PI3K Isoforms Research Articles

Polymorphonuclear myeloid-derived suppressor cells and phosphatidylinositol-3 kinase gamma are critical to tobacco-mimicking oral carcinogenesis in mice

BackgroundOral squamous cell carcinoma (OSCC) is a devastating disease most often associated with tobacco consumption that induces a field of mutations from which a tumor arises. Identification of ways to...

Quantum biochemistry description of PI3Kα enzyme bound to selective inhibitors

The PI3K class I is composed of four PI3K isoforms that serve as regulatory enzymes governing cellular metabolism, proliferation, and survival. The hyperactivation of PI3Kα is observed in various types of cancer and is linked to poor prognosis. Unfortunately, the development inhibitors selectively targeting one of the isoforms remains challenging, with only few agents in clinical use. The main difficulty arises from the high conservation among residues at the ATP-binding pocket across isoforms, which also serves as target pocket for inhibitors. In this work, molecular dynamics and quantum calculations were performed to investigate the molecular features guiding the binding of selective inhibitors, alpelisib and GDC-0326, into the ATP-binding pocket of PI3Kα. While molecular dynamics allowed crystallographic coordinates to relax, the interaction eergy between each amino acid residues and inhibitors was obtained by combining the Molecular Fractionation with Conjugated Caps scheme with Density Functional Theory calculations. In addition, the atomic charge of ligands in the bound and unbound (free) was calculated. Results indicated that the most relevant residues for the binding of alpelisib are Ile932, Glu859, Val851, Val850, Tyr836, Met922, Ile800, and Ile848, while the most important residues for the binding of GDC-0326 are Ile848, Ile800, Ile932, Gln859, Glu849, and Met922. In addition, residues Trp780, Ile800, Tyr836, Ile848, Gln859 Val850, Val851, Ile932 and Met922 are common hotspots for both inhibitors. Overall, the results from this work contribute to improving the understanding of the molecular mechanisms controlling selectivity and highlight important interactions to be considered during the rational design of new agents. Communicated by Ramaswamy H. Sarma

The Temporal and Spatial Changes of Autophagy and PI3K Isoforms in Different Neural Cells After Hypoxia/Reoxygenation Injury.

There are limited therapeutic options for patient with traumatic spinal cord injury (SCI). Phosphoinositide 3-kinase family (PI3Ks) are the key molecules for regulating cell autophagy, which is a possible way of treating SCI. As we know, PI3K family are composed of eight isoforms, which are distributed into three classes. While the role of PI3Ks in regulating autophagy is controversial and the effects may be in a cell-specific manner. Different isoforms do not distribute in neural cells consistently and it is not clear how the PI3K isoforms regulate and interact with autophagy. Therefore, we explored the distributions and expression of different PI3K isoforms in two key neural cells (PC12 cells and astrocytes). The results showed that the expression of LC3II/I and p62, which are the markers of autophagy, changed in different patterns in PC12 cells and astrocytes after hypoxia/reoxygenation injury (H/R). Furthermore, the mRNA level of eight PI3K isoforms did not change in the same way, and even for the same isoform the mRNA activities are different between PC12 cells and astrocytes. What is more, the results of western blot of PI3K isoforms after H/R were inconsistent with the relevant mRNA. Based on this study, the therapeutic effects of regulating autophagy on SCI are not confirmed definitely, and its molecular mechanisms may be related with different temporal and spatial patterns of activation and distributions of PI3K isoforms.

