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1
Hus, Iwona, Bartosz Puła, and Tadeusz Robak. "PI3K Inhibitors for the Treatment of Chronic Lymphocytic Leukemia: Current Status and Future Perspectives." Cancers 14, no. 6 (March 18, 2022): 1571. http://dx.doi.org/10.3390/cancers14061571.
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Phosphoinositide 3-kinases (PI3Ks) signaling regulates key cellular processes, such as growth, survival and apoptosis. Among the three classes of PI3K, class I is the most important for the development, differentiation and activation of B and T cells. Four isoforms are distinguished within class I (PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ). PI3Kδ expression is limited mainly to the B cells and their precursors, and blocking PI3K has been found to promote apoptosis of chronic lymphocytic leukemia (CLL) cells. Idelalisib, a selective PI3Kδ inhibitor, was the first-in-class PI3Ki introduced into CLL treatment. It showed efficacy in patients with del(17p)/TP53 mutation, unmutated IGHV status and refractory/relapsed disease. However, its side effects, such as autoimmune-mediated pneumonitis and colitis, infections and skin changes, limited its widespread use. The dual PI3Kδ/γ inhibitor duvelisib is approved for use in CLL patients but with similar toxicities to idelalisib. Umbralisib, a highly selective inhibitor of PI3Kδ and casein kinase-1ε (CK1ε), was found to be efficient and safe in monotherapy and in combination regimens in phase 3 trials in patients with CLL. Novel PI3Kis are under evaluation in early phase clinical trials. In this paper we present the mechanism of action, efficacy and toxicities of PI3Ki approved in the treatment of CLL and developed in clinical trials.
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Bohat, Ritu, Xiaofang Liang, Chunyu Xu, Yitao Tang, Jiakai Hou, Nicholas A. Egan, Leilei Shi, et al. "Abstract 4444: Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy." Cancer Research 83, no. 7_Supplement (April 4, 2023): 4444. http://dx.doi.org/10.1158/1538-7445.am2023-4444.
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Abstract Hyperactivation of the PI3K pathway has been reported to correlate with resistance to immune checkpoint blockade therapy (ICB) in melanoma, highlighting the therapeutic potential of combining PI3K inhibition (PI3Ki) with ICB. To maximize the clinical benefit of PI3Ki-based immune oncology (IO) combination, we characterized the role of PI3K isoforms in tumor and T cells and determined the immunological impacts of PI3Ki alone or in combination with ICB. Inhibitions of PI3K were achieved by either genetic knockdown (KD) or the bioactive compound in PTEN-present (B16/MC38), PTEN-absent (BP/D4M) tumor cell lines, and CD8+ T cells (Pmel-1). Following PI3Ki, we determined the activation status of the PI3K pathway (p-AKT level), transcriptional profile, and cellular function of these cells. We found both in vitro KD and pharmacological inhibition of either PI3Kα or PI3Kβ displayed a dramatic reduction of the PI3K pathway in tumor cells but moderate or no reduction in T cells, whereas the PI3K pathway significantly decreased in T cells with PI3Kγ or PI3Kδ inhibition. KD of PI3Kα or β isoforms drastically sensitized both D4M and MC38 tumors to αPD1 in vivo. We also observed that only PI3Kγ or PI3Kδ inhibition profoundly suppressed cytokine production and cytotoxicity of CD8+ T cell, suggesting that PI3Kα or PI3Kβ isoform inhibition can achieve tumor specific PI3Ki with limited impacts on T cell function. Furthermore, we used multiple syngeneic melanoma models to determine whether PI3K isoform inhibition can synergize the antitumor activity of ICB in vivo. In PTEN-present tumors, BYL719 (BYL, a PI3Kα inhibitor) synergized with αPD1 to delay tumor growth and extend survival (median survival of MC38-bearing mice in control (Ctrl), BYL, αPD1, and combination (Comb) groups: 30, 36, 33, and >45 respectively; p<0.05: Ctrl/BYL/αPD1 vs Comb). However, a limited combinatorial effect between GSK2636771(a PI3Kβ inhibitor) and αPD1 was observed in PTEN-present tumor models. Moreover, the combination of BYL and αPD1 exhibits superior antitumor activity in a spontaneous Braf-mutant, PTEN-loss melanoma model when compared with either reagent. Mechanistically, the combination of BYL and αPD1 improved CD8+ T cells tumor infiltration (14 days treatment, mean CD8+ number/mg of the tumor, Ctrl:1392.9, BYL:2073.9, αPD1:1545.2, Comb:4691.8; p<0.01: Ctrl/BYL/αPD1 vs Comb) and reduced MDSCs in MC38 tumors (p<0.05: Ctrl vs Comb). Multi-omics profiling of tumor cells with in vitro and in vivo PI3K isoform inhibition is ongoing. Collectively, our results demonstrate that PI3Kα inhibitor can potentiate T cell-mediated antitumor immune responses regardless of PTEN status, providing a strong rationale for the clinical development of the BYL-based IO combination. In collaboration with Novartis, MD Anderson Cancer Center will launch a Phase I/II trial of the FDA-approved BYL in combination with αPD1 in advanced melanoma and breast cancer patients. Citation Format: Ritu Bohat, Xiaofang Liang, Chunyu Xu, Yitao Tang, Jiakai Hou, Nicholas A. Egan, Leilei Shi, Ashley Guerrero, Roshni Jaffery, Elizabeth M. Burton, Han Liang, Hussein Tawbi, Michael A. Davies, Weiyi Peng. Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4444.
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Miller, Michelle, Philip Thompson, and Sandra Gabelli. "Structural Determinants of Isoform Selectivity in PI3K Inhibitors." Biomolecules 9, no. 3 (February 26, 2019): 82. http://dx.doi.org/10.3390/biom9030082.
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Phosphatidylinositol 3-kinases (PI3Ks) are important therapeutic targets for the treatment of cancer, thrombosis, and inflammatory and immune diseases. The four highly homologous Class I isoforms, PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ have unique, non-redundant physiological roles and as such, isoform selectivity has been a key consideration driving inhibitor design and development. In this review, we discuss the structural biology of PI3Ks and how our growing knowledge of structure has influenced the medicinal chemistry of PI3K inhibitors. We present an analysis of the available structure-selectivity-activity relationship data to highlight key insights into how the various regions of the PI3K binding site influence isoform selectivity. The picture that emerges is one that is far from simple and emphasizes the complex nature of protein-inhibitor binding, involving protein flexibility, energetics, water networks and interactions with non-conserved residues.
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Hawkins, P. T., K. E. Anderson, K. Davidson, and L. R. Stephens. "Signalling through Class I PI3Ks in mammalian cells." Biochemical Society Transactions 34, no. 5 (October 1, 2006): 647–62. http://dx.doi.org/10.1042/bst0340647.
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It is now accepted that activation of Class I PI3Ks (phosphoinositide 3-kinases) is one of the most important signal transduction pathways used by cell-surface receptors to control intracellular events. The receptors which access this pathway include those that recognize growth factors, hormones, antigens and inflammatory stimuli, and the cellular events known to be regulated include cell growth, survival, proliferation and movement. We have learnt a great deal about the family of Class I PI3K enzymes themselves and the structural adaptations which allow a variety of cell-surface receptors to regulate their activity. Class I PI3Ks synthesize the phospholipid PtdIns(3,4,5)P3 in the membranes in which they are activated, and it is now accepted that PtdIns(3,4,5)P3 and its dephosphorylation product PtdIns(3,4)P2 are messenger molecules which regulate the localization and function of multiple effectors by binding to their specific PH (pleckstrin homology) domains. The number of direct PtdIns(3,4,5)P3/PtdIns(3,4)P2 effectors which exist, even within a single cell, creates an extremely complex signalling web downstream of PI3K activation. Some key players are beginning to emerge, however, linking PI3K activity to specific cellular responses. These include small GTPases for the Rho and Arf families which regulate the cytoskeletal and membrane rearrangements required for cell movement, and PKB (protein kinase B), which has important regulatory inputs into the regulation of cell-cycle progression and survival. The importance of the PI3K signalling pathway in regulating the balance of decisions in cell growth, proliferation and survival is clear from the prevalence of oncogenes (e.g. PI3Kα) and tumour suppressors [e.g. the PtdIns(3,4,5)P3 3-phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10)] found in this pathway. The recent availability of transgenic mouse models with engineered defects in Class I PI3K signalling pathways, and the development of PI3K isoform-selective inhibitors by both academic and pharmaceutical research has highlighted the importance of specific isoforms of PI3K in whole-animal physiology and pathology, e.g. PI3Kα in growth and metabolic regulation, PI3Kβ in thrombosis, and PI3Kδ and PI3Kγ in inflammation and asthma. Thus the Class I PI3K signalling pathway is emerging as an exciting new area for the development of novel therapeutics.
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Garcia, Analia, Soochong Kim, Kamala Bhavaraju, Simone M. Schoenwaelder, and Satya P. Kunapuli. "Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways." Biochemical Journal 429, no. 2 (June 28, 2010): 369–77. http://dx.doi.org/10.1042/bj20100166.
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PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.
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Stypik, Mariola, Stanisław Michałek, Nina Orłowska, Marcin Zagozda, Maciej Dziachan, Martyna Banach, Paweł Turowski, et al. "Design, Synthesis, and Development of Pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part II—Benzimidazole Derivatives." Pharmaceuticals 15, no. 8 (July 27, 2022): 927. http://dx.doi.org/10.3390/ph15080927.
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Phosphoinositide 3-kinase (PI3K) is the family of lipid kinases participating in vital cellular processes such as cell proliferation, growth, migration, or cytokines production. Due to the high expression of these proteins in many human cells and their involvement in metabolism regulation, normal embryogenesis, or maintaining glucose homeostasis, the inhibition of PI3K (especially the first class which contains four subunits: α, β, γ, δ) is considered to be a promising therapeutic strategy for the treatment of inflammatory and autoimmune diseases such as systemic lupus erythematosus (SLE) or multiple sclerosis. In this work, we synthesized a library of benzimidazole derivatives of pyrazolo[1,5-a]pyrimidine representing a collection of new, potent, active, and selective inhibitors of PI3Kδ, displaying IC50 values ranging from 1.892 to 0.018 μM. Among all compounds obtained, CPL302415 (6) showed the highest activity (IC50 value of 18 nM for PI3Kδ), good selectivity (for PI3Kδ relative to other PI3K isoforms: PI3Kα/δ = 79; PI3Kβ/δ = 1415; PI3Kγ/δ = 939), and promising physicochemical properties. As a lead compound synthesized on a relatively large scale, this structure is considered a potential future candidate for clinical trials in SLE treatment.
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Diacovo, Thomas, Dosh Whye, Evgeni Efimenko, Jianchung Chen, Valeria Tosello, Kim De Keersmaecker, Adam Kashishian, et al. "Therapeutic Utility of PI3Kγ Inhibition in Leukemogenesis and Tumor Cell Survival." Blood 120, no. 21 (November 16, 2012): 1492. http://dx.doi.org/10.1182/blood.v120.21.1492.1492.
