Phosphatidylinositol 3-kinase Isoforms Research Articles

Abstract C045: Combinations of PI3K inhibitors with targeted oncology agents in multicellular spheroid models

Abstract Dysregulation of the phosphatidylinositol 3-kinase (PI3K) pathway plays a significant role in cancer, and inhibitors offer a promising strategy for targeted therapies. The selectivity of current inhibitors varies against different PI3K isoforms or subtypes. For example, alpelisib and inavolisib predominantly target the α-isoform, whereas duvelisib targets the δ- and γ-isoforms. By contrast, copanlisib is a pan-PI3K inhibitor, exhibiting activity against all isoforms. In combinations with other targeted agents, we investigated the activity of these four PI3K inhibitors against multicellular spheroids. The multicellular spheroids, grown from 60% malignant cells, 25% endothelial cells, and 15% mesenchymal stem cells, served as a model for human solid tumors that incorporates both malignant and stromal components. Thirty multicellular spheroid models were grown from various malignant cell types, including twenty-seven patient-derived cancer cell lines obtained from the NCI Patient-Derived Models Repository (https://pdmr.cancer.gov/) and three established cell lines from the NCI-60 human tumor cell line panel. After three days of spheroid growth, anticancer agents were introduced at concentrations up to their reported clinical Cmax value, if known. After seven days of drug exposure, cell viability was measured using the CellTiter-Glo 3D assay. Among the various drug combinations evaluated, simultaneous targeting of the PI3K and RAS pathways was highly effective, particularly against malignant cell lines with an activated RAS pathway. Notably, additive and/or synergistic activities were observed when either the α-isoform-specific PI3K inhibitors (alpelisib and inavolisib) or the pan-PI3K inhibitor (copanlisib) was combined with either the MEK inhibitor selumetinib, the ERK inhibitor ravoxertinib, or the type II pan-RAF kinase inhibitor DAY101. Additionally, combinations of the same three PI3K inhibitors with variant-specific inhibitors, such as vemurafenib (BRAF V600E), sotorasib (KRAS G12C), or MTRX-1133 (KRAS G12D) demonstrated selective activity against cell lines carrying the variant. Another effective combination with PI3K inhibitors was achieved with vertical inhibition of the PI3K pathway through the addition of the mTORC1/2 kinase inhibitor sapanisertib. Finally, combinations of a CDK4/6 inhibitor (abemaciclib or palbociclib) with a PI3K inhibitor demonstrated synergistic activities, although they were not as potent as other combinations. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas S Dexheimer, Thomas Silvers, Nathan P Coussens, Rabih Said, Beverly A Teicher, James H Doroshow. Combinations of PI3K inhibitors with targeted oncology agents in multicellular spheroid models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C045.

PI3Kδ Inhibitor Linperlisib As a ≥ 3-Line Treatment for Relapsed or Refractory Follicular Lymphoma: A Subgroup Analysis of a Phase II, Single-Arm, Open-Label Trial