Functional impact and molecular binding modes of drugs that target the PI3K isoform p110δ

Targeting the PI3K isoform p110δ against B cell malignancies is at the mainstay of PI3K inhibitor (PI3Ki) development. Therefore, we generated isogenic cell lines, which express wild type or mutant p110δ, for assessing the potency, isoform-selectivity and molecular interactions of various PI3Ki chemotypes. The affinity pocket mutation I777M maintains p110δ activity in the presence of idelalisib, as indicated by intracellular AKT phosphorylation, and rescues cell functions such as p110δ-dependent cell viability. Resistance owing to this substitution consistently affects the potency of p110δ-selective in contrast to most multi-targeted PI3Ki, thus distinguishing usually propeller-shaped and typically flat molecules. Accordingly, molecular dynamics simulations indicate that the I777M substitution disturbs conformational flexibility in the specificity or affinity pockets of p110δ that is necessary for binding idelalisib or ZSTK474, but not copanlisib. In summary, cell-based and molecular exploration provide comparative characterization of currently developed PI3Ki and structural insights for future PI3Ki design.

Abstract CT017: Pan-mutant and isoform selective PI3Kα inhibitor, RLY-2608, demonstrates selective targeting in a first-in-human study of PIK3CA-mutant solid tumor patients, ReDiscover trial

Abstract BACKGROUND: Oncogenic activation of PI3Kα via mutation of PIK3CA is the most common kinase driver event across solid tumors, particularly breast cancer (BC). Alpelisib, an orthosteric inhibitor, validated mutant-PI3Kα as a therapeutic target; however, toxicity from non-selective inhibition of WT PI3Kα (hyperglycemia) and other PI3K isoforms limits its clinical activity. RLY-2608, a novel oral allosteric PI3Kα inhibitor, is designed to overcome these limitations via mutant- and isoform-selective PI3Kα inhibition. We initiated a first-in-human study (NCT05216432) to define the MTD, safety, pharmacokinetics (PK), and anti-tumor activity of RLY-2608 in PIK3CA-mutant solid tumor patients (pts) and of RLY-2608 plus fulvestrant (combo) in pts with PIK3CA-mutant, HR+HER2-BC, who had received prior CDK4/6 inhibitor and endocrine therapy. METHODS: Adult pts with no prior PI3Ki/mTORi therapy received RLY-2608 QD or BID on a 4-week (wk) cycle following a Bayesian Optimal Interval dose escalation. Eligible BC pts received fulvestrant. PK, pharmacodynamics (ex vivo pAKT inhibition; tumor markers; ctDNAs), treatment emergent adverse events (TEAEs), and anti-tumor activity per RECIST 1.1 were assessed. RESULTS: As of 31Jan23, 40 pts (21 BC combo pts) with PIK3CA mutations (12 kinase, 18 helical, 10 other) received RLY-2608 at doses of 100-1600 mg/day. Median number of prior treatments was 4 (1-11). An MTD has not been reached and dose escalation continues. RLY-2608 PK were favorable with BID dosing providing low steady-state peak-to-trough variability for mutant-selective inhibition. This enabled dose-dependent increase in exposure and target inhibition with doses ≥600 mg BID and pAKT suppression (≥80%) in the expected therapeutic range with minimal/no impact on glucose homeostasis. Across arms, safety was consistent: most frequent TEAEs (>15%) were low grade (≤Gr 2) nausea (30%), fatigue (22.5%), headache (22.5%), hypokalemia (22.5%), and diarrhea (17.5%). No DLTs were observed, and no pts discontinued due to related AEs. Initial anti-tumor activity was observed in BC combo pts including declines in tumor markers, PIK3CA/ESR1 ctDNAs and radiographic tumor reduction in 7 of 10 RECIST measurable pts. At 600 mg BID, 6/6 BC combo pts had disease stabilization and remain on treatment, including all RECIST-measurable pts with tumor reduction. Overall, 17 (81%) BC combo pts remain on treatment with median duration of 12 (1-40) wks. CONCLUSION: Across PIK3CA genotypes, RLY-2608 demonstrated target inhibition and anti-tumor activity with minimal impact on glucose homeostasis. These proof-of-mechanism data indicate that RLY-2608 is the first allosteric, pan-mutant selective PI3Kα inhibitor and that RLY-2608 has broad therapeutic potential in PIK3CA-driven cancers. Citation Format: Andreas Varkaris, Komal Jhaveri, Cesar A. Perez, Janice S. Kim, Jason T. Henry, Vivek Subbiah, Alexander I. Spira, Lucía Sanz Gómez, Angel I. Guerrero-Zotano, Kari B. Wisinski, Xiaoyan Li, Gege Tan, Ramin Samadani, Tamieka Lauz Hunter, Wei Guo, Alison Timm, Djuro Karanovic, Beni B. Wolf, Erika P. Hamilton, Alison M. Schram. Pan-mutant and isoform selective PI3Kα inhibitor, RLY-2608, demonstrates selective targeting in a first-in-human study of PIK3CA-mutant solid tumor patients, ReDiscover trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT017.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Phosphatidylinositol kinases in GtoPdb v.2023.1