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Abstract Abstract 1492 Aberrant activation of the PI3K/Akt signaling pathway is a frequent event in cancer including various types of leukemia. Consequently, much emphasis has been placed on developing inhibitors that target this pathway. However, this would require an in depth knowledge of the role that specific class I PI3K isoforms (α, β, γ, δ)play in the pathogenesis of a particular hematological malignancy. For instance, PI3Kδ has been shown to be essential for the growth and survival of tumors derived from B cells such as chronic lymphocytic leukemia (CLL). Such knowledge has lead to development of the selective inhibitor GS-1101 (CAL-101) that has shown significant efficacy in clinical trials. Although PI3Kγ plays an important role in modulating the immune function of T cells, its role in leukemogenesis and tumor cell survival is poorly defined. Thus, it is unclear whether an inhibitor that also targets PI3Kγ would be of any benefit in hematological malignancies. T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer resulting from clonal proliferation of T lymphoid precursors. Previous reports suggest that hyperactivation of the PI3K/Akt signaling pathway is a common feature of this disease with the majority of cases due to the loss of function of the tumor suppressor PTEN. However, it remains to be determined whether any particular class I PI3K isoform predominates in T-ALL pathogenesis. We now report that in the absence of PTEN-mediated regulation in T cell progenitors that PI3Kγ can promote leukemogenesis even in the absence of its delta counterpart. However, inactivation of both isoforms was necessary for the suppression of tumor development in animals (< 20% dead at 220 days as compare to >85% for controls), suggesting that PI3Kα and/or PI3Kβ cannot adequately compensate for a deficiency in their γ/δ counterparts. The importance of PI3Kγ in tumor progression was established by the inability of the PI3Kδ selective inhibitor IC87114 to reduce tumor burden in mice (Fig. 1A). In contrast, treatment of PI3Kγ deficient tumors with the same inhibitor dramatically reduced disease in affected tissues (Fig. 1B). Based on these observations we developed an inhibitor, designated CAL-130, which targets both PI3Kγ and PI3Kδ in an attempt to exploit the addiction of PTEN null T-ALL tumors to both isoforms. IC50 values of this compound were 1.3 nM and 6.1 nM for p110δ and p110γ catalytic domains, respectively, as compared to 115 nM and 56 nM for p110α and p110β. Importantly, this small molecule does not inhibit additional intracellular signaling pathways (>300 kinases tested) that are critical for general cell function and survival. Oral administration of this compound to diseased mice (blast counts > 50 million/ml) for 7 days reduced tumor burden and extended median survival of treated animals to 45 day as compared 7.5 days for the control group (P<0.001). Of note, this inhibitor did not perturb plasma insulin or glucose levels in contrast to the metabolic perturbations associated with tissue-specific deficiencies in PI3Kα and PI3Kβ. The efficacy of this dual inhibitor was not limited to murine tumors as dual inhibition of PI3Kγ and PI3Kδ in primary human T-ALL cells displaying hyperactivation of this signaling pathway also reduced tumor cell survival by promoting activation of pro-apoptotic pathways. This work advances our understanding of the role that distinct PI3K isoforms play in development and survival of T-ALL and suggest that it may be possible to therapeutically exploit the addiction of this hematological malignancy to PI3Kγ and PI3Kδ. Moreover, by selectively targeting a signaling pathway key to tumor survival, it may be possible to limit toxicities associated with conventional chemotherapeutic agents that broadly affect metabolic pathways and DNA replication. Current studies are focused on evaluating the synergistic effect of PI3Kγ/δ blockade in combination with conventional chemotherapeutic agents used in the treatment of T-ALL. Disclosures: Kashishian: Gilead Sciences: Employment. Lannutti:Gilead Sciences Inc: Employment.
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Huang, Yi Elaine, Miho Iijima, Carole A. Parent, Satoru Funamoto, Richard A. Firtel, and Peter Devreotes. "Receptor-mediated Regulation of PI3Ks Confines PI(3,4,5)P3 to the Leading Edge of Chemotaxing Cells." Molecular Biology of the Cell 14, no. 5 (May 2003): 1913–22. http://dx.doi.org/10.1091/mbc.e02-10-0703.
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Recent studies have demonstrated that PH domains specific for PI(3,4,5)P3 accumulate at the leading edge of a number of migrating cells and that PI3Ks and PTEN associate with the membrane at the front and back, respectively, of chemotaxing Dictyostelium discoideum cells. However, the dependence of chemoattractant induced changes in PI(3,4,5)P3 on PI3K and PTEN activities have not been defined. We find that bulk PI(3,4,5)P3 levels increase transiently upon chemoattractant stimulation, and the changes are greater and more prolonged in pten– cells. PI3K activation increases within 5 s of chemoattractant addition and then declines to a low level of activity identically in wild-type and pten– cells. Reconstitution of the PI3K activation profile can be achieved by mixing membranes from stimulated pi3k1–/pi3k2– cells with cytosolic PI3Ks from unstimulated cells. These studies show that significant control of chemotaxis occurs upstream of the PI3Ks and that regulation of the PI3Ks and PTEN cooperate to shape the temporal and spatial localization of PI(3,4,5)P3.
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Lin, Shu, Zuwen Zhou, Rui Tan, Hua Xu, Huajie Zhang, Weipeng Zhang, Ling Chen, et al. "Abstract 5453: FCN-289, a novel, potent and selective PI3Kδ inhibitor for the treatment of B-cell malignancies." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5453. http://dx.doi.org/10.1158/1538-7445.am2022-5453.
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Abstract Phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway plays critical roles in cell growth, differentiation, motility, survival, and intracellular trafficking, and is one of the most frequently dysregulated pathways in human cancers including B-cell malignancies. There are 3 classes of PI3K, among which Class I PI3Ks including PI3Kα, β, γ, and δ isoforms are the mostly studied and plays key roles in physiological functions. PI3Kα has a role in insulin-dependent signaling, PI3Kβ functions in platelet aggregation, thrombosis and insulin signaling, and PI3Kγ/δ are expressed mainly in leukocytes and regulate lymphocyte activation, mast cell degranulation, and chemotaxis. Early PI3K inhibitors such as idelalisib are effective against B-cell malignancies such as chronic lymphocytic leukemia, but their clinical use is largely limited due to intolerable toxicities. More selective PI3Kδ inhibitors such as umbralisib (TGR-1202) demonstrates improved clinical efficacy and safety profile compared to current standard of care and was recently approved as a monotherapy for follicular lymphoma and marginal zone lymphoma. However, there is still unmet medical need for novel PI3Kδ inhibitors with improved safety profile and better efficacy to be used as monotherapy and in suitable combination strategies. Here we introduce FCN-289, a novel and oral next-generation PI3Kδ inhibitor. FCN-289 demonstrates potent kinase activity against PI3Kδ with single-digit nanomolar IC50 and remarkably improved selectivity over other PI3K isoforms compared with TGR-1202. FCN-289 exhibits significant anti-proliferating activity against various human diffuse large B-cell lymphoma (DLBCL)-derived cancer cell lines (OCI-LY10, TMD-8 and WSU-NHL) with superior activity compared with TGR-1202. Consistently, FCN-289 shows dose-dependent anti-tumor growth activity superior to that of TGR-1202 at the same and higher dose in TMD-8 DLBCL xenograft models. FCN-289 shows significantly improved anti-tumor activity when combined with BTK inhibitor ibrutinib in TMD-8 and OCI-LY10 DLBCL xenograft models. In non-clinical settings, FCN-289 exhibits good pharmacokinetic (PK) and safety properties with shorter Tmax and higher bioavailability in both rats and dogs, higher exposure in rats, improved CYP450 inhibition profile, and less plasma protein bound ratio compared with TGR-1202.Together, FCN-289 is a novel PI3Kδ inhibitor that possesses more potent in vitro and in vivo anti-cancer activities in B-cell malignancies-derived models with improved selectivity against other PI3K isoforms compared with TGR-1202. Combination with ibrutinib further improves anti-tumor activity compared with monotherapy. FCN-289 shows favorable PK and safety profiles compared with TGR-1202. Our findings highlight the therapeutic potential of FCN-289 as a novel targeted approach as monotherapy or in combination for treating B-cell malignancies. Citation Format: Shu Lin, Zuwen Zhou, Rui Tan, Hua Xu, Huajie Zhang, Weipeng Zhang, Ling Chen, Lijun Yang, Xingdong Zhao, Yanxin Liu, Zongyao Zou, Yuwei Gao, Jiashu Zhou, Weibo Wang. FCN-289, a novel, potent and selective PI3Kδ inhibitor for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5453.
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Tsolakos, N., T. N. Durrant, T. Chessa, S. M. Suire, D. Oxley, S. Kulkarni, J. Downward, et al. "Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors." Proceedings of the National Academy of Sciences 115, no. 48 (November 15, 2018): 12176–81. http://dx.doi.org/10.1073/pnas.1803446115.
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Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5–6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP3 and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing “excess p85,” p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.
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Xia, Liang, Yan Zhang, Jingbo Zhang, Songwen Lin, Kehui Zhang, Hua Tian, Yi Dong, and Heng Xu. "Identification of Novel Thiazolo[5,4-b]Pyridine Derivatives as Potent Phosphoinositide 3-Kinase Inhibitors." Molecules 25, no. 20 (October 12, 2020): 4630. http://dx.doi.org/10.3390/molecules25204630.
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A series of novel 2-pyridyl, 4-morpholinyl substituted thiazolo[5,4-b]pyridine analogues have been designed and synthesized in this paper. These thiazolo[5,4-b]pyridines were efficiently prepared in seven steps from commercially available substances in moderate to good yields. All of these N-heterocyclic compounds were characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) analysis and tested for phosphoinositide 3-kinase (PI3K) enzymatic assay. The results indicated that these N-heterocyclic compounds showed potent PI3K inhibitory activity, and the IC50 of a representative compound (19a) could reach to 3.6 nm. The structure−activity relationships (SAR) study showed that sulfonamide functionality was important for PI3Kα inhibitory activity, and 2-chloro-4-florophenyl sulfonamide (19b), or 5-chlorothiophene-2-sulfonamide (19c) showed potent inhibitory activity with a nanomolar IC50 value. The pyridyl attached to thiazolo[5,4-b]pyridine was another key structural unit for PI3Kα inhibitory potency, and replacement by phenyl lead to a significant decrease in activity. Enzymatic Inhibition results showed that compound 19a inhibited PI3Kα, PI3Kγ, or PI3Kδ with a nanomolar IC50 value, but its inhibitory activity on PI3Kβ was approximately 10-fold reduced. Further docking analysis revealed that the N-heterocyclic core of compound 19a was directly involved in the binding to the kinase through the key hydrogen bonds interaction.
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Yang, Bing, Bo Zhang, Qun Zhao, Jin Li, and Yujun Shi. "Development of New Thiophene-Containing Triaryl Pyrazoline Derivatives as PI3Kγ Inhibitors." Molecules 27, no. 8 (April 8, 2022): 2404. http://dx.doi.org/10.3390/molecules27082404.
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A series of new thiophene-containing triaryl pyrazoline derivatives, 3a–3t, were synthesized and evaluated regarding PI3K inhibition activity and anti-tumor potency based on a trial of introducing significant moieties, including pyrazoline and thiophene, and simplifying the parallel ring structures. Most of the tested compounds indicated potent PI3K inhibitory potency, with this series of compounds showing more potency for PI3Kγ than PI3Kα. The top hit 3s seemed more potent than the positive control LY294002 on inhibiting PI3Kγ (IC50 values: 0.066 μM versus 0.777 μM) and more selective from PI3Kα (Index values: 645 versus 1.74). It could be inferred that the combination of para- and meta-, as well as the modification of the electron-donating moieties, led to the improvement in potency. The anti-proliferation inhibitory activity and the enzymatic inhibition potency indicated consistent tendencies. The top hit 3s could inhibit the phosphorylation of Akt by inhibiting PI3K through the PI3K-Akt-mTOR pathway. The molecular docking simulation indicated that the binding pattern of 3s into PI3Kγ was preferable than that of PI3Kα, with more hydrogen bond, more π-involved interactions, and fewer π-sulfur interactions. The information in this work is referable for the further development of selective inhibitors for specific isoforms of PI3K.
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Paul, Juliane, Maurice Sojoun, Antje M. Wengner, Sabine Zitzmann-Kolbe, Andrea Sturz, Katja Haike, Magdalene H.-K. Koh, et al. "Simultaneous Inhibition of PI3Kδ and PI3Kα Induces ABC-DLBCL Regression Via Attenuation of BCR-Dependent and Independent Activation of NF-Kb and AKT." Blood 128, no. 22 (December 2, 2016): 1057. http://dx.doi.org/10.1182/blood.v128.22.1057.1057.