Introduction Follicular lymphoma (FL) is the most common subtype of indolent non-Hodgkin lymphoma (NHL), originating in follicular central B cells, and accounts for approximately 22% of all newly diagnosed cases of NHL. Chemo-immunotherapy is the standard treatment option for FL. Unfortunately, the progression of the disease to relapsed or refractory FL (r/r FL) is still inevitable, which raised concern about subsequent effective treatments. Several δ isoform of phosphatidylinositol 3-kinase (PI3K-δ) inhibitors have ever been approved for r/r FL after ≥2 lines of prior therapies. However, most of their indications for r/r FL were withdrawal for increased risk of death and serious side effects. The latest data from the Chinese phase II clinical trial of linperlisib (Trial registration ID: NCT04370405), a novel oral PI3Kδ-selective inhibitor, in patients with r/r FL who had received at least two systemic therapies were presented. In this trial, linperlisib demonstrated compelling efficacy and was generally well-tolerated in the treatment of r/r FL patients after ≥2 prior systemic therapies. Linperlisib was approved by China National Medical Products Administration (NMPA) for adult r/r FL previously treated with at least two system therapies in November 2022. The ≥3 lines of treatment options are still unmet for the r/r FL. In this setting, this subgroup analysis of the linperlisib phase II trial aimed to evaluate outcomes of linperlisib in later-line treatment of r/r FL. Methods This study included all the patients with r/r FL who had received at least two systemic therapies previously enrolled in the open-label, single-arm, phase II trial (linperlisib, 80mg, po, q.d., in a 28-day cycle), with overall response rate (ORR) as the primary endpoint. In this subgroup analysis, the patients were divided into two groups according to the treatment lines they received: >3 prior lines of treatment and ≤3 prior lines of treatment. All of the statistical analyses were descriptive. Results A total of 84 r/r FL patients from 25 sites in China from April 2019 to September 2020 were enrolled in this trial, with a cutoff date of September 30, 2021. The ORR was 79.8%, with statistical significance in a heavily pretreated FL patient population with a median of 4 prior therapies, with a well-tolerated safety profile. A total of 43 patients received >3 prior lines of treatment, while 41 patients received ≤3 prior lines of treatment. The median age between the two groups was similar (range: 29.0 - 78.0, 52.0 years for >3 prior lines vs. 48.0 years for ≤3 prior lines). The majority of patients had Ann Arbor staging III-IV (40 for >3 prior lines vs. 34 for ≤3 prior lines). The median number of prior regimens was 4 for >3 prior lines and 3 for ≤3 prior lines. Overall, there were 67 patients achieved responses, with ORR of 83.7% (36, 95%CI:69.3, 93.2) for >3 prior lines vs. 75.6% (31, 95% CI: 59.7, 87.6) for ≤3 prior lines based on independent review committee assessment. Compared with patients who received ≤3 prior lines, patients received ≤3 prior lines achieved higher disease control rate (41[95.3] vs. 37 [90.2%]). The median DOR was 12 months (95% CI: 7.3, 15.9) for >3 prior lines vs. 13 months (95% CI: 9.2, NE). The median PFS was 11.9 months (95% CI: 9.1,19.4) for >3 prior lines vs. 13.3 months (95% CI: 8.2, NE) for ≤3 prior lines. The OS of both groups was not reached. The most common treatment-related adverse events (≥20%, TRAEs) were neutropenia (21 [48.8%] for >3 prior lines vs. 18 [43.9%] for ≤3 prior lines), leukocyte count decreased (15 [34.9%] vs. 15 [36.6%]), alanine aminotransferase increased (10 [23.3%] vs. 9 [22.0%]), lymphocyte count decreased (9 [20.9%] vs. 5 [12.2%]), hypertriglyceridemia (9 [20.9%] vs. 11 [26.8%]), and hypercholesteremia (6 [24.0%] vs. 7 [11.9%]). The most common grade ≥3 TRAEs (≥10%) were neutropenia (9 [20.9%] vs. 4 [9.8%]), pulmonary inflammation (3 [7.0%] vs. 6 [14.6%]), infectious pneumonia (2 [4.7%] vs. 5 [12.2%]). Conclusions This subgroup analysis revealed that linperlisib could achieve benefit in r/r FL patients who received at least 2 prior system therapies regardless of treatment lines. The AEs were well-tolerated. Further trials with large-scale samples were warranted.

Synthesis of Novel 2,9-Disubstituted-6-morpholino Purine Derivatives Assisted by Virtual Screening and Modelling of Class I PI3K Isoforms.

The phosphatidylinositol-3 kinase (PI3K) pathway is one of the most frequently activated pathogenic signalling cascades in a wide variety of cancers. In the last 15 years, there has been an increase in the search for selective inhibitors of the four class I isoforms of PI3K, as they demonstrate better specificity and reduced toxicity in comparison to existing inhibitors. A ligand-based and target-based rational drug design strategy was employed to build a virtual library of 105 new compounds. Through this strategy, the four isoforms were compared regarding their activity pocket availability, amino acid sequences, and prone interactions. Additionally, a known active scaffold was used as a molecular base to design new derivatives. The virtual screening of the resultant library toward the four isoforms points to the obtention of 19 selective inhibitors for the PI3Kα and PI3Kγ targets. Three selective ligands, one for α-isoform and two for γ-isoform, present a ∆ (∆Gbinding) equal or greater than 1.5 Kcal/mol and were identified as the most promising candidates. A principal component analysis was used to establish correlations between the affinity data and some of the physicochemical and structural properties of the ligands. The binding modes and interactions established by the selective ligands in the active centre of the α and γ isoforms of PI3K were also investigated. After modelling studies, a synthetic approach to generate selective ligands was developed and applied in synthesising a set of derivatives that were obtained in good to excellent yield.

Simultaneous Inhibition of PI3Kgamma and PI3Kdelta Deteriorates T-cell Function With Implications for Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a common and incurable B-cell malignancy. Recent therapeutic approaches that target the B-cell receptor signaling pathway include inhibition of phosphatidylinositol-3-kinase (PI3K). The PI3K isoform delta is constitutively active in CLL, making it an attractive therapeutic target. However, the expression of PI3K isoforms is not exclusive to leukemic cells, as other immune cells in the tumor microenvironment also rely on PI3K activity. Subsequently, therapeutic inhibition of PI3K causes immune-related adverse events (irAEs). Here, we analyzed the impact of the clinically approved PI3Kδ inhibitors idelalisib and umbralisib, the PI3Kγ inhibitor eganelisib, and the dual-γ and -δ inhibitor duvelisib on the functional capacity of T cells. All investigated inhibitors reduced T-cell activation and proliferation in vitro, which is in line with PI3K being a crucial signaling component of the T-cell receptor signaling. Further, dual inhibition of PI3Kγ and PI3Kδ showed strong additive effects suggesting a role also for PI3Kγ in T cells. Extrapolation of this data to a clinical setting could provide an explanation for the observed irAEs in CLL patients undergoing treatment with PI3K inhibitors. Consequently, this highlights the need for a close monitoring of patients treated with PI3K inhibitors, and particularly duvelisib, due to their potentially increased risk of T-cell deficiencies and associated infections.

