may have therapeutic potential for the treatment of allergic airway inflammation. Supported by National Institutes of Health grants HL0793041 and AI35796.

Targeting Phosphoinositide-3-Kinase-δ with Theophylline Reverses Corticosteroid Insensitivity in Chronic Obstructive Pulmonary Disease

Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids. This has been linked to a reduction of histone deacetylase-2 as a result of oxidative stress and is reversed by theophylline. To determine the role of phosphoinositide-3-kinase-delta (PI3K-δ) on the development of corticosteroid insensitivity in COPD and under oxidative stress, and as a target for theophylline. Corticosteroid sensitivity was determined as the 50% inhibitory concentration of dexamethasone on tumor necrosis factor-α-induced interleukin-8 release in peripheral blood mononuclear cells from patients with COPD (n = 17) and compared with that of nonsmoking (n = 8) and smoking (n = 7) control subjects. The effect of theophylline and a selective PI3K-δ inhibitor (IC87114) on restoration of corticosteroid sensitivity was confirmed in cigarette smoke-exposed mice. Peripheral blood mononuclear cells of COPD (50% inhibitory concentration of dexamethasone: 156.8 ± 32.6 nM) were less corticosteroid sensitive than those of nonsmoking (41.2 ± 10.5 nM; P = 0.018) and smoking control subjects (47.5 ± 19.6 nM; P = 0.031). Corticosteroid insensitivity and reduced histone deacetylase-2 activity after oxidative stress were reversed by a non-selective PI3K inhibitor (LY294002) and low concentrations of theophylline. Theophylline was a potent selective inhibitor of oxidant-activated PI3K-δ, which was up-regulated in peripheral lung tissue of patients with COPD. Furthermore, cells with knock-down of PI3K-δ failed to develop corticosteroid insensitivity with oxidative stress. Both theophylline and IC87114, combined with dexamethasone, inhibited corticosteroid-insensitive lung inflammation in cigarette-smoke-exposed mice in vivo. Inhibition of oxidative stress dependent PI3K-δ activation by a selective inhibitor or theophylline provides a novel approach to reversing corticosteroid insensitivity in COPD.

Histone deacetylase-2 and airway disease

The increased expression of inflammatory genes in inflammatory lung diseases is regulated by acetylation of core histones, whereas histone deacetylase-2 (HDAC2) suppresses inflammatory gene expression. Corticosteroids suppress inflammatory genes in asthma by inhibiting histone acetyltransferase and in particular by recruiting HDAC2 to the nuclear factor-kappaB-activated inflammatory gene complex. This involves deacetylation of the acetylated glucocorticoid receptor. In COPD, severe asthma and asthmatics who smoke, HDAC2 is reduced, thus preventing corticosteroids from suppressing inflammation. The reduction in HDAC2 appears to be secondary to increased oxidative and nitrative stress in the lungs. Antioxidants and inhibitors of nitric oxide synthesis may therefore restore corticosteroid sensitivity in COPD, but this can also be achieved by low concentrations of theophylline and curcumin, which act as HDAC activators. Theophylline is a direct inhibitor of oxidant-activated phosphoinositide-3-kinase-delta, which is involved in inactivation of HDAC2. In the future selective PI3Kdelta inhibitors and more direct activators of HDAC2 may be used to treat corticosteroid-resistant inflammatory diseases of the lung, including COPD, severe asthma and asthma in smokers.

IC87114, a Selective Inhibitor of PI3Kδ Suppresses Joint Inflammation and Bone Erosion in Collagen-Induced Arthritis in Rat (50.14)