Phosphatidylinositol may be phosphorylated at either 3- or 4- positions on the inositol ring by PI 3-kinases or PI 4-kinases, respectively.Phosphatidylinositol 3-kinasesPhosphatidylinositol 3-kinases (PI3K, provisional nomenclature) catalyse the introduction of a phosphate into the 3-position of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol 4,5-bisphosphate (PIP2). There is evidence that PI3K can also phosphorylate serine/threonine residues on proteins. In addition to the classes described below, further serine/threonine protein kinases, including ATM (Q13315) and mTOR (P42345), have been described to phosphorylate phosphatidylinositol and have been termed PI3K-related kinases. Structurally, PI3Ks have common motifs of at least one C2, calcium-binding domain and helical domains, alongside structurally-conserved catalytic domains. wortmannin and LY 294002 are widely-used inhibitors of PI3K activities. wortmannin is irreversible and shows modest selectivity between Class I and Class II PI3K, while LY294002 is reversible and selective for Class I compared to Class II PI3K.Class I PI3Ks (EC 2.7.1.153) phosphorylate phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate and are heterodimeric, matching catalytic and regulatory subunits. Class IA PI3Ks include p110α, p110β and p110δ catalytic subunits, with predominantly p85 and p55 regulatory subunits. The single catalytic subunit that forms Class IB PI3K is p110γ. Class IA PI3Ks are more associated with receptor tyrosine kinase pathways, while the Class IB PI3K is linked more with GPCR signalling.Class II PI3Ks (EC 2.7.1.154) phosphorylate phosphatidylinositol to generate phosphatidylinositol 3-phosphate (and possibly phosphatidylinositol 4-phosphate to generate phosphatidylinositol 3,4-bisphosphate). Three monomeric members exist, PI3K-C2α, β and β, and include Ras-binding, Phox homology and two C2 domains.The only class III PI3K isoform (EC 2.7.1.137) is a heterodimer formed of a catalytic subunit (VPS34) and regulatory subunit (VPS15).Phosphatidylinositol 4-kinasesPhosphatidylinositol 4-kinases (EC 2.7.1.67) generate phosphatidylinositol 4-phosphate and may be divided into higher molecular weight type III and lower molecular weight type II forms.

Discovery of 4-oxo-4,5-dihydropyrazolo[1,5-a]quinoxaline-7-carboxamide derivatives as PI3Kα inhibitors via virtual screening and docking-based structure optimization

Compound 1 with pyrazolo[1,5-a]quinoxalin-4(5H)-one scaffold was identified as a PI3Kα inhibitor hit via virtual screening strategy. Additional similarity search and molecular docking based structural modification yielded a novel series of pyrazolo[1,5-a]quinoxalin-4(5H)-one derivatives. The most potent compound 49b exhibited remarkably improved PI3Kα inhibitory activity with IC50 value of 0.24 μM and moderate to good isoform selectivity over other class I PI3K isoforms. In addition, 49b significantly inhibited the proliferation of Kasumi-1 and T47D cells with IC50 value of 1.64 and 1.82 μM, respectively. Further PK study demonstrated that it has favorable pharmacokinetic profiles (AUC0-t = 3294.05 ng·h/mL at 5.0 mg/kg PO, F = 91.8%). All these data indicated that compound 49b was a promising PI3Kα inhibitor with beneficial drug-like properties and merited further development.