Targeting Class I-II-III PI3Ks in Cancer Therapy: Recent Advances in Tumor Biology and Preclinical Research.

Phosphatidylinositol-3-kinase (PI3K) enzymes, producing signaling phosphoinositides at plasma and intracellular membranes, are key in intracellular signaling and vesicular trafficking pathways. PI3K is a family of eight enzymes divided into three classes with various functions in physiology and largely deregulated in cancer. Here, we will review the recent evidence obtained during the last 5 years on the roles of PI3K class I, II and III isoforms in tumor biology and on the anti-tumoral action of PI3K inhibitors in preclinical cancer models. The dependency of tumors to PI3K isoforms is dictated by both genetics and context (e.g., the microenvironment). The understanding of class II/III isoforms in cancer development and progression remains scarce. Nonetheless, the limited available data are consistent and reveal that there is an interdependency between the pathways controlled by all PI3K class members in their role to promote cancer cell proliferation, survival, growth, migration and metabolism. It is unknown whether this feature contributes to partial treatment failure with isoform-selective PI3K inhibitors. Hence, a better understanding of class II/III functions to efficiently inhibit their positive and negative interactions with class I PI3Ks is needed. This research will provide the proof-of-concept to develop combination treatment strategies targeting several PI3K isoforms simultaneously.

Discovery of GDC-0077 (Inavolisib), a Highly Selective Inhibitor and Degrader of Mutant PI3Kα.

Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib, 32), which is now under evaluation in a Phase III clinical trial as a treatment for patients with PIK3CA-mutant breast cancer.

Functional specialization of different PI3K isoforms for the control of neuronal architecture, synaptic plasticity, and cognition.

Neuronal connectivity and activity-dependent synaptic plasticity are fundamental properties that support brain function and cognitive performance. Phosphatidylinositol 3-kinase (PI3K) intracellular signaling controls multiple mechanisms mediating neuronal growth, synaptic structure, and plasticity. However, it is still unclear how these pleiotropic functions are integrated at molecular and cellular levels. To address this issue, we used neuron-specific virally delivered Cre expression to delete either p110α or p110β (the two major catalytic isoforms of type I PI3K) from the hippocampus of adult mice. We found that dendritic and postsynaptic structures are almost exclusively supported by p110α activity, whereas p110β controls neurotransmitter release and metabotropic glutamate receptor-dependent long-term depression at the presynaptic terminal. In addition to these separate functions, p110α and p110β jointly contribute to N-methyl-d-aspartate receptor-dependent postsynaptic long-term potentiation. This molecular and functional specialization is reflected in different proteomes controlled by each isoform and in distinct behavioral alterations for learning/memory and sociability in mice lacking p110α or p110β.

Phenotypic Discovery of Triazolo[1,5-c]quinazolines as a First-In-Class Bone Morphogenetic Protein Amplifier Chemotype.

Phenotypic drug discovery (PDD) continues to fuel the research and development pipelines with first-in-class therapeutic modalities, but success rates critically depend on the quality of the underlying model system. Here, we employed a stem cell-based approach for the target-agnostic, yet pathway-centric discovery of small-molecule cytokine signaling activators to act as morphogens during development and regeneration. Unbiased screening identified triazolo[1,5-c]quinazolines as a new-in-class in vitro and in vivo active amplifier of the bone morphogenetic protein (BMP) pathway. Cellular BMP outputs were stimulated via enhanced and sustained availability of BMP-Smad proteins, strictly dependent on a minimal BMP input. Holistic target deconvolution unveiled a unique mechanism of dual targeting of casein kinase 1 and phosphatidyl inositol 3-kinase isoforms as key effectors for efficient amplification of osteogenic BMP signaling. This work underscores the asset of PDD to discover unrecognized polypharmacology signatures, in this case significantly expanding the chemical and druggable space of BMP modulators.