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Abstract Introduction: ABC-DLBCL is a subtype of DLBCL with less favorable clinical outcomes to the standard of care (SoC) therapies. Constitutive activation of NF-κB by various genetic alterations in ABC-DLBCL has been identified as one of the key mechanisms driving chemotherapy resistance. Inhibition of B cell receptor (BCR) signaling with BTK (Bruton's tyrosine kinase) inhibitor ibrutinib demonstrated encouraging clinical responses in ABC-DLBCL. However, patients with CD79wt/MyD88mut, or CARD11mut did not respond to ibrutinib, indicating the need for new therapies targeting ibrutinib refractory ABC-DLBCL. Recent approval of PI3Kδ selective inhibitor idelalisib for the treatment of indolent NHL (iNHL) as monotherapy highlighted selective inhibition of PI3Kδ as an effective therapeutic strategy. However, idelalisib did not show clinical activity in DLBCL in a Phase I expansion cohort study. Here we report immunohistochemical (IHC) analysis of clinical tumor samples from follicular lymphoma (FL) and DLBCL patients and a series of in vitro and in vivo mechanistic and functional studies to explore the importance of PI3K isoforms in regulating key signaling pathways in ABC-DLBCL. Methods: Expression of PI3K isoforms and PTEN was assessed by IHC and western blot from a panel of 45 FL and 45 DLBCL primary tumors. Effects of PI3K inhibitors (PI3Kδ-selective inhibitor idelalisib, PI3Kα-selective inhibitor BLY-719, PI3Kβ-selective inhibitor TGX-221, predominant PI3Kα/δ inhibitor copanlisib and BTK inhibitor ibrutinib) on nuclear NF-κB activation were determined using stable cell lines expressing NF-κB-luciferase reporter (obtained by lentiviral infection), IHC staining of p65 NF-κB, expression of CCL4, IL-6, and IL-10 by RT-PCR and protein production by ELISA assays. In vitro and in vivo mechanisms of action were addressed by assessing the activities of the key survival signaling pathways. In vitro and in vivo anti-tumor activities were investigated using cell lines and patient derived xenograft ABC-DLBCL models representing the key molecular features of BCR-dependent and independent ABC-DLBCL. Results: Although PI3Kδ was predominantly expressed in both FL and DLBCL, high PI3Kα expression was more prevalent in DLBCL (60% vs 18%), a patient population resistant to PI3Kδ-selective inhibition in the clinic. Simultaneous inhibition of PI3Kα and PI3Kδ with BYL-719 plus idelalisib or copanlisib alone dramatically enhanced anti-tumor profile in ABC-DLBCL models compared to selective inhibition of PI3Kδ, PI3Kα or BTK. The anti-tumor activity was associated with suppression of p-AKT and a novel mechanism of blocking NF-κB activation driven by CD79mut, CARD11mut, A20mut or MyD88mutin vitro and in vivo. Suppression of NF-kB activation by PI3K inhibition is independent from AKT, but involves a novel mechanism of modulating c-IAP expression. Inhibition of PI3Kα/δ resulted in complete tumor regression in an ibrutinib-resistant MyD88mut-LY0257 patient-derived (PDx) ABC-DLBCL model. Furthermore, rebound activation of BTK and AKT was identified as a mechanism limiting CD79mut ABC-DLBCL to show robust response to PI3K and BTK inhibitors, respectively, as single agents in vivo. Combination of ibrutinib with PI3Kα/δ inhibitor copanlisib dosed intermittently iv was well tolerated and produced complete tumor regression in CD79Bmut TMD-8 cell line and Ly2298 PDx ABC-DLBCL models. Conclusions: High expression of PI3Kα in addition to PI3Kδ in ABC-DLBCL is associated with intrinsic resistance to PI3Kδ selective inhibition. Simultaneous inhibition of PI3Kα/δ by copanlisib modulates not only the PI3K/AKT pathway but also BCR-dependent and independent activation of nuclear NF-κB via a novel AKT-independent mechanism, indicating a promising utility for the treatment of clinically relevant ibrutinib-resistant ABC-DLBCL patients with CD79wt/MyD88mut, A20mut, or CARD11mut tumor genotypes. Combination of PI3Kα/δ and BTK inhibitors demonstrated promising potential for ibrutinib-responsive ABC-DLBCL to achieve complete tumor regression by blocking rebound activation of BTK and AKT. Thus, our findings presented here provide additional insights on intrinsic and acquired resistance mechanisms to selective PI3Kδ and BTK inhibitors and provide rationale for clinical development of PI3K inhibitors with specific isoform profile in combination for the treatment of ABC-DLBCL. Disclosures Paul: Bayer AG: Employment. Sojoun:Bayer AG: Employment. Wengner:Bayer AG: Employment. Zitzmann-Kolbe:Bayer AG: Employment. Sturz:Bayer AG: Employment. Haike:Bayer AG: Employment. Martin:Bayer AG: Employment. Mumberg:Bayer AG: Employment. Ziegelbauer:Bayer AG: Employment. Liu:Bayer AG: Employment.
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Bresnick, Anne R., and Jonathan M. Backer. "PI3Kβ—A Versatile Transducer for GPCR, RTK, and Small GTPase Signaling." Endocrinology 160, no. 3 (January 2, 2019): 536–55. http://dx.doi.org/10.1210/en.2018-00843.
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AbstractThe phosphoinositide 3-kinase (PI3K) family includes eight distinct catalytic subunits and seven regulatory subunits. Only two PI3Ks are directly regulated downstream from G protein–coupled receptors (GPCRs): the class I enzymes PI3Kβ and PI3Kγ. Both enzymes produce phosphatidylinositol 3,4,5-trisposphate in vivo and are regulated by both heterotrimeric G proteins and small GTPases from the Ras or Rho families. However, PI3Kβ is also regulated by direct interactions with receptor tyrosine kinases (RTKs) and their tyrosine phosphorylated substrates, and similar to the class II and III PI3Ks, it binds activated Rab5. The unusually complex regulation of PI3Kβ by small and trimeric G proteins and RTKs leads to a rich landscape of signaling responses at the cellular and organismic levels. This review focuses first on the regulation of PI3Kβ activity in vitro and in cells, and then summarizes the biology of PI3Kβ signaling in distinct tissues and in human disease.
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Arcucci, Silvia, Fernanda Ramos-Delgado, Coralie Cayron, Nicole Therville, Marie-Pierre Gratacap, Céline Basset, Benoit Thibault, and Julie Guillermet-Guibert. "Organismal roles for the PI3Kα and β isoforms: their specificity, redundancy or cooperation is context-dependent." Biochemical Journal 478, no. 6 (March 19, 2021): 1199–225. http://dx.doi.org/10.1042/bcj20210004.
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PI3Ks are important lipid kinases that produce phosphoinositides phosphorylated in position 3 of the inositol ring. There are three classes of PI3Ks: class I PI3Ks produce PIP3 at plasma membrane level. Although D. melanogaster and C. elegans have only one form of class I PI3K, vertebrates have four class I PI3Ks called isoforms despite being encoded by four different genes. Hence, duplication of these genes coincides with the acquisition of coordinated multi-organ development. Of the class I PI3Ks, PI3Kα and PI3Kβ, encoded by PIK3CA and PIK3CB, are ubiquitously expressed. They present similar putative protein domains and share PI(4,5)P2 lipid substrate specificity. Fifteen years after publication of their first isoform-selective pharmacological inhibitors and genetically engineered mouse models (GEMMs) that mimic their complete and specific pharmacological inhibition, we review the knowledge gathered in relation to the redundant and selective roles of PI3Kα and PI3Kβ. Recent data suggest that, further to their redundancy, they cooperate for the integration of organ-specific and context-specific signal cues, to orchestrate organ development, physiology, and disease. This knowledge reinforces the importance of isoform-selective inhibitors in clinical settings.
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Hutter, Grit, Yvonne Zimmermann, Anna-Katharina Zoellner, Philip Irrgang, Oliver Weigert, Wolfgang Hiddemann, and Martin Dreyling. "Combination of PI3K and PDPK1 Inhibitors Is Highly Effective in Mantle Cell Lymphoma." Blood 124, no. 21 (December 6, 2014): 3123. http://dx.doi.org/10.1182/blood.v124.21.3123.3123.
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Abstract Introduction: Mantle cell lymphoma (MCL), is a distinct lymphoma subtype with an aggressive clinical course and a median survival of 3-5 years. New emerging strategies include especially inhibitors of the PI3K/Akt/mTOR pathway which is constitutively activated in MCL and plays a critical role in tumor growth and survival. In the present study we analysed four different PI3K-Inhibitors (IPI-145, CAL101, A66 and TGX221) targeting different isoforms (PI3Kδ/γ, PI3Kδ, PI3Kα and PI3Kβ) for their effectiveness in MCL. Methods: MCL cell lines (Z-138, Mino-1, Granta-519, Jeko-1, Rec-1, Maver-1) were exposed to different PI3K-Inhibitors (IPI-145, CAL101, A66 and TGX221) with or without murine feeder layer (M210B4). The effect of drugs was evaluated by cell count (trypan-blue staining), cell metabolism (WST-assay or ONE-GloTMLuciferase assay for luciferized MCL cell lines), cell cycle (FACS) and apoptosis (Annexin V PE/7-AAD staining). Subsequently, combinations of PI3K-inhibitors and other target molecules (PDPK1: BX912, OSU03012, GSK2334470) were analysed. Western blot and mRNA analyses were performed after exposure to various inhibitors of the PI3K/Akt/mTOR pathway and correlated to sensitivity of cell lines. Finally, efficiency of drug combinations were confirmed in primary patient samples. Results: The PI3K inhibitor IPI145 (8,2%-62,4%) was most effective in MCL cell lines followed by A66 (0%-41,7%), TGX221 (0%-33,8%) and CAL101 (0%-28,2%) suggesting a higher efficiency by inhibiting multiple isoforms of PI3K. In patient samples IPI145 revealed also the highest cytotoxicity followed by CAL101, TGX221 and A66 (72,4%; 67,9%; 67,1% and 43,7%, respectively). Comparing various combinations of PI3K-inhibitors in MCL cell lines the simultaneous inhibition of α (A66) and δ (CAL101) isoforms achieved the highest reduction of cell count (65,2%). However, combined inhibition of both of PI3K δ and PDPK1 (BX912), led to a more efficient G2 arrest and higher rate of apoptosis due to the simultaneous dephosphorylation of members of the mTOR- and MAPK-pathways. Accordingly, the observed cytotoxicity was even higher (59%-87%) than for dual inhibition of PI3K (23%-60%). Currently, Western blot analyses are being expanded to further identify the differential mechanisms of PI3K inhibitor combinations. In addition, the presence of a murine feeder layer enhanced the effect of PI3K inhibitors in MCL cells nidated into the feederlayer because of higher levels of Akt phosphorylation possibly due to interaction with the microenvironment. Accordingly, this effect was not detectable in the supernatant cells. Conclusion: Based on our comparative analysis of PI3K inhibitors targeting of a and d isoforms appears to be the most efficient mechanism in MCL. However, this impact could be further increased by simultaneous inhibition of PDPK1. Western blot results suggest that these results are due to the inhibition of complementary pathways. Interestingly, the murine feeder layer enhanced the effect of PI3K inhibitors emphasizing the role of the microenvironment in the molecular mechanisms of targeted strategies. Disclosures Dreyling: Gilead: Gilead: speaker's honoraria Other.
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Meadows, Sarah, Sorensen Rick, Yahiaoui Anella, Jia Liu, Li Li, Peng Yue, Christophe Queva, and Stacey Tannheimer. "Up-Regulation of the PI3K Signaling Pathway Mediates Resistance to Idelalisib." Blood 126, no. 23 (December 3, 2015): 3707. http://dx.doi.org/10.1182/blood.v126.23.3707.3707.