Abstract 4444: Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy

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.

Abstract 5162: Discovery of INCB098377: a potent inhibitor of phosphoinositide 3-kinase gamma (PI3Kγ)

Abstract Immune checkpoint blockade has shown impressive efficacy in patients with inflamed tumors, although minimal activity has been observed in tumors lacking T cells. Myeloid cells are one of the most abundant cell types in both inflamed and non-inflamed tumors, and may contribute to immune checkpoint blockade resistance. The plasticity of macrophages enables them to directly and indirectly modulate T cell responses, and directly kill tumor cells via phagocytosis. This suggests that targeting myeloid cells could be an effective therapeutic approach. Class I PI3Ks are a family of dual specificity lipid and protein kinases. Unlike other class I PI3Ks, PI3Kγ is predominantly expressed in myeloid cells. PI3Kγ has been shown to be a key mediator that drives the immunosuppressive macrophage program by stimulating AKT/mTOR signaling and promote C/EBPβ expression while inhibiting NF-кB activity (Keneda MM. Nature. 2016;17:437-442). Here, we present the discovery and characterization of INCB098377, a potent and selective PI3Kγ inhibitor. Specific inhibition of PI3Kγ with INCB098377 may induce anti-tumor activity by reshaping the tumor immune microenvironment. In cell-based assays, INCB098377 has an IC50 of 1.4 nM and is greater than 100-fold selective over other PI3K isoforms. It also shows a favorable PK profile in several animal species. Treatment of M2 polarized macrophages with INCB098377 resulted in changes towards a more pro-inflammatory phenotype. CD163 and CD206 were decreased, whereas HLA-DR and co-stimulatory CD80/86 molecules were increased. MHC-I expression was unchanged, suggesting a role for these macrophages in MHC-II-mediated antigen presentation. Furthermore, INCB098377 treatment reduced macrophage-mediated immunosuppression and restored T cell proliferation in M2 polarized macrophages co-cultured with allogeneic human T cells. In vivo, significant tumor growth inhibition was observed with once-daily dosing of 10 mg/kg INCB098377 in both syngeneic and humanized mouse tumor models without toxicity. Moreover, efficacy was observed in inflamed and non-inflamed tumor models. Consistent with the proposed mechanism of action, INCB098377 inhibited phospho-AKT levels in vivo and in human PBMCs. Treatment with INCB098377 induced pro-inflammatory responses without macrophage depletion suggests that robust tumor microenvironment changes are responsible for observed anti-tumor efficacy. In addition, INCB098377 inhibited neutrophil migration in the Carrageenan-induced paw inflammation model. INCB098377, a potent and selective inhibitor of PI3Kγ, shows effective anti-tumor activity in a variety of mouse and humanized cancer models through the inhibition of immunosuppressive cells trafficking into the tumor, modulation of myeloid cell function, and enhancement of T cell proliferation. Acknowledgments: Diana Alvarez Arias and Stephen Douglass contributed equally to this study. Citation Format: Diana A. Arias, Stephen Douglass, Lisa Truong, Qian Wang, Kathy H. Wang, Gengjie Yang, Michael Hansbury, Sybil O’Connor, Kevin Bowman, Robert Collins, Matthew Stubbs, Leslie Hall, Christina Stevens, Christopher Maddage, Brent Douty, Maryanne Covington, Lynn Leffet, Eddy Yue, Andrew Combs, Sunkyu Kim, Niu Shin, Holly Koblish, Rodrigo Hess. Discovery of INCB098377: a potent inhibitor of phosphoinositide 3-kinase gamma (PI3Kγ). [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 5162.