Exploring PI3Kγ binding preference with Eganelisib, Duvelisib, and Idelalisib via energetic, pharmacophore and dissociation pathway analyses

Phosphatidylinositol 3-kinase (PI3K) is the central regulator of cellular functions and is suggested as a target for various diseases; thus, effective PI3K inhibitors provide a promising opportunity for the pharmaceutical intervention of many diseases. Among them, PI3Kγ has received more attention because of its essential role in immune signaling. However, the development of novel selective PI3Kγ inhibitors is a major challenge due to the high sequence homology across the class I PI3K isoforms. Therefore, understanding the substrate specificity and receptor-ligand interaction of PI3Kγ would be an appropriate strategy for the rational design of potent γ-selective inhibitors. In this study, by combining various molecular modeling approaches (including classic and enhanced sampling molecular dynamics (MD) simulations, end-point binding free energy calculations, and pharmacophore models), three quinolinone core-containing inhibitors, Idelalisib/CAL-101, Duvelisib/IPI-145, and Eganelisib/IPI-549, were employed to reveal the selective binding mechanisms targeting PI3Kγ. The classic MD and free energy calculations highlight the significant interaction and some key residues for the selective binding against PI3Kγ. Furthermore, the dissociation pathway analysis based on umbrella sampling simulations reveals that hydrophobic interactions are dominant for binding of the three ligands during the dissociation processes, and cooperation between the P-loop and the ligands always exists in the binding/dissociation process. Finally, the pharmacophore model revealed that IPI-549 contains a unique hydrophobic feature, and PI3Kγ exhibits an important hydrogen bond donor feature of hydrogen amide. These findings may provide some important information for the rational design and optimization of PI3Kγ-selective inhibitors.

ZAP70 Activation Compensates for Loss of Class IA PI3K Isoforms Through Activation of the JAK–STAT3 Pathway

Tyrosine kinases have crucial functions in cell signaling and proliferation. The phosphatidylinositol 3-kinase (PI3K) pathway is frequently deregulated in human cancer and is an essential regulator of cellular proliferation. We aimed to determine which tyrosine kinases contribute to resistance elicited by PI3K silencing and inhibition. To mimic catalytic inactivation of p110α/β, specific p110α (BYL719) and p110β (KIN193) inhibitors were used in addition to genetic knock-out in in vitro assays. Cell viability was assessed using crystal violet staining, whereas cellular transformation ability was analyzed by soft-agar growth assays. Activated zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70) generated resistance to PI3K inhibition. This resistance was via activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) axis. We demonstrated that activated ZAP70 has a high transforming capability associated with the formation of malignant phenotype in untransformed cells and has the potential to be a tumor-initiating factor in cancer cells. ZAP70 may be a potent driver of proliferation and transformation in untransformed cells and is implicated in resistance to PI3K inhibitors in cancer cells.

Computational study reveals substituted benzimidazole derivatives' binding selectivity to PI3Kδ and PI3Kγ.

Phosphatidylinositol 3-kinase (PI3K) is a key regulatory kinase in the PI3K/AKT/mTOR signaling pathway, which is involved in the regulation of cell proliferation, differentiation, apoptosis, and angiogenesis. Class IA PI3K isoforms γ and δ share a highly homologous ATP binding site and are distinguished by only a few residues around the binding site. Subtype-selective inhibitors have been proven to have great advantages in tumor treatment. Preliminary studies have obtained PI3K inhibitors bearing a benzimidazole structural motif with a certain selectivity for PI3Kδ and PI3Kγ subtypes. On this basis, we investigated the selective inhibitory mechanism of PI3Kδ and PI3Kγ using four developed inhibitors via molecular docking, molecular dynamics, binding free energy calculations, and residue energy decomposition. This study could provide references for the further development of PI3K-isoform-selective inhibitors.

IGF-1 regulates astrocytic phagocytosis and inflammation through the p110α isoform of PI3K in a sex-specific manner.

Insulin‐like growth factor‐I (IGF‐I) signaling plays a key role in neuroinflammation. Here we show that IGF‐1 also regulates phagocytosis of reactive astrocytes through p110α isoform of phosphatidylinositol 3‐kinase (PI3K), differentially in both sexes. Systemic bacterial lipopolysaccharide (LPS)‐treatment increased the expression of GFAP, a reactive astrocyte marker, in the cortex of mice in both sexes and was blocked by IGF‐1 only in males. In primary astrocytes, LPS enhanced the mRNA expression of Toll‐like receptors (TLR2,4) and proinflammatory factors: inducible nitric oxide synthase (iNOS), chemokine interferon‐γ‐inducible protein‐10 (IP‐10) and cytokines (IL‐1β, IL‐6, and IL‐10) in male and female. Treatment with IGF‐1 counteracted TLR4 but not TLR2, iNOS, and IP10 expression in both sexes and cytokines expression in males. Furthermore, reactive astrocyte phagocytosis was modulated by IGF‐1 only in male astrocytes. IGF‐1 was also able to increase AKT‐phosphorylation only in male astrocytes. PI3K inhibitors, AG66, TGX‐221, and CAL‐101, with selectivity toward catalytic p110α, p110β, and p110δ isoforms respectively, reduced AKT‐phosphorylation in males. All isoforms interact physically with IGF‐1‐receptor in both sexes. However, the expression of p110α is higher in males while the expression of IGF‐1‐receptor is similar in male and female. AG66 suppressed the IGF‐1 effect on cytokine expression and counteracted the IGF‐1‐produced phagocytosis decrease in male reactive astrocytes. Results suggest that sex‐differences in the effect of IGF‐1 on the AKT‐phosphorylation could be due to a lower expression of the p110α in female and that IGF‐1‐effects on the inflammatory response and phagocytosis of male reactive astrocytes are mediated by p110α/PI3K subunit.

