SMER28 attenuates PI3K/mTOR signaling by direct inhibition of PI3K p110 delta

SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and consequently growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction of phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3Kδ, and to a lesser extent p110γ. The biological relevance of our observations was underscored by interference of SMER28 with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor typrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited.

Phosphoinositide 3-Kinase p110 Delta Differentially Restrains and Directs Naïve Versus Effector CD8+ T Cell Transcriptional Programs

Phosphoinositide 3-kinase p110 delta (PI3K p110δ) is pivotal for CD8+ T cell immune responses. The current study explores PI3K p110δ induction and repression of antigen receptor and cytokine regulated programs to inform how PI3K p110δ directs CD8+ T cell fate. The studies force a revision of the concept that PI3K p110δ controls metabolic pathways in T cells and reveal major differences in PI3K p110δ regulated transcriptional programs between naïve and effector cytotoxic T cells (CTL). These differences include differential control of the expression of cytolytic effector molecules and costimulatory receptors. Key insights from the work include that PI3K p110δ signalling pathways repress expression of the critical inhibitory receptors CTLA4 and SLAMF6 in CTL. Moreover, in both naïve and effector T cells the dominant role for PI3K p110δ is to restrain the production of the chemokines that orchestrate communication between adaptive and innate immune cells. The study provides a comprehensive resource for understanding how PI3K p110δ uses multiple processes mediated by Protein Kinase B/AKT, FOXO1 dependent and independent mechanisms and mitogen-activated protein kinases (MAPK) to direct CD8+ T cell fate.

Translating PI3K-Delta Inhibitors to the Clinic in Chronic Lymphocytic Leukemia: The Story of CAL-101 (GS1101)

Overview: Targeted therapy with imatinib has transformed the treatment of chronic myeloid leukemia (CML). Unlike CML, chronic lymphocytic leukemia (CLL) lacks a common genetic aberration but does demonstrate constitutive activation of PI3-kinase (PI3K) as compared to normal B cells. This constitutively active PI3K in CLL likely relates to tonic B-cell receptor signaling that is present across a wide variety of B-cell malignancies. Although PI3K is quite proximal and represents an ideal target to pharmacologically modulate, the complexity of this pathway on which many normal functions are dependent had for many years been problematic. The p110 delta isoform of PI3K is relatively specific to hematopoietic cells, and elegant mouse studies where p110 delta was genetically inactivated demonstrated only a selective B-cell defect. Subsequent development of a potent, selective p110 delta inhibitor prompted translation into the clinic for the treatment of CLL and low-grade non-Hodgkin lymphoma (NHL). From the first patient treated where a dramatic early nodal response was noted, considerable excitement has developed for this class of drugs in CLL and NHL. We will summarize the development process of CAL-101 (now GS1101) in the treatment of chronic lymphoid malignancies such as CLL.