Abstract 4928: Gedatolisib, a well-tolerated pan-PI3K/mTOR inhibitor, exhibits potent therapeutic effects on endometrial cancer models regardless of their PI3K pathway mutational status

Abstract Background: The PI3K, AKT, and mTOR (PAM) pathway is one of the most commonly activated oncogenic pathways in gynecological cancers. Loss of PTEN function and PIK3CA activating mutations are especially frequent in endometrial cancer (EC). Targeting the PAM pathway, in combination with other cooperative oncogenic pathways (e.g., the estrogen pathway), is a promising EC treatment strategy. Currently, everolimus, an mTORC1 inhibitor, in combination with letrozole, an aromatase inhibitor, is one of the available hormonal therapy options for patients with advanced endometrioid EC. Most PAM inhibitors (PAM-i) selectively spare (or weakly inhibit) one or more key PAM pathway components, which can lead to drug resistance. To minimize drug resistance, a more comprehensive inhibition of the PI3K isoforms and downstream mTOR complexes may be required. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, mTORC1, and mTORC2, can potentially be an effective and well tolerated therapy for patients with EC. Methods: A panel of endometrial, ovarian and cervical cancer cell lines with different PI3K pathway mutational status were assayed for their sensitivity to gedatolisib and other PAM inhibitors (PI3K-α: alpelisib, pan-PI3K: copanlisib; AKT: capivasertib; mTORC1: everolimus; pan-PI3K/mTOR: gedatolisib). Cell viability, cell proliferation, and flow cytometry analytical assays were conducted. Xenograft studies evaluating gedatolisib in EC cell lines were also performed. Results: Gedatolisib strongly inhibited PAM pathway activation, and reduced cell cycle progression and cell viability in the endometrial, ovarian, and cervical cancer cell lines. Compared to the other PAM-i, Gedatolisib exhibited superior anti-proliferative potency and efficacy in almost all the cell lines tested, regardless of the PI3K pathway mutational status or cancer type. In EC xenografts, gedatolisib substantively inhibited tumor growth irrespective of the cell line models’ PI3K pathway mutational status. Conclusions: We demonstrated that, gedatolisib induces superior anti-proliferative activity compared to the other PAM pathway inhibitors, regardless of the PI3K pathway mutational status of the cancer cell lines evaluated. Gedatolisib as a single agent and in combination with other therapies has previously reported promising preliminary clinical efficacy and safety in various solid tumor types, including EC. In summary, nonclinical and preliminary clinical data supports the development of gedatolisib in combination with hormonal therapy for treatment of patients with EC. Citation Format: Stefano Rossetti, Aaron Broege, Ian MacNeil, Ben Rich, Jhomary Molden, Brian Sullivan, Lance Laing. Gedatolisib, a well-tolerated pan-PI3K/mTOR inhibitor, exhibits potent therapeutic effects on endometrial cancer models regardless of their PI3K pathway mutational status. [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 4928.

Abstract 639: Prevention of pancreatitis and subsequent pancreatic cancer and fibrosis development by PI3Kγ inhibition