Comparing the PI3K Inhibitors in Hematological Malignancies: Review of Faers

Background:Phosphatidylinositol 3-kinase inhibitors (PI3KI) are a novel class of drugs that are small molecular inhibitors of various isoforms of phosphatidylinositol 3-kinase (PI3K). Over the past decade, various PI3KI have been approved in follicular lymphoma, marginal zone lymphoma, and chronic lymphocytic leukemia. While Copanlisib inhibits P13K-alpha and P13K-delta isoforms, idelalisib inhibits P13K-delta, duvelisib inhibits PI3K-delta and PI3K-gamma, and the recently approved agent umbralisib targets PI3K-delta and casein kinase 1 epsilon with improved selectivity for the PI3K-delta. All the agents except Copanlisib are given orally. Immune-mediated adverse effects like colitis, marrow suppression with infections, and dermatological toxicities are known complications of PI3KI. We did a retrospective analysis of the adverse effects (AE) of the PI3KI in the FDA Adverse Event Reporting System (FAERS).Methods:FAERS public dashboard is a resource through which information related to AEs of treatments reported to the FDA is made available to the public. We investigated the common immune-mediated, infectious, hepatic, and dermatological toxicities of the three PI3KI, idelalisib, copanlisib, and duvelisib for the years 2018 to March 2021. Umbralisib has not much-reported data in FAERS.Results:The data regarding various adverse effects are summarized in Table 1. Idelalisib has the most reported data. Diarrhea is reported to be most common with duvelisib (21.83% of total AE). Though diarrhea is common, colitis is uncommon and least reported with copanlisib (1.15%). Among the patients with colitis, death occurred in 27.2% with idelalisib, 15.3% with duvelisib and 50% with copanlisib. Pneumonitis is most reported with copanlisib (3.45%). Among the patients with pneumonitis, death occurred in 34.7% with idelalisib, none with duvelisib and 50% with copanlisib. Rash is most reported with copanlisib (6.32%) and the incidence of serious dermatological toxicity is uncommon in all the 3 drugs. Rates of neutropenia and thrombocytopenia are overall similar between the drugs except for a slightly higher rate in copanlisib including febrile neutropenia. Infections including pneumonia, urinary tract infection, PJP pneumonia, and CMV reactivation are overall comparable between the 3 drugs. Among the patients with various infections, death occurred in 24.1% with idelalisib, 14.5% with duvelisib and 16.9% with copanlisib. The rate of hepatotoxicity is also comparable between the drugs. Among the patients with hepatic failure, death occurred in 33.3% with idelalisib, 100% with duvelisib and copanlisib.Conclusion:The overall rates of serious adverse effects are comparable between the 3 PI3KI, though diarrhea is most common with duvelisib, pneumonitis, and rash with copanlisib. As these drugs are mainly used in the relapsed refractory setting, many more years of follow-up are needed to get a better idea of real-world data as we have more experience with these drugs. [Display omitted] DisclosuresMaster: Blue Bird Bio: Current holder of individual stocks in a privately-held company.

Disrupted PI3K subunit p110α signaling protects against pulmonary hypertension and reverses established disease in rodents.

Enhanced signaling via RTKs in pulmonary hypertension (PH) impedes current treatment options because it perpetuates proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Here, we demonstrated hyperphosphorylation of multiple RTKs in diseased human vessels and increased activation of their common downstream effector phosphatidylinositol 3'-kinase (PI3K), which thus emerged as an attractive therapeutic target. Systematic characterization of class IA catalytic PI3K isoforms identified p110α as the key regulator of pathogenic signaling pathways and PASMC responses (proliferation, migration, survival) downstream of multiple RTKs. Smooth muscle cell-specific genetic ablation or pharmacological inhibition of p110α prevented onset and progression of pulmonary hypertension (PH) as well as right heart hypertrophy in vivo and even reversed established vascular remodeling and PH in various animal models. These effects were attributable to both inhibition of vascular proliferation and induction of apoptosis. Since this pathway is abundantly activated in human disease, p110α represents a central target in PH.