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Abstract Background: Idelalisib (Zydelig®), a potent and selective PI3Kδ inhibitor, was recently approved for the treatment of relapsed CLL, SLL and FL. PI3Kδ is critical for malignant B-cell proliferation, survival and homing, and inhibition results in a rapid decrease in lymphadenopathy and clinical response (Yang et al., Clin Can Res 2015). Although idelalisib is very potent in mitigating disease, most patients experience incomplete response and ultimately progress. To understand the mechanism of resistance, we screened ABC-DLBCL cell lines for sensitivity to idelalisib and selected TMD8, an ABC-DLBCL cell line, for the generation of idelalisib resistant cells. We report on the mechanisms of resistance and on downstream biomarkers associated with sensitivity to idelalisib in lymphoma cell lines. Methods: Growth inhibition to idelalisib or other inhibitors was assessed using CellTiterGlo viability assay (Promega) at 96 h. Idelalisib resistant line (TMD8R) was generated by continuous exposure to1μM idelalisib (~2x Cmax corrected for protein binding). A DMSO passage matched line was generated as control (TMD8S). Clonal isolates from pools were generated through 2 rounds of limiting dilution. Cell lines were analyzed by whole exome sequencing (WES, GeneWiz), RNASeq (Expression Analysis) and phosphoproteomics (MultiPathway PTMScan Direct, Cell Signaling Technologies). Protein expression was measured using Simple Western (Protein Simple) and SDS/PAGE Western blot. Results: TMD8 were sensitive to idelalisib and the pan PI3K inhibitor (GDC-0941) with an EC50 of 42 and 27nM, respectively, but not to PI3Kα (BYL-719) or PI3Kβ (AZD-6482) inhibitors, indicating that cell viability is mostly driven by PI3Kδ. TMD8 cells with acquired resistance to idelalisib (TMD8R) showed a loss of sensitivity to idelalisib, with a growth inhibition of 19% vs. 92% at 1μM as compared to TMD8S. No mutation in PIK3CD or other PI3K pathway members, including PTEN, was found in TMD8R clones by WES. In 8/8 TMD8R clones a dramatic reduction of PTEN protein expression was observed (9-fold) despite normal expression of PTEN mRNA by RNAseq (Figure 1A). Additionally 8/8 TMD8R clones showed a modest up-regulation of PIK3CG mRNA (2-fold) and protein (3-5 fold, Figure 1B). PIK3CD remained the most prevalent PI3K isoform expressed in all TMD8R clones (Figure 1C). TMD8R were cross-resistant to the dual PI3Kδ/γ inhibitor duvelisib (EC50 > 4 uM for TMD8R vs. EC50 = 58 nM for TMD8S). RNAseq analysis of idelalisib sensitive and resistant ABC-DLBCL cell lines showed that idelalisib treatment led to c-Myc mRNA down regulation in sensitive cell lines (TMD8 and Ri-1) but not in the resistant cell lines (U2932 and SU-DHL-8). In addition, expression of c-Myc target genes was unchanged in the TMDR. Analysis of phosphoproteins showed PI3K and MAPK pathway up-regulation in TMDR, compared to TMD8S, with increased expression of p-Akt T308, p-Akt S473, p-PDK1 S241, p-GSK3β S9 and p-ERK T202/Y204 (11-, 6-, 2-, 1- and 8-fold, respectively), providing a potential mechanism for the loss of c-Myc down regulation in resistant cells (Wen-Bin Tsai et al., Cancer Res, 2012). Other B-cell receptor signaling proteins (p-BTK Y223, p-SYK Y525/526, and p-STAT-5 Y694) were not detected or unchanged. TMD8R cells were cross resistant to the BTK inhibitors ibrutinib and ONO/GS-4059. Akt (MK-2206) or PDK1 (GSK233440) inhibitors were less potent in TMD8R vs. TMD8S cells, yet in combination with idelalisib at 1μM, their potency was restored to the level observed in TMD8S, indicating that resistant cells retain some dependency on the PI3Kδ. Conclusions: Resistance to idelalisib in the ABC-DLBCL cell line TMD8 was not acquired through de novo mutations. Rather loss of PTEN protein expression, modest up regulation of PI3Kγ, activation of the PI3K and MAPK pathway and loss of c-Myc down regulation by idelalisib were identified as potential mechanism of resistance. Treatment with agents targeting the PI3K pathway in combination with idelalisib may overcome resistance. Figure 1. Profiling of TMD8R Cells Reveal Increased PI3Kγ and PTEN loss. (A) Decreased expression of PTEN (9 fold) in TMD8R clones as compared to TMD8S (B) Overexpression of PI3Kγ (3-5 fold) in TMD8R clones as compared to TMD8S (C) PI3Kδ isoform is the highest expressed isoform in both TMD8S and TMD8R clones. Figure 1. Profiling of TMD8R Cells Reveal Increased PI3Kγ and PTEN loss. (A) Decreased expression of PTEN (9 fold) in TMD8R clones as compared to TMD8S (B) Overexpression of PI3Kγ (3-5 fold) in TMD8R clones as compared to TMD8S (C) PI3Kδ isoform is the highest expressed isoform in both TMD8S and TMD8R clones. Disclosures Meadows: Gilead Sciences: Employment, Other: Share holder. Rick:Gilead Sciences: Employment, Other: stock holder. Anella:Gilead Sciences: Employment, Other: Stock holder. Liu:Gilead Sciences: Employment, Other: stock holder. Li:Gilead Sciences: Employment, Other: Share holder. Yue:Gilead Sciences: Employment, Other: Share holder. Queva:Gilead Sciences: Other: Stock holder. Tannheimer:Gilead Sciences: Employment, Other: Share holder.
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Barberis, Laura, and Emilio Hirsch. "Targeting phosphoinositide 3-kinase γ to fight inflammation and more." Thrombosis and Haemostasis 99, no. 02 (2008): 279–85. http://dx.doi.org/10.1160/th07-10-0632.
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SummaryThe family of class I phosphoinositide-3-kinase (PI3K) is composed of four lipid kinases involved at multiple levels in innate and adaptive immune responses. Class I PI3Ks are divided into two subclasses, IA and IB, sharing a similar catalytic core. Whereas class IA PI3Ks are primarily activated by receptor tyrosine kinases, the unique element of class IB PI3K (PI3Kγ) is activated by G protein coupled receptors (GPCRs), like chemokine receptors. PI3Kγ is mainly expressed in leukocytes where it plays a significant role in chemotaxis. Here, we report recent advances in the analysis of the role of PI3Kγ in leukocytes and in endothelial cells. Results, derived from studies based on both pharmacological and genetic approaches, confirm PI3Kγ as an attractive target for drug discovery. PI3Kγ specific inhibition has gained increasing attention for the treatment of allergic, autoimmune and inflammatory diseases. Development of inhibitors has already provided series of hits, whose efficacy is currently under scrutiny worldwide.
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Narkhede, Mayur, and Bruce D. Cheson. "Copanlisib in the treatment of non-Hodgkin lymphoma." Future Oncology 16, no. 26 (September 2020): 1947–55. http://dx.doi.org/10.2217/fon-2020-0195.
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B-cell receptor signaling is important in the pathogenesis of non-Hodgkin lymphoma. The PI3K pathway is activated by B-cell receptor signaling. Recently, several PI3K inhibitors have been in development for the treatment of indolent non-Hodgkin lymphomas. Copanlisib is a PI3Kα and PI3Kδ inhibitor that has been approved for its use as third-line therapy in the treatment of relapsed or refractory follicular lymphoma. The two other PI3k inhibitors approved by the US FDA in this setting are idelalisib and duvelisib. In this review, we compare the efficacy and adverse event profile of these different PI3K inhibitors and discuss the advantages and challenges of using copanlisib along with a guide on managing routinely encountered adverse events in the clinics.
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Thijssen, Rachel, Gregor van Bochove, Martin FM de Rooij, Johanna ter Burg, Marcel Spaargaren, Coumaran Egile, Marie Jose Kersten, Eric Eldering, and Arnon P. Kater. "Combined Inhibition of Phosphatidylinositol 3-Kinase (PI3K) Isoform α and δ By the Pan-Class I PI3K Inhibitor SAR245409 (XL765) in Primary Chronic Lymphocytic Leukemia Cells Blocks Survival, Adhesion and Proliferation." Blood 124, no. 21 (December 6, 2014): 4691. http://dx.doi.org/10.1182/blood.v124.21.4691.4691.
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Abstract CLL cells are highly dependent on B- cell receptor (BCR) signaling and on stimuli from the microenvironment for survival and proliferation. New drugs targeting PI3K downstream of BCR signaling have emerged as promising treatment options for patients with CLL. Among four PI3K catalytic subunits, the PI3Kd isoform is crucial for downstream BCR signaling, but the relative importance of the PI3Kα isoform in CLL is less clear. Impressive clinical activity of idelalisib in CLL and indolent NHL patients was recently reported. Idelalisib, a PI3Kd specific inhibitor, inhibits chemotaxis and adhesion of leukemia cells, resulting in rapid lymphocytosis followed by a decrease in lymphadenopathy. However, idelalisib has no direct impact on leukemic cell survival [1], raising the potential risk of residual clones responsible for the development of resistance. In this study, we evaluated the impact of a pan-class I PI3K inhibitor (SAR245409/XL765), a PI3Kα-specific inhibitor (BYL719) and a PI3Kd specific inhibitor (idelalisib) on PI3K/mTOR signaling, apoptosis, cell adhesion, CD40-induced survival and proliferation in primary patient derived leukemic cells. Phosphorylation of the downstream effector of mTOR, S6RP, was completely blocked by SAR245409 but not by BYL719 or idelalisib. SAR245409 induced apoptosis in unstimulated CLL cells (IC50= 0.86µM) in contrast to BYL719 or idelalisib (IC50 >10µM), demonstrating that targeting multiple PI3K isoforms is required to completely block the PI3K/Akt/mTOR pathway (table 1). Importantly, SAR245409 also induced apoptosis in p53 or ATM dysfunctional CLL samples. SAR245409, as well as idelalisib, and in contrast to BYL719 completely inhibited BCR-mediated adhesion to fibronectin [2]. Similarly, SAR245409 inhibited CD40L-mediated survival [3], and induced upregulation of the pro-apoptotic protein BIM. All 3 PI3K inhibitors inhibited CD40 ligation + IL-21-mediated CLL proliferation [4]. This study revealed that the pan-class I PI3K inhibitor SAR245409 is more cytotoxic to primary CLL cells than PI3Kα or PI3Kd specific inhibitors. Furthermore, combined inhibition of PI3Kα and d can block signaling pathways that are critical for CLL survival, adhesion and proliferation in the LN microenvironment (see table 1). This work provides a rationale for the evaluation of SAR245409 in CLL patients either as monotherapy or in combination therapies. [1] Hoellenriegel et al. The phospoinositide 3'-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood 2011;(118):3603-3612 [2] de Rooij et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;(119):2590-2594. [3] Smit et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;(109):1660-1668. [4] Pascutti et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood 2013;(122):3010-3019. Table 1. The effect of the PI3Kd inhibitor idelalisib, PI3Kα inhibitor BYL719 or pan PI3K inhibitor SAR245409 on CLL cells in functional assays PI3Kd inhibitor PI3Kα inhibitor pan PI3K inhibitor Cytotoxicity (IC50)1 >10µM >10µM 0.86µM Inhibition of adhesion2 48%** 21% 43%** Activation Inhibition of CD40L-induced survival3 14% 0% 54%* Inhibition of CD40L+IL21 induced proliferation4 47%* 35%* 51%* 1 CLL cells were incubated with 0.001-10 μM idelalisib (n=18), BYL719 (n=6) or SAR245409 (n=28) for 48 hours. Viability was assessed by DiOC6/PI staining.2 CLLcells pretreated with 1 µM idelalisib, BYL719, or SAR245409 were stimulated with α-IgM and allowed to adhere to fibronectin-coated surfaces (n=5). 3 CLL cells were cultured on fibroblast expressing CD40L in the absence or presence of 1 µM of idelalisib, BYL719, or SAR245409 for 3 days. Apoptosis was assessed by DiOC6/PI staining (n=8).4 CFSE labelledCLL cells were cultured on fibroblast expressing CD40L with IL-21 and co-treated with 1 µM idelalisib, BYL719, or SAR245409. After 4 days, CFSE was measured by FACS (n=11)2-4 The one sample T test was used to determine the significance of differences between means of treated samples and normalized values of untreated samples (100%). * p <0,05;** p<0,01 Disclosures Egile: Sanofi: Employment. Kersten:Sanofi: Research Funding. Kater:Sanofi: Research Funding.
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Figueiredo, Ana M., Pilar Villacampa, Rodrigo Diéguez-Hurtado, Juan José Lozano, Piotr Kobialka, Ana Rosa Cortazar, Anabel Martinez-Romero, et al. "Phosphoinositide 3-Kinase–Regulated Pericyte Maturation Governs Vascular Remodeling." Circulation 142, no. 7 (August 18, 2020): 688–704. http://dx.doi.org/10.1161/circulationaha.119.042354.
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Background: Pericytes regulate vessel stabilization and function, and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. Methods: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed Pdgfrb(BAC)-CreER T2 mice into RiboTag flox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single-cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models that allow selective inactivation of PI3Kα and PI3Kβ isoforms and their negative regulator phosphate and tensin homolog deleted on chromosome 10 (PTEN) in mural cells. Results: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling, pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that PI3Kβ, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kβ inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. Conclusions: Our results identify new molecular and morphological traits associated with pericyte maturation and uncover PI3Kβ activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kβ activity.
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Newman, Debra K. "PI3Kβ goes to the head of its class." Blood 114, no. 10 (September 3, 2009): 2011–12. http://dx.doi.org/10.1182/blood-2009-06-228551.
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Abstract In this issue of Blood, Canobbio and colleagues report on the unique and redundant contributions of 2 isoforms of PI3K, PI3Kβ and PI3Kγ, to platelet activation by GPCRs and ITAM–coupled receptors.1
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Yano, Naohiro, Vlad Ianus, Ting C. Zhao, Andy Tseng, James F. Padbury, and Yi-Tang Tseng. "A novel signaling pathway for β-adrenergic receptor-mediated activation of phosphoinositide 3-kinase in H9c2 cardiomyocytes." American Journal of Physiology-Heart and Circulatory Physiology 293, no. 1 (July 2007): H385—H393. http://dx.doi.org/10.1152/ajpheart.01318.2006.