Abstract Anti-inflammatory, immunosuppressive macrophages play significant roles in promoting both tumorigenesis and fibrosis. Wound-healing type macrophages accumulate in sites of tissue injury, such as the cirrhotic liver, where they can induce oxidative damage and secrete immunosuppressive and pro-fibrotic factors that lead to oncogenesis. These innate immune cells are also major cellular producers of PDGFB and TGF-β1, which stimulate fibroblasts to secrete extracellular matrix proteins, such as collagen, and which also promote metastatic progression of neoplastic diseases. Chronic liver and pancreatic inflammation are significant risk factors for the development of hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC), respectively. We have determined that the myeloid cell specific PI3K isoform PI3Kγ promotes the accumulation of myeloid cells and their immune suppressive polarization within the tumor microenvironment (TME), and that this kinase thereby promotes tumor growth in animal models of cancer, including PDAC. Genetic and pharmacological inhibition of PI3Kγ suppresses tumor growth and reduces fibrosis that is associated with PDAC and other tumors. Targeting immune suppressive myeloid cells represent an emerging approach within the field of cancer immunotherapy and fibrosis treatment; indeed, inhibition of PI3Kγ by the therapeutic eganelisib has shown beneficial effects, including increased overall and progression-free survival in clinical trials for melanoma, head and neck carcinoma, urothelial carcinoma and triple negative breast carcinoma. In this project, we are exploringWe examined the effect of whether PI3Kγ inhibition can in cancer prevention using mouse models of prevent cancer development by suppressing damaging chronic hepatic and pancreatic inflammation, associated fibrosis, and progression towards tumorigenesis using models of HCC and PDAC. We found that PI3Kγ inhibition prevented prevents acute and chronic inflammation and that promotes normal tissue loss and development of fibrosis in mouse models of chronic pancreatitis and liver inflammation. Studies are underway to determine iOur studies indicate thatf PI3Kγ inhibition can also prevent the development of pancreatic and liver carcinoma that results fromin response to chronic inflammation and fibrosis. Our These studies thus far suggest the a potential role of for PI3Kγ inhibition in the prevention of cancer development by preventing the chronic inflammation that leads towards fibrosis and carcinogenesis. The results of these studies could be applied to the future design of clinical trials with using clinical PI3Kγ inhibitors. Citation Format: Anghesom Ghebremedhin, Judith Varner, Marc Paradise. Prevention of pancreatitis and subsequent pancreatic cancer and fibrosis development by PI3Kγ inhibition [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 639.

Abstract 1176: Quantitative integration of PIK3CA hotspots and paralogy identifies occult oncogenic mutants in PIK3CD

Abstract The majority of somatic variants identified by tumor genomic profiling are low frequency alterations of unknown biologic and clinical significance. To facilitate the systematic identification of likely oncogenic versus benign somatic mutations, there has been a growing interest in harnessing additional computationally amenable metrics such as sequence paralogy. Here, we developed a novel paralogy-directed computational methodology called Enriched Paralogy to identify Occult Cancer Hotspots (EPOCH), which calculates the significance of mutational recurrence at aligned positions in evolutionarily related genes to prioritize likely oncogenic mutations for in depth functional characterization. We validated the utility of EPOCH using sequencing data from a cohort of 52,676 tumor/normal pairs, with a focus on identifying likely oncogenic mutations in the well characterized PIK3CA (p110α) class 1a PI3K isoform and the lesser characterized PIK3CD (p110δ) isoform. p110δ mutants nominated as likely oncogenic by EPOCH induced AKT pathway activation, enhanced anchorage-independent colony formation, and demonstrated allele-specific dependencies on p85 regulatory protein binding. The potency of PI3K inhibitors against p110δ mutants was at best equal to or less than wildtype p110δ. In sum, we present the novel algorithm EPOCH that integrates paralogy to extend the utility of current recurrence-based precision oncology predictions and to automate the functional interpretation of less common mutations. Our work identifies novel oncogenic and targetable p110δ mutants and supports the inclusion of PIK3CD in targeted sequencing panels despite the relative rarity of mutations in this gene. The therapeutic studies herein also provide the rationale for the development and clinical testing of isoform- or mutant-specific p110δ inhibitors for patients with rare, yet druggable, oncogenic p110δ mutations. Citation Format: Arijh Elzein, Alexander N. Gorelick, Ino de Bruijn, Barry S. Taylor, Aphrothiti J. Hanrahan, David B. Solit. Quantitative integration of PIK3CA hotspots and paralogy identifies occult oncogenic mutants in PIK3CD [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 1176.