Abstract 1377: Volume scanning, a rational approach to covalent PI3Kα inhibitors

Abstract Inhibitors of the phosphatidylinositol 3-kinase (PI3K) - protein kinase B (PKB/Akt) - mechanistic target of rapamycin (mTOR) axis are considered valuable assets in cancer therapy. A considerable effort has been dedicated to the development of drugs targeting class I PI3Ks, which are evaluated in preclinical and clinical studies.[1-5] Here we present a strategy to convert a phase II clinical candidate, a pan-PI3K inhibitor (PQR309, bimiralisib)[5,6], into a highly selective, covalent PI3Kα inhibitor with the aim to minimize off-target and on-target metabolic side effects of PI3K inhibitor cancer therapy. We exploited a rational approach to increase target selectivity by covalently targeting PI3Kα at the non-conserved nucleophilic Cys862. A combination of warhead activity design, proximity screening and an optimized orientation allowed a tight control of reversible inhibitor binding in combination with an isoform-specific covalent reaction. To avoid off-target reactions, all warheads' reactivities were determined and optimize for selectivity and of Cys862 modification. An extensive Structure Activity Relationship (SAR) study was performed and a wide range of linear and restricted rotation linkers were introduced. A comprehensive understanding of the kinetics of irreversible inhibition acquired by kinetic TR-FRET assays and subsequent determination of kchem, kinact and calculated Ki allowed the establishment of a SAR, for compound selection with minimal off-target reactivity and high PI3Kα selectivity. X-ray crystallography and MS-based proteomics validated the covalent modification of Cys862. Our pilot compounds exceed specificity and potency over an experimental dimethyl-substituted enone, CNX-1351.[7] Moreover, our compounds display increased stability in rat liver microsomal assays and outperform the rapidly metabolized CNX-1351. Our strategy to investigate and tune warheads' reactivity represents a major step forward in the rational design of covalent chemical tools, overcoming the serendipity in the discovery of irreversible compounds. Moreover, we provide highly selective chemical tools to dissect PI3K isoform signaling in physiology and disease. A clarification of the role of the different PI3K isoforms in insulin signaling allows to address the challenges in isoform selectivity and to develop PI3K inhibitors showing ideal isoform specificity. [1] Rageot D et. al. J Med Chem. 2019, 62 (13), 6241-6261. [2] Borsari C et. al. ACS Med Chem Lett. 2019, 10 (10), 1473-1479. [3] Marone R et al. Biochim Biophys Acta 2008, 1784 (1), 159-185. [4] Wymann MP and Schneiter R Nat Rev Mol Cell Biol. 2008, 9 (2), 162-176. [5] Beaufils F et. al. J Med Chem. 2017, 60 (17), 7524-7538. [6] Wicki A et. al. Eur J Cancer. 2018, 96, 6-16. [7] Nacht M et. al. J Med Chem. 2013, 56 (3), 712-721. Citation Format: Chiara Borsari, Erhan Keles, Jacob McPhail, Alexander Schäfer, Rohitha Sriramaratnam, Martina De Pascale, Matthias Gstaiger, John Burke, Matthias P. Wymann. Volume scanning, a rational approach to covalent PI3Kα inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1377.

Idelalisib-Rituximab Results in Considerable Toxicity but Induces Durable Clinical Remissions in Patients with TP53 Disrupted B Cell Prolymphocytic Leukemia