Abstract PI3Kδ plays an essential role in antigen-receptor signaling, proliferation, activation and function of lymphocytes. Mice deficient in PI3Kδ activity show substantial reduction in immunoglobulin levels, partial impairment in chemoattractant-induced neutrophil migration as well as defects in signaling and function of macrophages. Collagen-induced arthritis (CIA), a commonly used model for studying antirheumatic drugs, requires participation of both B and T cells to initiate disease and reproduces many of the pathogenic mechanisms detected in human rheumatoid arthritis. In this study, we have investigated the ability of IC87114, a selective PI3Kδ inhibitor, to reverse the rheumatoid arthritic like state in the CIA model of arthritis. Clinical arthritis was initiated by immunizing animals with collagen followed by a booster dose on day 7. IC87114 or control treatment was initiated when at least one hind paw was significantly inflamed and continued for an 18-day treatment course. IC87114-treatment of arthritic rats reduced the progression and severity of clinical arthritis that was evident within 6 days after initiation of therapy. IC87114-treatment significantly reduced the level of anti-collagen antibodies. Arthritic rats treated with IC87114 had significantly lower radiographic scores compared with control, indicating that treatment with IC87114 was effective in protecting bone integrity. Histopathological evaluation demonstrated that IC87114 was effective in reducing the histological changes induced by rheumatoid arthritis. The effect of IC87114-treatment on these parameters together suggests a therapeutic potential for PI3Kδ-selective compounds to ameliorate rheumatoid arthritis.

Role of PI3Kδ and PI3Kγ in inflammatory arthritis and tissue localization of neutrophils

The p110delta isoform of class I phosphoinositide 3-kinase (PI3K) plays a major role in B cell receptor signaling, while its p110gamma counterpart is thought to predominate in leukocyte chemotaxis. Consequently, emphasis has been placed on developing PI3Kgamma selective inhibitors to treat disease states that result from inappropriate tissue accumulation of leukocytes. We now demonstrate that PI3Kdelta blockade is effective in treating an autoimmune disorder in which neutrophil infiltration is required for tissue injury. Using the K/BxN serum transfer model of arthritis, in which neutrophils and leukotriene B(4) (LTB(4)) participate, we show that genetic deletion or selective inhibition of PI3Kdelta diminishes joint erosion to a level comparable to its PI3Kgamma counterpart. Moreover, the induction and progression of joint destruction was profoundly reduced in the absence of both PI3K isoforms and correlated with a limited ability of neutrophils to migrate into tissue in response to LTB(4). However, the dynamic interplay between these isoforms is not pervasive, as fMLP-induced neutrophil extravasation was primarily reliant on PI3Kgamma. Our results not only demonstrate that blockade of PI3Kdelta has potential therapeutic value in the treatment of chronic inflammatory conditions, but also provide evidence that dual inhibition of these lipid kinases may yield superior clinical results.

Mechanisms and implications of phosphoinositide 3-kinase δ in promoting neutrophil trafficking into inflamed tissue

AbstractThe phosphoinositide 3-kinase (PI3K) catalytic subunit p110δ is expressed in neutrophils and is thought to play a role in their accumulation at sites of inflammation by contributing to chemoattractant-directed migration. We report here that p110δ is present in endothelial cells and participates in neutrophil trafficking by modulating the proadhesive state of these cells in response to tumor necrosis factor α (TNFα). Specifically, administration of the selective inhibitor of PI3Kδ, IC87114, to animals reduced neutrophil tethering to and increased rolling velocities on cytokine-activated microvessels in a manner similar to that observed in mice deficient in p110δ. These results were confirmed in vitro as inhibition of this isoform in endothelium, but not neutrophils, diminished cell attachment in flow. A role for PI3Kδ in TNFα-induced signaling is demonstrated by a reduction in Akt-phosphorylation and phosphatidylinositol-dependent kinase 1 (PDK1) enzyme activity upon treatment of this cell type with IC87114. p110δ expressed in neutrophils also contributes to trafficking as demonstrated by the impaired movement of these cells across inflamed venules in animals in which this catalytic subunit was blocked or genetically deleted, results corroborated in transwell migration assays. Thus, PI3Kδ may be a reasonable therapeutic target in specific inflammatory conditions as blockade of its activity reduces neutrophil influx into tissues by diminishing their attachment to and migration across vascular endothelium. (Blood. 2004;103:3448-3456)

Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement.

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3K delta in neutrophil chemotaxis, we have developed a potent and selective PI3K delta inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K delta activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K delta, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K delta can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.