Design, synthesis and bioactivity evaluation of a series of quinazolinone derivatives as potent PI3Kγ antagonist

Targeting PI3Kγ would be a useful strategy for treating inflammatory and cancer diseases. However, the development of selective inhibitors of PI3Kγ is very challenging due to the high structural and sequence homology with other PI3K isoforms. A series of quinazolinone derivatives were designed, synthesized and biologically evaluated as PI3Kγ-selective inhibitors. Among all the 28 compounds, compound 9b was found to be the most potent selective inhibitor with IC50 values of 13.11 nM against PI3Kγ kinase. Additionally, compound 9b could generate toxicity on leukemia cells in a panel of 12 different of cancer cell lines with the IC50 value of 2.41 ± 0.11 μM on Jurkat cell. Preliminary mechanism studies indicated that compound 9b through inhibit the activity of PI3K-AKT in human and murine leukemia cells, and activated phosphorylated p38 and phosphorylated ERK presented potent antiproliferative activity, which provided a potent small molecule for further cancer therapy.

Roles of PI3Kγ and PI3Kδ in mantle cell lymphoma proliferation and migration contributing to efficacy of the PI3Kγ/δ inhibitor duvelisib

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma that is incurable with existing therapies, and therefore presents a significant unmet clinical need. The ability of this disease to overcome therapy, including those that target the B cell receptor pathway which has a pathogenic role in MCL, highlights the need to develop new treatment strategies. Herein, we demonstrate that a distinguishing feature of lymph node resident MCL cells is the expression of phosphatidylinositol 3-kinase γ (PI3Kγ), a PI3K isoform that is not highly expressed in other B cells or B-cell malignancies. By exploring the role of PI3K in MCL using different PI3K isoform inhibitors, we provide evidence that duvelisib, a dual PI3Kδ/γ inhibitor, has a greater effect than PI3Kδ- and PI3Kγ-selective inhibitors in blocking the proliferation of primary MCL cells and MCL cell lines, and in inhibiting tumour growth in a mouse xenograft model. In addition, we demonstrated that PI3Kδ/γ signalling is critical for migration of primary MCL cells and cell lines. Our data indicates that aberrant expression of PI3Kγ is a critical feature of MCL pathogenesis. Thus, we suggest that the dual PI3Kδ/γ duvelisib would be effective for the treatment of mantle cell lymphoma.

Abstract OT3-08-01: A phase 1 trial of LOXO-783, a potent, highly mutant-selective, brain-penetrant allosteric PI3Kα H1047R inhibitor in PIK3CA H1047R-mutant advanced breast cancer (aBC) and other solid tumors (PIKASSO-01, trial in progress)