BackgroundB-cell prolymphocytic leukaemia (B-PLL) is a rare disorder representing 1% of lymphocytic leukemias. With a median age of 69 years, pts characteristically present with B symptoms, marked lymphocytosis, cytopenias, massive splenomegaly and minimal lymphadenopathy. The largest trial of B-PLL included only 14 patients (pts) (Döhner et al, 1993). There are no randomized trials published in B-PLL. The median overall survival is 3 years. 13q14, 11q23 and 17p deletions are common by fluorescence- in-situ hybridisation (FISH) (Lens et al, 1999). TP53 mutations are noted in >50%, conferring resistance to immunochemotherapy (Dearden 2012). In the absence of international guidelines or trial data, clinicians typically employ fludarabine, cyclophosphamide and rituximab (FCR) or bendamustine-R (BR) in TP53- intact pts with allogeneic stem cell transplant (allo-SCT) consolidation in eligible, chemotherapy-sensitive pts (Castagna et al, 2005). For TP53 -disrupted B-PLL, alemtuzumab has efficacy but is associated with serious infectious toxicity.The B-cell-receptor signaling pathway plays a critical role in CLL pathogenesis, although little is known regarding its importance in B-PLL (Bernal et al, 2001). This pathway is partly mediated by the activation of the delta isoform of phosphatidylinositol 3-kinase (PI3Kδ) which is highly expressed in lymphoid cells (Herishanu et al, 2011). Idelalisib is a first-in-class, potent, oral, selective PI3Kδ inhibitor. Initial results of the first 5 pts in the compassionate use programme describe clear efficacy of idelalisib-rituximab (I-R) in TP53- disrupted B-PLL (Eyre et al, 2016). We update this series with considerably longer follow up and additional pts.Methods8 pts received I-R in a UK-wide compassionate use programme supported by Gilead. Data was retrospectively collected for adverse events (AEs), clinical and radiological responses. Rituximab (375 mg/m2) was given 2-weekly (cycle (C) 1-2) and then 4-weekly (C3-6). Idelalisib 150 mg b.d. was given continuously with dose interruption and reduction as required for toxicity and was continued in pts with response after C6 as monotherapy. Response was ascertained radiologically and clinically according to institutional practice. AEs were reported prospectively to Gilead. FISH for p53 deletion was performed in all pts. Sanger sequencing was performed in 3 pts. Standard infectious prophylaxis was routinely added at the time that the safety signal was announced.ResultsThe median age was 64.5 years (range: 57-76 years). 7 were male and 1 was female. The median time from diagnosis to first treatment was 9.5 months (range: 2-60 months). 1 pt had a prior splenectomy, but all others had small volume lymphadenopathy and splenomegaly prior to I-R. TP53 deletions by FISH were found in 7 pts. No known additional mutations were noted. Standard cytogenetics were not performed. 4 pts were treatment naïve, 1 pt received prior methylprednisolone, 1 pt BR, gemcitabine-cisplatin and alemtuzumab, 1 pt alemtuzumab and 1 pt R-CVP and then BR. 5 pts completed 6C x 28-day I-R. 3 pts stopped early: 1 pt died of an unrelated lung cancer in C2, and 2 pts stopped due to idelalisib-related grade (G) 4 colitis at C4.All 8 pts responded to I-R. The single pt who died early of lung cancer obtained a partial response. All other 7 pts obtained complete remission (CR) when assessed by examination, FBC, radiological and FISH. Of those 7 pts, only 1 progression has occurred. This pt subsequently responded to ibrutinib. 1 pt (treatment naïve) died in CR of G5 pneumonitis at 10 months on therapy. There are 5 ongoing CRs at a median of 18 months (range 17-29 months). 2 of these 5 have stopped idelalisib due to toxicity (both G3-4 colitis), 2 remain on monotherapy, and 1 has undergoing consolidation with an allo-SCT having stopped idelalisib (G3 diarrhoea). The median follow up for all pts is 18 months (range 1-29 months).ConclusionsI-R displays rapid, deep and durable remissions in TP53 -disrupted B-PLL. The toxicity profile of idelalisib is well described and is clearly demonstrated in this cohort. Pneumonitis, colitis and CMV reactivation were particularly noteworthy. Of interest, remissions were durable in the 2 pts that stopped therapy following colitis for 13 and 14 months respectively. Our data supports importance of compassionate use programme to study novel agents in rare disorders. Updated response and toxicity data will be presented. [Display omitted] DisclosuresFox:Gilead: Consultancy, Honoraria, Other: travel expenses, Speakers Bureau. Bloor:Roche: Honoraria; Gilead: Consultancy, Other: travel expenses; AbbVie: Honoraria, Other: travel expenses; Janssen: Other: travel expenses, Speakers Bureau. Dungawalla:Gilead: Honoraria. Shankara:Gilead: Honoraria, Other: Travel expenses, Speakers Bureau. Schuh:Abbvie: Honoraria; Janssen: Honoraria; Abbvie: Honoraria; Gilead: Honoraria, Research Funding; Roche: Honoraria; Novartis: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Gilead: Consultancy; Roche: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

The PI-3-Kinase P110α Catalytic Subunit of T Lymphocytes Modulates Collagen-Induced Arthritis.

The phosphatidylinositol 3-kinase (PI3K) family of enzymes plays a determinant role in inflammation and autoimmune responses. However, the implication of the different isoforms of catalytic subunits in these processes is not clear. Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory disease that entails innate and adaptive immune response elements in which PI3K is a potential hub for immune modulation. In a mouse transgenic model with T-cell-specific deletion of p110α catalytic chain (p110α−/−ΔT), we show the modulation of collagen-induced arthritis (CIA) by this isoform of PI3K. In established arthritis, p110α−/−ΔT mice show decreased prevalence of illness than their control siblings, higher IgG1 titers and lower levels of IL-6 in serum, together with decreased ex vivo Collagen II (CII)-induced proliferation, IL-17A secretion and proportion of naive T cells in the lymph nodes. In a pre-arthritis phase, at 13 days post-Ag, T-cell-specific deletion of p110α chain induced an increased, less pathogenic IgG1/IgG2a antibodies ratio; changes in the fraction of naive and effector CD4+ subpopulations; and an increased number of CXCR5+ T cells in the draining lymph nodes of the p110α−/−ΔT mice. Strikingly, T-cell blasts in vitro obtained from non-immunized p110α−/−ΔT mice showed an increased expression of CXCR5, CD44 and ICOS surface markers and defective ICOS-induced signaling towards Akt phosphorylation. These results, plus the accumulation of cells in the lymph nodes in the early phase of the process, could explain the diminished illness incidence and prevalence in the p110α−/−ΔT mice and suggests a modulation of CIA by the p110α catalytic chain of PI3K, opening new avenues of intervention in T-cell-directed therapies to autoimmune diseases.