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Stimulation of cardiac β-adrenergic receptors (β-AR) activates both the Gs- and Gi-coupled signaling cascades, including the phosphoinositide 3 kinase (PI3K) pathway, that have important physiological implications. Multiple isoforms of PI3K exist in the heart. The goals of this study were to examine the intracellular signaling pathways linking β-AR to PI3K and to identify the PI3K isoform mediating this transactivation in a cardiac context. Acute β-AR stimulation with isoproterenol resulted in increased tyrosine kinase-associated PI3K activity and phosphorylation of Akt and p70S6K in H9c2 cardiomyocytes. Cotreatment with ICI-118,551, but not CGP-20712, abolished the increase in PI3K activity, suggesting a β2-AR-mediated event. PI3K activation was also abrogated by cotreatment with pertussis toxin, 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolol[3,4-d]pyrimidine (PP2, a selective Src-family tyrosine kinases inhibitor), or AG-1296 [selective platelet-derived growth factor receptor (PDGFR) inhibitor] but not with an inhibitor for protein kinase A, protein kinase C, Ras, adenylyl cyclase, epidermal growth factor receptor, or insulin-like growth factor-1 receptor. β-AR stimulation induced an increase in tyrosine phosphorylation of PDGFR, which was abolished by inhibition of Src either by PP2 or small interfering RNA. Moreover, H9c2 cardiomyocytes stably transfected with a vector expressing a Gβγ sequestrant peptide derived from the COOH-terminus of β-AR kinase-1 failed to activate PI3K after β-AR stimulation, suggesting Gβγ is required for the transactivation. Furthermore, acute β-AR stimulation in vivo resulted in increases in PDGFR-associated PI3K and PI3Kα isoform activities but not the activities of other isoforms (PI3Kβ, -δ, -γ) in adult mouse heart. Taken together, these data provide in vitro and in vivo evidence for a novel mechanism of β-AR-mediated transactivation of cardiac PI3Kα via sequential involvement of Gαi/Gβγ, Src, and PDGFR.
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RUBIO, Ignacio, Pablo RODRIGUEZ-VICIANA, Julian DOWNWARD, and Reinhard WETZKER. "Interaction of Ras with phosphoinositide 3-kinase γ." Biochemical Journal 326, no. 3 (September 15, 1997): 891–95. http://dx.doi.org/10.1042/bj3260891.
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Phosphoinositide 3-kinase γ (PI3Kγ) can be activated in vitro by both α and βγ subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kα. Here we demonstrate the binding of Ras to PI3Kγ in vitro. An N-terminal region of PI3Kγ was identified as a binding site for Ras. After co-expression with PI3Kγ in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kα by Ras in the same cells.
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HEHL, Solveig, Borislav STOYANOV, Wolf OEHRL, Roland SCHÖNHERR, Reinhard WETZKER, and Stefan H. HEINEMANN. "Phosphoinositide 3-kinase-γ induces Xenopus oocyte maturation via lipid kinase activity." Biochemical Journal 360, no. 3 (December 10, 2001): 691–98. http://dx.doi.org/10.1042/bj3600691.
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Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the γ-isoform of PI3K (PI3Kγ) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kγ (PI3Kγ-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kγ-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kγ-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kγ-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kγ in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect.
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Borsari, Chiara, and Matthias P. Wymann. "Targeting Phosphoinositide 3-Kinase – Five Decades of Chemical Space Exploration." CHIMIA 75, no. 12 (December 9, 2021): 1037. http://dx.doi.org/10.2533/chimia.2021.1037.
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Phosphoinositide 3-kinase (PI3K) takes a key role in a plethora of physiologic processes and controls cell growth, metabolism, immunity, cardiovascular and neurological function, and more. The discovery of wortmannin as the first potent PI3K inhibitor (PI3Ki) in the 1990s provided rapid identification of PI3K-dependent processes, which drove the assembly of the PI3K/protein kinase B (PKB/Akt)/target of rapamycin (mTOR) pathway. Genetic mouse models and first PI3K isoform-specific inhibitors pinpointed putative therapeutic applications. The recognition of PI3K as target for cancer therapy drove subsequently drug development. Here we provide a brief journey through the emerging roles of PI3K to the development of clinical PI3Ki candidates.
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Varkaris, Andreas, Ferran Fece de la Cruz, Elizabeth Martin, Bryanna Nordern, Nicholas Chevalier, Ignaty Leshchiner, Anastasia Stavridi, et al. "Abstract GS03-10: Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations." Cancer Research 84, no. 9_Supplement (May 2, 2024): GS03–10—GS03–10. http://dx.doi.org/10.1158/1538-7445.sabcs23-gs03-10.
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Abstract Background: PIK3CA mutations occur in ~40% of HR-positive breast cancers, where alpelisib, an orthosteric PI3Kα inhibitor, is FDA-approved in combination with fulvestrant. Although prior studies have identified potential resistance mechanisms, such as PTEN loss, clinical acquired resistance to orthosteric PI3Kα inhibitors and the role of next-generation allosteric PI3Kα inhibitors remain poorly understood. Methods: To identify on-target and off-target alterations potentially mediating resistance to PI3Kα inhibitors, we used a targeted next-generation sequencing assay (Guardant360; Guardant Health) to analyze ctDNA in serially collected plasma samples from 32 patients with PIK3CA-mutated advanced HR-positive, HER2-negative breast cancer treated with alpelisib and inavolisib. In addition, we performed whole exome sequencing (WES) of 100 tissue samples collected from 8 autopsy series from patients with metastatic, PIK3CA-mutant HR-positive, HER2-negative breast cancer previously treated with PI3Kα inhibitors. Acquired alterations were prioritized through a combination of structural modeling and free-energy perturbation simulation and validated in genomically engineered PIK3CA mutant breast cancer cell lines T47D (PIK3CA H1047R-mutant) or MCF7 (PIK3CA E545K-mutant). Results: We observed that 50% of patients acquire genomic alterations within the PI3K-pathway, including PTEN loss and activating AKT1 mutations. Notably, while secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Ka-inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket including PIK3CA Q859K and PIK3CA W780R. Some mutations had differential effects on PI3Ka-selective vs. pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Ka-inhibitor RLY-2608. Conclusion: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Ka inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Ka inhibitor. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers. Citation Format: Andreas Varkaris, Ferran Fece de la Cruz, Elizabeth Martin, Bryanna Nordern, Nicholas Chevalier, Ignaty Leshchiner, Anastasia Stavridi, Janice Kim, Alkistis Papatheodoridi, Hakan Gunaydin, Brian Danysh, Laxmi Parida, Ioannis Sanidas, Yongli Ji, Kayao Lau, Gerburg Wulf, Aditya Bardia, Laura Spring, Steven Isakoff, Jochen Lennerz, Levi Pierce, Ermira Pazolli, Gad Getz, Ryan Corcoran, Dejan Juric. Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-10.
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Bony, Claire, Serge Roche, Ueno Shuichi, Takehiko Sasaki, Michael A. Crackower, Josef Penninger, Hiroyuki Mano, and Michel Pucéat. "A Specific Role of Phosphatidylinositol 3–Kinase γ." Journal of Cell Biology 152, no. 4 (February 19, 2001): 717–28. http://dx.doi.org/10.1083/jcb.152.4.717.
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Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase–dependent pathway that stimulates PLCγ and generates IP3, a breakdown product of phosphatidylinositol 4,5–bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca2+ spiking). PIP2 is phosphorylated on 3′ by phosphoinositide 3–kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5–trisphosphate, regulates activity of PLCγ. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCγ, Ca2+ homeostasis. However, although PI3Kα and -β have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kγ. We report that neonatal rat cardiac cells in culture express PI3Kα, -β, and -γ. The purinergic agonist predominantly activates PI3Kγ. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCγ as well as IP3 generation in ATP-stimulated cells. Furthermore, an anti-PI3Kγ, but not an anti-PI3Kβ, injected in the cells prevents the effect of ATP on cell Ca2+ spiking. A dominant negative mutant of PI3Kγ transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca2+ spiking of wild-type but not of PI3Kγ2/2 embryonic stem cell–derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCγ. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca2+ spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kγ antibody and a dominant negative mutant of PI3Kγ injected or transfected into cells prevent the latter event. We conclude that PI3Kγ activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca2+ spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kγ to fully activate PLCγ in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP3 generation and thus cardiac autonomic activity.
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Blunt, Matthew D., Matthew J. Carter, Marta Larrayoz, Maria Montserrat Aguilar, Sarah Murphy, Mark Reynolds, Thomas Tipton, et al. "The Dual PI3K/mTOR Inhibitor PF-04691502 Induces Substantial Apoptosis in Chronic Lymphocytic Leukemia Cells in Vitro and Prolongs Survival in the Eµ-TCL1 Mouse Model." Blood 124, no. 21 (December 6, 2014): 832. http://dx.doi.org/10.1182/blood.v124.21.832.832.
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Abstract Pharmacological inhibition of specific B cell receptor signalling pathways within chronic lymphocytic leukemia (CLL) cells offers the potential for improved therapeutic options with reduced off target toxicity. Idelalisib, the PI3Kδ selective inhibitor, has been approved for CLL and significantly improved overall survival among patients with relapsed CLL in combination with rituximab compared to rituximab alone. In addition to PI3Kδ however, there are three other Class I PI3K isoforms, PI3Kα, PI3Kβ and PI3Kγ, with PI3Kα known to have a role in CLL survival and chemotaxis. In neutrophils, functional redundancy between PI3K isoforms is evident, with inhibition of at least three PI3K isoforms required for maximal apoptosis. Inhibition of mTOR is known to induce cell cycle arrest and apoptosis in CLL cells, however prolonged inhibition of mTOR results in activation of a positive feedback loop resulting in PI3K\Akt reactivation. To overcome these caveats, pan PI3K inhibition alongside mTOR inhibition may achieve superior cytotoxicity against CLL cells compared to PI3Kδ or mTOR inhibition alone. We therefore sought to investigate the effect of a dual pan PI3K and mTOR inhibitor, PF-04691502, on primary CLL cells and in the Eµ-TCL1 mouse model of CLL. Twenty five primary CLL samples were treated with PF-04691502 and downstream signalling and subsequent apoptosis assessed by immunoblotting and Annexin V/PI staining respectively. In primary CLL cells, PF-04691502 induced 80% apoptosis after 24 hours at 10µM concentration. PF-04691502 had an IC50 value of 1µM as assessed by Annexin V/PI staining, with minimal toxicity to normal human B or T cells and a trend towards more specific killing in unmutated CLL (p=0.09) compared to mutated CLL. PF-04691502 inhibited both soluble and immobilised anti-IgM induced signalling and overcame anti-IgM induced survival signals. PF-04691502 abrogated CXCL12 induced signalling and impaired subsequent CLL migration towards the chemokine in a transwell chemotaxis assay. Finally, PF-04691502 was able to overcome protection from co-culture with stroma inducing significant apoptosis of CLL cells when added continuously and in wash out experiments. To assess the effect of PF-04691502 in vivo, twenty Eµ-TCL1 mice were inoculated with tumour cells and after day 21 dosed once daily with vehicle, 5mg/kg or 10mg/kg PF-04691502 for a further 14 days. Mice were assessed for leukemic cell counts, spleen size and white blood cell count throughout the duration of the experiment. The effect of PF-04691502 on B cell receptor and chemokine receptor induced signalling in ex vivo Eµ-TCL1 cells was assessed by immunoblotting. Near identical results to the CLL data above were observed. Using Eµ-TCL1 cells isolated from the spleen, PF-04691502 was shown to profoundly inhibit anti-IgM and CXCL12 induced signalling and chemotaxis as well as induce substantial apoptosis as measured by Annexin V/PI staining. Therefore, we subsequently assessed the effect of PF-04691502 in vivo using this model. Mice treated with PF-04691502 displayed a transient increase in leukemic cell numbers in the blood persisting for 1-4 days, followed by a reduction to levels significantly below that of the vehicle control. The whole white blood cell count remained stable in the PF-04691502 treated mice during the course of treatment, whilst the vehicle treated mice showed significant increases in tumour load up to 40×106 cells/ml 33 days post inoculation. Spleen sizes increased steadily over time in the control mice, whilst PF-04691502 treatment prevented this. After 33 days, PF-04691502 treated mice had spleen sizes comparable to non tumor-recipient control animals. In addition, CD5+ B220+ Eµ-TCL1 cells were significantly reduced in the bone marrow, spleen and lymph nodes following PF-04691502 treatment compared to control mice (p=0.0198; p< 0.0001; p=0.0151 respectively). These results demonstrate that PF-04691502 induces substantial apoptosis of primary CLL cells in vitro and significantly prolongs survival in an in vivo murine model of CLL. Such data indicate that dual pan PI3K/mTOR inhibitors may prove efficacious in the treatment of CLL patients. Disclosures No relevant conflicts of interest to declare.
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Liu, Lixin, Kamal D. Puri, Josef M. Penninger, and Paul Kubes. "Leukocyte PI3Kγ and PI3Kδ have temporally distinct roles for leukocyte recruitment in vivo." Blood 110, no. 4 (August 15, 2007): 1191–98. http://dx.doi.org/10.1182/blood-2006-11-060103.