The Role of PI3K Signaling in Cell Trafficking of Multiple Myeloma

Abstract 1804 Introduction: Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is a fundamental pathway for the regulation of cell proliferation, survival, adhesion, migration, and metabolism in a variety of physiological and pathological processes. The pathway and its downstream effectors are frequently activated in patients with acute leukemias, chronic leukemias, various types of lymphomas, and multiple myeloma (MM). In this study we investigated the expression of the different PI3K catalytic subunits in MM and effect of three different PI3K inhibitors on the interaction of MM cells with the bone marrow (BM) microenvironment. Methods and Results: First we characterized the baseline expression of the different PI3K-p110-alpha, beta, gamma and delta catalytic subunits in MM cell lines (MM1s, OPM1, OPM2, H929, RPMI, INA6, U266, and U266LR7) by immnunobloting. PI3K-p110-alpha was highly expressed in MM1s and RPMI8226; PI3K-p110-beta was highly expressed in all cell lines; PI3K-p110-gamma was highly expressed in OPM1 and OPM2; and PI3K-p110-delta was highly expressed in MM1s and INA6. Furthermore, we investigated BKM120, a novel pan-PI3K inhibitor (Novartis, Swizerland). MM cells (MM1s, H929, OPM1, and OPM2) were treated with increasing concentrations of BKM120 (0, 100, 250, 500 and 1000 nM) for 4 hrs, labeled with Calcein-AM, and applied to fibronectin adhesion plate, or to 96-well plate pre-coated with stroma. Cells were incubated for 1 hr at 37C, non-adherent cells were washed and MM adhesion was measured by fluorescence-reader. BKM120 reduced the adhesion of all MM cell lines to fibronectin and stromal cells in a dose-dependent manner. Mechanistically, BKM120 decreased the activation of adhesion-related signaling in MM cells induced by co-culture with stroma including pFAK, pSRC pCoffilin and pMyosin light chain, as detected by immunoblotting. Moreover, BKM120 inhibited the downstream signaling of PI3K: p-Akt, p-P70S6, and p-S6R and regulated the survival of MM cells with or without co-culture with Bone Marrow stroma (IC50- 1uM 48hrs) and caused cell cycle arrest, as detected by PI staining and analyzed by flow cytometry, and decreased the expression of cyclin D1, p-Rb and increased the expression of P27 and P21, as detected by immunoblotting. Furthermore, we compared the activity of BKM120 to other PI3K inhibitors NVP-BEZ-235, a dual PI3K/mTOR inhibitor (Selleck, Houston, TX); and CAL101, a potent PI3K-p110-delta inhibitor (Selleck, Houston, TX); we examined the effect of different PI3K inhibitors (BKM120 500nM, CAL101 500nM or NVP-BEZ-235 100nM) on adhesion of MM1s cells to fibronectin, and found that the three inhibitors decreased the adhesion. Similarly, down-regulation of the expression of the PI3K catalytic subunits in MM1s, using siRNA decreased the adhesion of MM cells to fibronectin. In addition, we tested the effect of inhibition of PI3K on chemotaxis of MM cells. MM1s cells treated with BKM120 500nM, CAL101 500nM or NVP-BEZ-235 100nM for 4hrs, or with knockdown of PI3K by siRNA were applied to the upper chamber of a Boyden-migration assay, and allowed to migrate towards media with or without SDF1 30nM or conditioned media from MM stroma in the lower chamber for 4hrs. Interestingly, BKM120, NVP-BEZ-235 and the PI3K knock down increased significantly the chemotaxis of MM cells towards SDF1 and BM stromal, while CAL-101 had no effect on chemotaxis. These results were in accord with the effect of the drugs on the surface expression of CXCR4; as both BKM120 and NVP-BEZ-235, but not CAL101, increased the expression of CXCR4 in MM cells. Conclusion: we characterized the basal expression of the different PI3K catalytic subunits in MM cells lines, and showed that BKM120 inhibited PI3K signaling including proliferation and cell cycle in MM cells. BKM120 inhibited MM adhesion; an effect which was observed in NVP-BEZ-235 and CAL101, while only BKM120 and NVP-BEZ-235 increased the chemotaxis and CXCR4 surface expression of MM cells. These findings suggest that BKM120 can be used for regulation of MM cell trafficking in vivo by disrupting adhesion of MM cells to the BM and inducting of mobilization, leading to increased sensitivity to therapeutic agents. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Blockade of p110delta Isoform Activity of Phosphoinositide 3-Kinase Inhibits Blast Cell Proliferation in Acute Myeloblastic Leukemia.

AMLs constitute an heterogeneous group of hematopoietic stem cell disorders of rather poor prognosis. Abnormal activation of signaling pathways including STAT, MAPK and NF-KB have been described in AMLs and could constitute new targets for therapy. In this study, we focused on the PI3K/AKT and mTOR/P70S6K activity in primary blast cells purified from the bone marrow of patients with primary (n = 54) or secondary (n =10) AMLs. Patients with de novo AMLs were issued from the AML2001 French Multicenter Trial directed by the GOELAMS. The FAB classification was the following: M0 (n = 8), M1 (n = 10) M2 (n = 21), M4 (n = 10), M4eo (n = 2), M5 (n = 11), unknown AML (n = 2). In 58% of these patients, a constitutive activation of PI3K/AKT was detected in blast cells by western blot analysis showing AKT and FOXO3a phosphorylation, and confirmed by immunofluorescence confocal microscopy and flow cytometry analysis in CD34+ blast cells. Constitutive GAB1/2 phosphorylation was detected in all PI3K+ patients. However, we did not find any correlation between PI3K activation and FLT3 gene mutations. PTEN and SHIP-1 expression was normal in all tested PI3K+ patients. Proliferation (3H thymidine incorporation) was significantly increased in PI3K+ samples compared to PI3K- samples (p=0.001). The mTOR/P70S6K pathway was activated and the mTOR inhibitor rapamycin selectively reduced proliferation of PI3K+ samples. We determined the expression of the alpha, beta and delta isoforms of the catalytic sub-unit of PI3K (p110) in leukemic cells and found that p110 delta was the only consistently expressed isoform. In 8 PI3K+ samples tested, IC87114 (ICOS Corporation), a specific p110delta inhibitor compound used at 10μM totally suppressed AKT and FOXO3a phosphorylation. IC87114 inhibited proliferation of PI3K+ leukemic cells whereas it did not induce apoptosis. IC87114 did not significantly impaired the proliferation of PI3K- blast cells. Interestingly, IC87114 did not inhibit the proliferation or clonogenicity of CD34+ cord blood cells cultured in SCF, FLT3L and TPO used as controls. Overall, our results report, a/ a constitutive activation of PI3K in 58% of AML, b/ no correlation between FLT3 gene mutations and PI3K positivity, c/ no implication of PTEN or SHIP-1 phosphatases in PI3K activation, d/ a major role of the p110 delta isoform of PI3K in leukemic cell proliferation, e/ a potential therapeutic value of inhibiting mTOR and P110 delta activity.