Integrated molecular modeling techniques to reveal selective mechanisms of inhibitors to PI3Kδ with marketed Idelalisib

Phosphatidylinositol-3-kinase (PI3K) is important for cell proliferation, differentiation, and apoptosis, and the diverse physiological roles of different PI3K isoforms have highlighted the significance of the development of PI3Kδ inhibitors. A large number of PI3Kδ inhibitors have been reported after the FDA approval of Idelalisib, but the clinical use of Idelalisib was limited because of its serious side effects. Therefore, great efforts have been made on the development of PI3Kδ inhibitors with higher selectivity and lower toxicity, but there is no new PI3Kδ inhibitor coming into the market so far. Even so, as the first listed PI3K inhibitor, Idelalisib could be used as an effective tool to investigate the selective inhibition mechanism of PI3Kδ. Thus, in this study, a modeling strategy integrated 3D-QSAR, pharmacophore model, and molecular dynamics simulation was employed to reveal the key chemical characteristics of Idelalisib analogs and the binding pattern between the inhibitors and PI3Kδ. First, the CoMFA model with high statistical significance was built to reveal the general structure-activity relationships. And then, a reliable pharmacophore model with a robust discrimination capability was constructed to expound the main chemical characteristics of the PI3Kδ inhibitors. Finally, molecular dynamics simulation was conducted to explore the binding modes and some key residues refer to δ-selective binding were highlighted with binding-free energy calculation. In summary, these models and results would provide some effective help for the discovery or the rational design of novel PI3Kδ inhibitors.

Inhibition of P110α and P110δ catalytic subunits of PI3 kinase reverses impaired arterial healing after injury in hypercholesterolemic male mice.

Endothelial cell (EC) migration is critical for healing arterial injuries, such as those that occur with angioplasty. Impaired re-endothelialization following arterial injury contributes to vessel thrombogenicity, intimal hyperplasia, and restenosis. Oxidized lipid products, including lysophosphatidylcholine (lysoPC), induce canonical transient receptor potential 6 (TRPC6) externalization leading to increased [Ca2+]i, activation of calpains, and alterations of the EC cytoskeletal structure that inhibit migration. The p110α and p110δ catalytic subunit isoforms of phosphatidylinositol 3-kinase (PI3K) regulate lysoPC-induced TRPC6 externalization in vitro. The goal of this study was to assess the in vivo relevance of those in vitro findings to arterial healing following a denuding injury in hypercholesterolemic mice treated with pharmacologic inhibitors of the p110α and p110δ isoforms of PI3K and a general PI3K inhibitor. Pharmacologic inhibition of the p110α or the p110δ isoform of PI3K partially preserves healing in hypercholesterolemic male mice, similar to a general PI3K inhibitor. Interestingly, the p110α, p110δ, and the general PI3K inhibitor do not improve arterial healing after injury in hypercholesterolemic female mice. These results indicate a potential new role for isoform-specific PI3K inhibitors in male patients following arterial injury/intervention. The results also identify significant sex differences in the response to PI3K inhibition in the cardiovascular system, where female sex generally has a cardioprotective effect. This study provides a foundation to investigate the mechanism for the sex differences in response to PI3K inhibition to develop a more generally applicable treatment option.

Class II phosphatidylinositol 3-kinase isoforms in vesicular trafficking

Phosphatidylinositol 3-kinases (PI3Ks) are critical regulators of many cellular processes including cell survival, proliferation, migration, cytoskeletal reorganization, and intracellular vesicular trafficking. They are a family of lipid kinases that phosphorylate membrane phosphoinositide lipids at the 3′ position of their inositol rings, and in mammals they are divided into three classes. The role of the class III PI3K Vps34 is well-established, but recent evidence suggests the physiological significance of class II PI3K isoforms in vesicular trafficking. This review focuses on the recently discovered functions of the distinct PI3K-C2α and PI3K-C2β class II PI3K isoforms in clathrin-mediated endocytosis and consequent endosomal signaling, and discusses recently reported data on class II PI3K isoforms in different physiological contexts in comparison with class I and III isoforms.