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Abstract Phosphoinositide 3-kinases (PI3Ks) have been considered important in leukocyte motility. PI3Kγ, the class IB PI3K, expressed prominently in leukocytes and also in endothelial cells, mediates leukocyte functional responses induced by chemoattractants. To reveal its role in leukocyte recruitment, we used intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in PI3Kγ-deficient (PI3Kγ-/-) mice. We report here that PI3Kγ deficiency had no significant effects on leukocyte rolling flux or rolling velocity and minor effects on adhesion (30% to 35%) in response to CXC chemokine MIP-2 (CXCL2) or KC (CXCL1). However, leukocyte emigration was severely impaired in PI3Kγ-/- mice in an early (first 90 minutes) response to MIP-2 or KC. Chimeric mice receiving bone marrow transplants revealed that this early response was entirely dependent upon PI3Kγ in neutrophils but not parenchymal cells (endothelium and others). Identical responses were observed when endogenous chemokine production was induced by TNFα; leukocyte emigration was reduced in PI3Kγ-/- mice. More prolonged responses to MIP-2 (for 4 to 5 hours) or TNFα (6 to 8 hours) were almost entirely PI3Kγ independent and largely dependent on PI3Kδ. Our results reveal that leukocyte emigration response to CXC chemokines is entirely dependent upon PI3Kγ or PI3Kδ, but these are nonoverlapping, temporally distinct events in inflamed tissues in vivo.
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Carson, Jeffrey D., Glenn Van Aller, Ruth Lehr, Robert H. Sinnamon, Robert B. Kirkpatrick, Kurt R. Auger, Dashyant Dhanak, et al. "Effects of oncogenic p110α subunit mutations on the lipid kinase activity of phosphoinositide 3-kinase." Biochemical Journal 409, no. 2 (December 21, 2007): 519–24. http://dx.doi.org/10.1042/bj20070681.
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The PIK3CA gene, encoding the p110α catalytic subunit of Class IA PI3Ks (phosphoinositide 3-kinases), is frequently mutated in many human tumours. The three most common tumour-derived alleles of p110α, H1047R, E542K and E545K, were shown to potently activate PI3K signalling in human epithelial cells. In the present study, we examine the biochemical activity of the recombinantly purified PI3K oncogenic mutants. The kinetic characterizations of the wt (wild-type) and the three ‘hot spot’ PI3K mutants show that the mutants all have approx. 2-fold increase in lipid kinase activities. Interestingly, the phosphorylated IRS-1 (insulin receptor substrate-1) protein shows activation of the lipid kinase activity for the wt and H1047R but not E542K and E545K PI3Kα, suggesting that these mutations represent different mechanisms of lipid kinase activation and hence transforming activity in cancer cells.
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Wyatt, Garhett, Rachel Steinmetz, Traci Lyons, and Weston Porter. "Abstract PO5-05-08: LOSS OF SINGLEMINDED 2S RESULTS IN A PI3K SUBUNIT SWITCH WHICH DRIVES THERAPEUTIC RESISTANCE IN ESTROGEN RECEPTOR POSITIVE BREAST CANCER." Cancer Research 84, no. 9_Supplement (May 2, 2024): PO5–05–08—PO5–05–08. http://dx.doi.org/10.1158/1538-7445.sabcs23-po5-05-08.
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Abstract Estrogen receptor (ER) + breast cancer (BC) comprises over 70% of BC cases and are targeted via ER modulated therapies. Despite this, ER+BC patients can experience recurrence within 20 years and the majority of BC related deaths can be attributed to metastatic ER+BC. These distant metastases are commonly diagnosed as endocrine therapy resistant. Thus, there is an unmet need to identify novel biomarkers for treating ER+ patients with metastases. We have identified a tumor suppressor gene, singleminded 2s (SIM2s), expressed in breast epithelial cells that inhibits epithelial to mesenchymal transition and metastasis, and is downregulated in the progression of breast disease. In ER+BC cell lines, loss of SIM2s results in upregulation of mesenchymal markers and increased PI3K/Akt signaling. Dysregulation of the PI3K/Akt signaling pathway in ER+BC is involved with tumor progression and acquired therapeutic resistance. Our study suggests loss of SIM2s confers resistance in ER+BC through a PI3K subunit switch resulting in upregulation of pro-survival signaling. MCF7 SIM2 knock out cells exhibit increased expression of mesenchymal markers and undergo a phenotypic change compared to wild type cells. Utilizing a migration/invasion assay, MCF7 SIM2 knock out cells exemplify an increase in invasion potential compared to wild type cells. Furthermore, changes in PI3K subunit expression were observed via western blot and real time qPCR analysis. Clonogenic assays revealed an acquired resistance to PI3Kα inhibition, but a susceptibility to PI3Kδ inhibition. This is a significant finding as a current standard of care for patients with ER+ breast cancer recurrence is PI3Kα inhibition. PI3Kδ is an already approved therapeutic target in chronic lymphocytic leukemia, thus PI3Kδ may present a new therapeutically targetable opportunity for ER+BC recurrence. Elucidating the mechanism for acquired therapeutic resistance is an integral avenue for understanding how breast cancer progresses and improving the prognosis of ER+BC patients. Citation Format: Garhett Wyatt, Rachel Steinmetz, Traci Lyons, Weston Porter. LOSS OF SINGLEMINDED 2S RESULTS IN A PI3K SUBUNIT SWITCH WHICH DRIVES THERAPEUTIC RESISTANCE IN ESTROGEN RECEPTOR POSITIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-08.
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Cao, Biyin, Jingyu Zhu, Man Wang, Yang Yu, Huixin Qi, Kunkun Han, Zubin Zhang, et al. "A Novel PI3K Inhibitor Identified By a High Throughput Virtual Screen Displays Potent Activity Against Multiple Myeloma." Blood 124, no. 21 (December 6, 2014): 4722. http://dx.doi.org/10.1182/blood.v124.21.4722.4722.
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Abstract The phosphatidylinositol-3-kinase (PI3K) has been developed as a promising target for the treatment of many cancers, including hematological malignancies, such as lymphoma, leukemia, and multiple myeloma (MM). Recent clinical trials have demonstrated Idelalisib, a specific PI3Kdelta inhibitor, reached a total 57% of response rate in relapsed indolent lymphoma and 72% of overall complete rate in relapsed chronic lymphocytic leukemia. MM remains fatal as a cancer of plasma cells because of lack of efficient drugs. Therefore, we tried to find novel PI3K inhibitors for MM therapy. To this end, we performed a high throughput virtual screen against 800,000 small molecule compounds using PI3Kγ as the analytic model and identified C98, a function unknown compound, as a potent inhibitor of PI3Ks. The cell-free enzymatic studies showed that C98 inhibited all class I PI3Ks at nano- or low micromolar concentrations but had no effects on AKT or mTOR activity. Molecular docking analysis revealed that C98 interfered with the ATP-binding pockets of PI3Ks by forming H-bonds and arene-H interactions with specific amino residues through which C98 specifically inhibited PI3K/AKT/mTOR signaling pathway, but had no effects on other kinases and proteins including IGF-1R, ERK, p38, c-Src, and STAT3. PTEN is a key negative modulator of the PI3K signaling pathway, but KIF did not show any induction of PTEN, thus further demonstrating that KIF is a selective PI3K inhibitor. Inhibition of PI3K by C98 led to myeloma cell apoptosis as shown activated caspase cascade signaling. Furthermore, oral administration of C98 delayed tumor growth in two independent human myeloma xenograft models in nude mice but did not show overt toxicity. Pharmacokinetic analysis revealed that C98 can be found in MM tissues at an average concentration up to 622.6 ng/g, suggesting that C98 can penetrate into the tumor tissues where it exerts its anti-myeloma activity. It also suggests C98 has a reliable oral activity. Therefore, through a high throughput virtual screen we identified a novel PI3K inhibitor that is orally active against multiple myeloma with great potential for further development. Disclosures No relevant conflicts of interest to declare.
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Sadasivan, Chandu, Pavel Zhabyeyev, Dina Labib, James A. White, D. Ian Paterson, and Gavin Y. Oudit. "Cardiovascular toxicity of PI3Kα inhibitors." Clinical Science 134, no. 19 (October 2020): 2595–622. http://dx.doi.org/10.1042/cs20200302.
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Abstract The phosphoinositide 3-kinases (PI3Ks) are a family of intracellular lipid kinases that phosphorylate the 3′-hydroxyl group of inositol membrane lipids, resulting in the production of phosphatidylinositol 3,4,5-trisphosphate from phosphatidylinositol 4,5-bisphosphate. This results in downstream effects, including cell growth, proliferation, and migration. The heart expresses three PI3K class I enzyme isoforms (α, β, and γ), and these enzymes play a role in cardiac cellular survival, myocardial hypertrophy, myocardial contractility, excitation, and mechanotransduction. The PI3K pathway is associated with various disease processes but is particularly important to human cancers since many gain-of-function mutations in this pathway occur in various cancers. Despite the development, testing, and regulatory approval of PI3K inhibitors in recent years, there are still significant challenges when creating and utilizing these drugs, including concerns of adverse effects on the heart. There is a growing body of evidence from preclinical studies revealing that PI3Ks play a crucial cardioprotective role, and thus inhibition of this pathway could lead to cardiac dysfunction, electrical remodeling, vascular damage, and ultimately, cardiovascular disease. This review will focus on PI3Kα, including the mechanisms underlying the adverse cardiovascular effects resulting from PI3Kα inhibition and the potential clinical implications of treating patients with these drugs, such as increased arrhythmia burden, biventricular cardiac dysfunction, and impaired recovery from cardiotoxicity. Recommendations for future directions for preclinical and clinical work are made, highlighting the possible role of PI3Kα inhibition in the progression of cancer-related cachexia and female sex and pre-existing comorbidities as independent risk factors for cardiac abnormalities after cancer treatment.
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Chawsheen, Mahmoud A., and Hazem A. Al-Bustany. "Docking Study to Predict the Efficacy of Phosphatidylinositol 3-Kinase α Inhibitors." ARO-THE SCIENTIFIC JOURNAL OF KOYA UNIVERSITY 7, no. 2 (December 20, 2019): 47–52. http://dx.doi.org/10.14500/aro.10565.
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The phosphatidylinositol 3-kinase (PI3K) family comprises lipid kinases that cross-link signals between living cells and their surroundings. PI3Ks are classified into several groups and isoforms with specific characteristics and functions. Genes encoding PI3Ks are mutated in several types of cancer, and their isoforms have varying capacity in promoting cell signaling and cancer progression. Many compounds have been introduced as PI3Kα inhibitors, but not all of them have the same inhibitory effects. For successful PI3K-related biomedical experiments, it is vital to select the most specific and potent compounds with the highest inhibitory effects for targeting this kinase. In this study, we investigate 28 well-recognized PI3Kα inhibitors through predicting their specificity and potency using the docking software AutoDock Vina. Our data showed that PF 05212384 had the highest docking score (−9.2 kcal/mol), and 3-methyladenine had the lowest docking score (−4.8 kcal/mol). Our data also showed different types of interactions and bonds formed between the inhibitors and protein residues. In conclusion, PF 05212384 and AZD 6482 compounds are the best candidates for targeting PI3Kα. In addition to hydrophobic interactions in the PI3Kα binding pocket, the formation of hydrogen bonds between these inhibitors and binding pocket residues was confirmed.
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Ascione, Liliana, Paola Zagami, Eleonora Nicolò, Edoardo Crimini, Giuseppe Curigliano, and Carmen Criscitiello. "PIK3CAMutations in Breast Cancer Subtypes Other Than HR-Positive/HER2-Negative." Journal of Personalized Medicine 12, no. 11 (October 31, 2022): 1793. http://dx.doi.org/10.3390/jpm12111793.
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The phosphoinositide 3-kinase (PI3K) pathway plays a key role in cancer, influencing growth, proliferation, and survival of tumor cells. PIK3CA mutations are generally oncogenic and responsible for uncontrolled cellular growth. PI3K inhibitors (PI3Ki) can inhibit the PI3K/AKT/mTOR pathway, although burdened by not easily manageable toxicity. Among PI3Ki, alpelisib, a selective p110α inhibitor, is approved for the treatment of hormone receptor (HR)+/HER2- PIK3CA mutant metastatic breast cancer (BC) that has progressed to a first line endocrine therapy. PIK3CA mutations are also present in triple negative BC (TNBC) and HER2+ BC, although the role of PI3K inhibition is not well established in these subtypes. In this review, we go through the PI3K/AKT/mTOR pathway, describing most common mutations found in PI3K genes and how they can be detected. We describe the available biological and clinical evidence of PIK3CA mutations in breast cancers other than HR+/HER2-, summarizing clinical trials investigating PI3Ki in these subtypes.
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Maffei, Angelo, Giuseppe Lembo, and Daniela Carnevale. "PI3Kinases in Diabetes Mellitus and Its Related Complications." International Journal of Molecular Sciences 19, no. 12 (December 18, 2018): 4098. http://dx.doi.org/10.3390/ijms19124098.
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Recent studies have shown that phosphoinositide 3-kinases (PI3Ks) have become the target of many pharmacological treatments, both in clinical trials and in clinical practice. PI3Ks play an important role in glucose regulation, and this suggests their possible involvement in the onset of diabetes mellitus. In this review, we gather our knowledge regarding the effects of PI3K isoforms on glucose regulation in several organs and on the most clinically-relevant complications of diabetes mellitus, such as cardiomyopathy, vasculopathy, nephropathy, and neurological disease. For instance, PI3K α has been proven to be protective against diabetes-induced heart failure, while PI3K γ inhibition is protective against the disease onset. In vessels, PI3K γ can generate oxidative stress, while PI3K β inhibition is anti-thrombotic. Finally, we describe the role of PI3Ks in Alzheimer’s disease and ADHD, discussing the relevance for diabetic patients. Given the high prevalence of diabetes mellitus, the multiple effects here described should be taken into account for the development and validation of drugs acting on PI3Ks.
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Koyasu, S. "Role of class IA phosphoinositide 3-kinase in B lymphocyte development and functions." Biochemical Society Transactions 32, no. 2 (April 1, 2004): 320–25. http://dx.doi.org/10.1042/bst0320320.
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PI3K (phosphoinositide 3-kinase) family members control a variety of cellular responses, such as cell growth, survival, cytoskeletal remodelling and the trafficking of intracellular organelles, in many cell types, including lymphocytes. It has been difficult to evaluate the roles of distinct PI3Ks in immune responses, because specific inhibitors for each PI3K are lacking and most stimuli activate multiple PI3Ks. The development of gene-targeted mice has now allowed the elucidation of roles played in vivo by PI3K species in the immune system. Studies on mice deficient in catalytic as well as regulatory subunits of class IA PI3Ks have shown the importance of this class of PI3K in B lineage cells. Here I discuss the role of class IA PI3Ks in B lymphocyte development and B cell antigen receptor-mediated signal transduction.
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Chen, Yu-Chen Enya, Melinda Lea Burgess, Antje Blumenthal, Sally Mapp, Peter Mollee, Devinder Gill, and Nicholas Andrew Saunders. "Activation of Fc Gamma Receptor-Dependent Responses to Therapeutic Antibodies By Nurse like Cells Requires PI3Kdelta." Blood 132, Supplement 1 (November 29, 2018): 3128. http://dx.doi.org/10.1182/blood-2018-99-109719.
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Abstract There is a continued reliance on antibody therapies for treating chronic lymphocytic leukaemia (CLL). Whilst the inclusion of antibody therapies in many standard treatment regimes results in good outcomes, acquired resistance remains a significant clinical challenge for many CLL patients resulting in insensitivity to the antibody treatment. Thus, understanding the mechanisms driving treatment resistance is likely to lead to therapies to reverse resistance and improve patient outcomes. Earlier studies from our laboratory have shown that resistance to therapeutic antibodies, in CLL, is due to a reduced ability of monocyte derived macrophages (MDMs) to participate in FcγR-dependent antitumour responses (e.g. ADCC and antibody-dependent phagocytosis (ADP). In this regard, we recently showed that SYK and BTK activation are downstream of the FcγRs (Oncogene, 36(17):2366-2376, 2017). Moreover, we showed that signalling through the FcγR pathway was reduced in antibody-resistant MDMs and could be reversed using inhibitors of SHIP1. These studies indicated that knowledge of FcγR signalling could exploited to reverse resistance. Unfortunately, knowledge of the exact signalling events controlling FcγR activity in MDMs from CLL patients is unclear. In this study we investigated the involvement of PI3K isoforms in FcγR-dependent ADCC and ADP in MDMs from CLL patients. PI3K isoforms have been shown to be important pathway regulators for immune-receptor function in various immune cells such as T cells, B cells and NK cells as well as in cancerous cells. In the first instance, I examined the expression of PI3K isoforms in MDMs from CLL patients. This showed that PI3Kα, β, and δ are expressed in MDMs whereas PI3Kγ is below the limit of detection. Next, I examined the involvement of the different PI3K isoforms to contribute to FcγR-dependent ADCC by MDMs. For this we used a suite of isoform-selective inhibitors to target each PI3K isoform and examined their effect on ADCC responses by MDMs. The PI3Kδ-selective inhibitor, idelalisib and the pan PI3K inhibitor BKM120 (Buparlisib) were able to inhibit ADCC responses to the CD20-targeting therapeutic antibody, obinutuzumab. Similarly, both buparlisib and idelalisib were able to inhibit AKT phosphorylation at concentrations that also inhibited ADCC. In contrast. None of the other isoform-selective inhibitors were able to suppress ADCC responses to obinutuzumab. These studies have been repeated using isoform-specific siRNAs. This is the first report to show that PI3Kδ is involved in FcγR signalling in MDMs from CLL patients or in MDMs from any tumour type. Based on these findings we conclude that PI3Kδ is a critical effector molecule for antitumour responses to therapeutic antibodies in CLL. Disclosures Mollee: Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gill:Pharma: Membership on an entity's Board of Directors or advisory committees.
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Okkenhaug, K., A. Bilancio, J. L. Emery, and B. Vanhaesebroeck. "Phosphoinositide 3-kinase in T cell activation and survival." Biochemical Society Transactions 32, no. 2 (April 1, 2004): 332–35. http://dx.doi.org/10.1042/bst0320332.
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PI3Ks (phosphoinositide 3-kinases) regulate diverse signalling pathways involved in growth, proliferation, survival, differentiation and metabolism. In T cells, PI3Ks can be activated by a number of different receptors, including the TcR (T cell receptor), co-stimulatory receptors, cytokine receptors and chemokine receptors. However, the specific roles of PI3Ks downstream of these receptors vary. An inactivating mutation in the leucocyte-specific PI3K isoform p110 δ results in impaired TcR-dependent proliferation under circumstances where CD28 co-stimulation is blocked or not required. Recruitment and activation of PI3K by CD28 promotes survival by inducing increased expression of Bcl-XL. However, CD28 engages additional signals that regulate proliferation and interleukin-2 production independently of PI3K. Thus a model emerges whereby PI3K is involved in both TcR and CD28 signalling, but each receptor may only exploit a subset of the signalling pathways potentially controlled by PI3K activation.
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Consonni, Alessandra, Lina Cipolla, Gianni Guidetti, Ilaria Canobbio, Elisa Ciraolo, Emilio Hirsch, Marco Falasca, Mitsuhiko Okigaki, Cesare Balduini, and Mauro Torti. "Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling." Blood 119, no. 3 (January 19, 2012): 847–56. http://dx.doi.org/10.1182/blood-2011-07-364992.
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Abstract Integrin α2β1–mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kβ, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kβ (PI3KβKD), but occurred normally in PI3KγKD platelets. Integrin α2β1 failed to stimulate PI3Kβ in platelets from phospholipase Cγ2 (PLCγ2)–knockout mice, and we found that intracellular Ca2+ linked PLCγ2 to PI3Kβ activation. Integrin α2β1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLCγ2 and intracellular Ca2+. Whereas activation of Pyk2 occurred normally in PI3KβKD platelets, stimulation of PI3Kβ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3Kβ was required for α2β1–mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin αIIbβ3 were reduced after inhibition of PI3Kβ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3Kβ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3Kβ downstream of integrin α2β1, and document a novel role for Pyk2 and PI3Kβ in integrin α2β1 promoted inside-out activation of integrin αIIbβ3 and thrombus formation.
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Laurent, Pierre-Alexandre, Cédric Garcia, Marie-Pierre Gratacap, Bart Vanhaesebroeck, Pierre Sié, Bernard Payrastre, and Anne-Dominique Terrisse. "The class I phosphoinositide 3-kinases α and β control antiphospholipid antibodies-induced platelet activation." Thrombosis and Haemostasis 115, no. 06 (2016): 1138–46. http://dx.doi.org/10.1160/th15-08-0661.
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SummaryAntiphospholipid syndrome (APS) is an autoimmune disease characterised by the presence of antiphospholipid antibodies (aPL) associated with increased thrombotic risk and pregnancy morbidity. Although aPL are heterogeneous auto-antibodies, the major pathogenic target is the plasma protein β2-glycoprotein 1. The molecular mechanisms of platelet activation by aPL remain poorly understood. Here, we explored the role of the class IA phosphoinositide 3-kinase (PI3K) α and β isoforms in platelet activation by aPL. Compared to control IgG from healthy individuals, the IgG fraction isolated from patients with APS potentiates platelet aggregation induced by low dose of thrombin in vitro and increases platelet adhesion and thrombus growth on a collagen matrix under arterial shear rate through a mechanism involving glycoprotein Ib (GPIb) and Toll Like Receptor 2 (TLR-2). Using isoforms-selective pharmacological PI3K inhibitors and mice with megakaryocyte/platelet lineage-specific inactivation of class IA PI3K isoforms, we demonstrate a critical role of the PI3Kβ and PI3Kα isoforms in platelet activation induced by aPL. Our data show that aPL potentiate platelet activation through GPIbα and TLR-2 via a mechanism involving the class IA PI3Kα and β isoforms, which represent new potential therapeutic targets in the prevention or treatment of thrombotic events in patients with APS.
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Park, Jei Hyoung, Kyoung Jin Nho, Ji Young Lee, Yung Joon Yoo, Woo Jin Park, Chunghee Cho, and Do Han Kim. "Anti-Ischemic Effects of PIK3IP1 Are Mediated through Its Interactions with the ETA-PI3Kγ-AKT Axis." Cells 11, no. 14 (July 11, 2022): 2162. http://dx.doi.org/10.3390/cells11142162.
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Oxidative stress, caused by the accumulation of reactive oxygen species (ROS) during acute myocardial infarction (AMI), is one of the main factors leading to myocardial cell damage and programmed cell death. Phosphatidylinositol-3-kinase-AKT (PI3K-AKT) signaling is essential for regulating cell proliferation, differentiation, and apoptosis. Phosphoinositide-3-kinase (PI3K)-interacting protein 1 (PIK3IP1) is an intrinsic inhibitor of PI3K in various tissues, but its functional role during AMI remains unknown. In this study, the anti-ischemic role of PIK3IP1 in an in vitro AMI setting was evaluated using H9c2 cells. The MTT assay demonstrated that cell viability decreased significantly via treatment with H2O2 (200–500 μM). The TUNEL assay results revealed substantial cellular apoptosis following treatment with 200 μM H2O2. Under the same conditions, the expression levels of hypoxia-inducible factor (HIF-1α), endothelin-1 (ET-1), bcl-2-like protein 4 (BAX), and cleaved caspase-3 were elevated, whereas those of PIK3IP1, LC3II, p53, and Bcl-2 decreased significantly. PIK3IP1 overexpression inhibited H2O2-induced and PI3K-mediated apoptosis; however, PIK3IP1 knockdown reversed this effect, suggesting that PIK3IP1 functions as an anti-apoptotic molecule. To identify both the upstream and downstream molecules associated with PIK3IP1, ET-1 receptor type-specific antagonists (BQ-123 and BQ-788) and PI3K subtype-specific antagonists (LY294002 and IPI-549) were used to determine the participating isoforms. Co-immunoprecipitation was performed to identify the binding partners of PIK3IP1. Our results demonstrated that ROS-induced cardiac cell death may occur through the ETA-PI3Kγ-AKT axis, and that PIK3IP1 inhibits binding with both ETA and PI3Kγ. Taken together, these findings reveal that PIK3IP1 plays an anti-ischemic role by reducing the likelihood of programmed cell death via interaction with the ETA-PI3Kr-AKT axis.
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Jain, Neeraj, Lalit Sehgal, R. Eric Davis, Stephen Joseph Shuttleworth, and Felipe Samaniego. "Felipe Samaniego." Blood 128, no. 22 (December 2, 2016): 5145. http://dx.doi.org/10.1182/blood.v128.22.5145.5145.
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Abstract Background: Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of the patients develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. Several reports indicated that the BTK inhibitor ibrutinib successfully blocks B-cell receptor signaling and shows clinical benefit in leukemia and lymphomas, including mantle cell lymphoma [23422267] and DLBCL [26193343], for which ibrutinib is FDA-approved. In phase I/II clinical trials, ibrutinib elicited an overall response rate of 68% in patients with relapsed/refractory MCL. However, in spite of these encouraging results, responses are variable and generally incomplete, acquired resistance is common, and recurrence is anticipated [26430726]. We undertook a study of factors underlying acquired ibrutinib resistance (IR) in initially ibrutinib-sensitive DLBCL cell lines. Methods: IR DLBCL cell lines were generated by continuous culture of parental (PT) cell lines in increasing concentrations of ibrutinib, up to a maximum concentration of 10µM. Once established, IR cell lines were removed from ibrutinib, expanded, and cultured under the same conditions as the PT cell lines for further experiments. Gene expression profiling (GEP) of IR and PT populations was performed on Agilent 4 x 44K gene microarrays. Results: Of five ABC DLBCL cell lines tested, two (OCI-LY3, U2932) were initially resistant to ibrutinib (IC50>10µM).Three (TMD8, OCI-LY10, HBL1) were initially sensitive (IC50 < 10 nM), but chronic exposure to ibrutinib generated syngeneic versions with IC50 > 5µM. In comparison to PT versions of these cell lines, IR cells did not form clumps in suspension cultures, displayed irregular cell morphology, elevated colony formation ability in methylcellulose matrix, and had higher proliferation rate. Western blots and GEP data showed increased expression by IR cell lines of IAP family members survivin, cIAP2, and oncogenic BCL2 and BCL6. Reduced B-cell receptor signaling, and enhanced PI3K-Akt activity was identified in IR cell lines. Analysis of PI3K isoforms revealed up-regulation of PI3Kα and PI3Kβ with decreased expression of PI3Kδ and PTEN (PI3K negative regulator). Given the enhanced PI3K isoform expression with IR, we treated cell lines with KA2237, a PI3Kβ/δ isoform targeting drug, and observed reduced metabolic activity (survival) of IR cells compared to PT cell lines. Conclusion: This study highlights that changes in a regulator (PTEN) and mediator (p110β) of PI3K/AKT signaling have important roles in the development of ibrutinib resistance in DLBCL. Treatment with KA2237 may provide a better outcome for ibrutinib-resistant DLBCL. Disclosures Samaniego: Karus Therapuetics: Research Funding.
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Brosinsky, Paulin, Julia Bornbaum, Björn Warga, Lisa Schulz, Klaus-Dieter Schlüter, Alessandra Ghigo, Emilio Hirsch, Rainer Schulz, Gerhild Euler, and Jacqueline Heger. "PI3K as Mediator of Apoptosis and Contractile Dysfunction in TGFβ1-Stimulated Cardiomyocytes." Biology 10, no. 7 (July 16, 2021): 670. http://dx.doi.org/10.3390/biology10070670.
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Background: TGFβ1 is a growth factor that plays a major role in the remodeling process of the heart by inducing cardiomyocyte dysfunction and apoptosis, as well as fibrosis thereby restricting heart function. TGFβ1 mediates its effect via the TGFβ receptor I (ALK5) and the activation of SMAD transcription factors, but TGFβ1 is also known as activator of phosphoinositide-3-kinase (PI3K) via the non-SMAD signaling pathway. The aim of this study was to investigate whether PI3K is also involved in TGFβ1–induced cardiomyocytes apoptosis and contractile dysfunction. Methods and Results: Incubation of isolated ventricular cardiomyocytes with TGFβ1 resulted in impaired contractile function. Pre-incubation of cells with the PI3K inhibitor Ly294002 or the ALK5 inhibitor SB431542 attenuated the decreased cell shortening in TGFβ1–stimulated cells. Additionally, TGFβ-induced apoptosis was significantly reduced by the PI3K inhibitor Ly294002. Administration of a PI3Kγ-specific inhibitor AS605240 abolished the TGFβ effect on apoptosis and cell shortening. This was also confirmed in cardiomyocytes from PI3Kγ KO mice. Induction of SMAD binding activity and the TGFβ target gene collagen 1 could be blocked by the PI3K inhibitor Ly294002, but not by the specific PI3Kγ inhibitor AS605240. Conclusions: TGFβ1-induced SMAD activation, cardiomyocyte apoptosis, and impaired cell shortening are mediated via both, the ALK5 receptor and PI3K, in adult cardiomyocytes. PI3Kγ specifically contributes to apoptosis induction and impairment of contractile function independent of SMAD signaling.
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Zieger, M., W. Oehrl, R. Wetzker, P. Henklein, G. Nowak, and R. Kaufmann. "Different Signaling Pathways Are Involved in CCKB Receptor-Mediated MAP Kinase Activation in COS-7 Cells." Biological Chemistry 381, no. 8 (August 6, 2000): 763–68. http://dx.doi.org/10.1515/bc.2000.097.
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Abstract Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokininB (CCKB) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCKB receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCKB receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kγ did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutant of the regulatory p85 subunit induced significant inhibition of CCK-dependent ERK activity. These results indicate an involvement of PI3K class 1A species α, β or/and δ in signal transduction via CCKB receptors. In addition, protein kinase C (PKC)-dependent signaling pathways contribute to CCKB-mediated MAP kinase signaling as shown by inhibition of CCK-8-induced ERK activation by the PKC inhibitor bisindolylmaleimide.
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Kaur, Harjeet, Chang Shin Park, Jodee M. Lewis, and Jason M. Haugh. "Quantitative model of Ras–phosphoinositide 3-kinase signalling cross-talk based on co-operative molecular assembly." Biochemical Journal 393, no. 1 (December 12, 2005): 235–43. http://dx.doi.org/10.1042/bj20051022.
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In growth-factor-stimulated signal transduction, cell-surface receptors recruit PI3Ks (phosphoinositide 3-kinases) and Ras-specific GEFs (guanine nucleotide-exchange factors) to the plasma membrane, where they produce 3′-phosphorylated phosphoinositide lipids and Ras-GTP respectively. As a direct example of pathway networking, Ras-GTP also recruits and activates PI3Ks. To refine the mechanism of Ras–PI3K cross-talk and analyse its quantitative implications, we offer a theoretical model describing the assembly of complexes involving receptors, PI3K and Ras-GTP. While the model poses the possibility that a ternary receptor–PI3K–Ras complex forms in two steps, it also encompasses the possibility that receptor–PI3K and Ras–PI3K interactions are competitive. In support of this analysis, experiments with platelet-derived growth factor-stimulated fibroblasts revealed that Ras apparently enhances the affinity of PI3K for receptors; in the context of the model, this suggests that a ternary complex does indeed form, with the second step greatly enhanced through membrane localization and possibly allosteric effects. The apparent contribution of Ras to PI3K activation depends strongly on the quantities and binding affinities of the interacting molecules, which vary across different cell types and stimuli, and thus the model could be used to predict conditions under which PI3K signalling is sensitive to interventions targeting Ras.
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Uche, Uzodinma, and Lawrence Kane. "PIK3IP1- A novel negative regulator of PI3K (IRM10P.743)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 129.10. http://dx.doi.org/10.4049/jimmunol.192.supp.129.10.
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Abstract Phosphatidylinositide-3-Kinases (PI3Ks) are a family of lipid kinases that play important intracellular signaling roles in cellular functions such as cell proliferation, motility, growth, intracellular trafficking, differentiation and survival. PI3K produces PIP3 which further facilitates the activation of downstream effectors such as Akt and PDK1. These effectors facilitate the cellular processes associated with PI3K activity. Conversely, because of the nature of PI3Ks roles, dysregulation of PI3K is implicated in many cancers. PTEN, SHIP and INPP4B are negative regulators of PI3K activity that function downstream of PI3K and have been shown to act as tumor suppressors. Recently, PI3K Interacting Protein 1 (PIK3IP1), a novel negative regulator that functions upstream and proximal to PI3K, has been identified. PIK3IP1, is a transmembrane protein that down-regulates PI3K activity, leading to reduced Akt phosphorylation. Our lab recently showed that PIK3IP1 can inhibit TCR signaling and TCR activation. To better understand the role of PIK3IP1 in T cells, we have begun to carefully define its expression pattern in various T cell subsets. In addition, we have developed a PIK3IP1 ecto-domain Fc fusion protein, which we are using to define possible ligands for PIK3IP1. Finally, we have obtained knockout mice lacking expression of PIK3IP1, to better define the requirements for PIK3IP1 in primary T cells.
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Stokes, Clare A., and Alison M. Condliffe. "Phosphoinositide 3-kinase δ (PI3Kδ) in respiratory disease." Biochemical Society Transactions 46, no. 2 (March 9, 2018): 361–69. http://dx.doi.org/10.1042/bst20170467.
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Defining features of chronic airway diseases include abnormal and persistent inflammatory processes, impaired airway epithelial integrity and function, and increased susceptibility to recurrent respiratory infections. Phosphoinositide 3-kinases (PI3K) are lipid kinases, which contribute to multiple physiological and pathological processes within the airway, with abnormal PI3K signalling contributing to the pathogenesis of several respiratory diseases. Consequently, the potential benefit of targeting PI3K isoforms has received considerable attention, being viewed as a viable therapeutic option in inflammatory and infectious lung disorders. The class I PI3K isoform, PI3Kδ (Phosphoinositide 3-kinases δ) is of particular interest given its multiple roles in modulating innate and adaptive immune cell functions, airway inflammation and corticosteroid sensitivity. In this mini-review, we explore the role of PI3Kδ in airway inflammation and infection, focusing on oxidative stress, ER stress, histone deacetylase 2 and neutrophil function. We also describe the importance of PI3Kδ in adaptive immune cell function, as highlighted by the recently described Activated PI3K Delta Syndrome, and draw attention to some of the potential clinical applications and benefits of targeting this molecule.
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Sundararaj, Kamala, Rachel Burge, Samaneh Saberikashani, Lucas Bialousow, Amanda Linke, Merissa Smith, Michael C. Ostrowski, John P. O'Bryan, and G. Aaron Hobbs. "Abstract PR11: KRAS mutant-specific interactions reveal mechanisms in pancreatic cancer tumorigenesis and metabolic function." Cancer Research 84, no. 2_Supplement (January 16, 2024): PR11. http://dx.doi.org/10.1158/1538-7445.panca2023-pr11.
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Abstract Mutational activation of the KRAS proto-oncogene is the initiating mutational event in pancreatic ductal adenocarcinoma (PDAC) and ~90% of patients harbor KRAS mutations. PDAC is marred by a 12% five-year overall survival rate, and developing effective therapeutic strategies remains a priority. Accumulating evidence suggests that all KRAS mutations are not created equal. While the KRASG12R mutation is rare in lung and colorectal cancers (<1%), it is the third most common KRAS mutation in PDAC, accounting for approximately 20% of all cases. However, KRASG12R cannot interact with the lipid kinase PI3Ka, a well-characterized RAS effector necessary for KRAS-driven tumorigenesis. Previous studies in mice models of cancer have demonstrated that ablation of the KRAS:PI3Ka interaction limits tumorigenesis. Despite the inability of KRASG12R to activate PI3Ka directly, AKT signaling is robustly activated in KRASG12R-mutant PDAC. The mechanisms that allow the KRASG12R mutant to overcome the inability to activate PI3K and promote PDAC are unclear. We recently developed a Ptf1a-CREERT2;KrasLSL-G12R genetically engineered mouse model to study KRASG12R in an in vivo context. This model does not develop pancreas lesions or tumors. We have uncovered two unique characteristics of human PDAC that we hypothesize allows for the KRASG12R mutation to produce tumors only in human pancreatic tissue. First, we have found that all four PI3K isoforms are overexpressed in human PDAC, and the PI3Kd and PI3Kg isoforms are specifically upregulated in KRASG12R-mutant PDAC. Second, PTEN is oxidized in PDAC. PTEN oxidation results in an intramolecular disulfide bond, which inhibits the phosphatase activity of PTEN and leads to hyperactivated PI3K signaling. Critically, mouse pancreas tissue only expresses the PI3Ka/b isoforms and PTEN is in the reduced state, demonstrating two significant differences between mouse models and human disease. Additionally, we have determined that PTEN becomes fully oxidized in nutrient-restricted medium (low glucose/glutamine), a common strategy employed to mimic the pancreatic tumor microenvironment in cell culture. Using PTEN oxidation-resistant variants, we demonstrate that KRASG12R-mutant PDAC cell lines are reliant on oxidized (inactivated) PTEN for proliferation. To confirm that PTEN inactivation can aid KRASG12R-mediated tumorigenesis in mouse models, we generated at KrasG12R/+;Ptenfl/fl genetically engineered mouse model, and the results of this model will be presented herein. Thus, our data demonstrate that increased PI3K isoform expression, coupled with PTEN oxidation, creates a unique environment that allows KRASG12R to initiate and promote pancreatic tumorigenesis. As PTEN oxidation overcomes the need for KRAS to activate PI3K signaling in PDAC, these results indicate that directly targeting KRAS alone will be insufficient at reducing tumor growth in humans and clinically successful therapeutic strategies will have to develop alternative methods to reduce PI3K signaling in addition to directly targeting KRAS activity. Citation Format: Kamala Sundararaj, Rachel Burge, Samaneh Saberikashani, Lucas Bialousow, Amanda Linke, Merissa Smith, Michael C. Ostrowski, John P. O'Bryan, G. Aaron Hobbs. KRAS mutant-specific interactions reveal mechanisms in pancreatic cancer tumorigenesis and metabolic function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR11.