Abstract Background: Phosphoinositide 3-kinase alpha (PI3Kα) H1047R mutations are activating oncogenic events that occur in ~15% of breast cancers (BC). Available PI3Kα inhibitors target both wild-type (WT) and mutant PI3Kα and, as a result, their efficacy may be limited by on-target WT PI3Kα-mediated toxicities, including hyperglycemia, skin rash, and diarrhea. LOXO-783 is an oral, potent and highly mutant-selective, brain-penetrant allosteric PI3Kα H1047R inhibitor. Preclinically, LOXO-783 is highly selective for PI3Kα H1047R over WT PI3Kα and other PI3K isoforms and induces single-agent tumor regressions in ER+, HER2- PI3Kα H1047R-mutant BC models without causing hyperglycemia or increases in plasma insulin/C-peptide. LOXO-783 also demonstrates brain penetration in vivo with dose-dependent tumor growth inhibition in brain metastasis models. This trial investigates LOXO-783 alone and in combination with other anticancer therapies in patients with PIK3CA H1047R-mutant aBC and other solid tumors. Trial Design: This global, first-in-human phase 1a/b study of LOXO-783 includes dose escalation of LOXO-783 monotherapy followed by dose expansion of LOXO-783 alone and in combination with other anticancer therapies (Table). Monotherapy dose escalation will be evaluated using a modified toxicity probability interval-2 (mTPI-2) design. In dose expansion (Parts A-E), each combination cohort will include a safety lead-in of 3-6 patients. Men and premenopausal women with ER+ aBC must receive concomitant treatment with a GnRH agonist. An optional pharmacodynamic (PD) biomarker sub-study will be conducted at select dose levels during dose escalation in patients with ER+, HER2- aBC with soft tissue disease amenable to safe repeat tumor biopsies. Eligibility criteria: Eligible patients must have PIK3CA H1047R-mutant aBC or other solid tumors with measurable disease or non-measurable bone-only disease (aBC patients only). In dose escalation, patients may have received up to 5 prior regimens. In dose expansion, prior therapy requirements are outlined in the Table below. Key exclusion criteria include prior inhibitor(s) of PI3K/AKT/mTOR (except in dose escalation or in select patients with prior intolerance of these inhibitors), colorectal cancer, endometrial cancer with concurrent PI3K/AKT/mTOR and/or RAS/RAF alterations and diabetes mellitus (DM) requiring medication (except in Part C). Study objectives: Recommended phase 2 dose (RP2D) determination; safety and tolerability assessment, PK and PD evaluation, objective response rate and clinical benefit rate assessment per RECIST v1.1. Recruitment is ongoing (PIKASSO-01, NCT05307705). Table. Dose Expansion (Phase 1b) Citation Format: Dejan Juric, Komal Jhaveri, Muralidhar Beeram, Erika Hamilton, Vincent Chau, Matthew P. Hanley, Shiyao Liu, Lillian M. Smyth, Funda Meric-Bernstam. A phase 1 trial of LOXO-783, a potent, highly mutant-selective, brain-penetrant allosteric PI3Kα H1047R inhibitor in PIK3CA H1047R-mutant advanced breast cancer (aBC) and other solid tumors (PIKASSO-01, trial in progress) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-08-01.

Abstract P4-08-02: LOXO-783: A potent, highly mutant selective and brain-penetrant allosteric PI3Kα H1047R inhibitor in combination with standard of care (SOC) treatments in preclinical PI3Kα H1047R-mutant breast cancer models

Abstract Background Phosphoinositide 3-kinase alpha (PI3Kα) H1047R mutations are activating oncogenic events that occur in ~15% of breast cancers (BC). Early generation PI3Kα inhibitors target both wild-type (WT) and mutant PI3Kα and, as a result, their efficacy may be limited by on-target WT PI3Kα-mediated toxicities, including hyperglycemia, skin rash, and diarrhea. LOXO-783 is an oral, potent and highly mutant-selective, brain-penetrant allosteric PI3Kα H1047R inhibitor that is currently in phase 1 testing. Preclinically, LOXO-783 as a single agent is highly selective for PI3Kα H1047R over WT PI3Kα and other PI3K isoforms, and induces single-agent tumor regressions in ER+, HER2- PI3Kα H1047R-mutant breast cancer models without causing hyperglycemia or increases in plasma insulin/C-peptide. LOXO-783 also demonstrates brain penetration in vivo with dose-dependent tumor growth inhibition in brain metastasis models. Here we report the efficacy of LOXO-783 with SOC treatments in preclinical breast cancer models. Methods Cell proliferation assays and in vivo studies to evaluate combination effects were performed in various PI3Ka H1047R mutant HR+, HER2- and triple negative breast cancer models. For each combination in vitro, a combination index (CI) based on the Loewe Additivity Method was calculated (CI>2 antagonism, 0.5>CI< 2 additivity, CI< 0.5 synergy). For the in vivo studies, the Bliss Independence Method was used to evaluate the statistical significance of the combination effects. Results Combining LOXO-783 with either fulvestrant (FUL; CI at 50% inhibition = 0.28) or imlunestrant (CI at 50% inhibition = 0.43) showed increased efficacy in cell proliferation assays using the HR+, HER2-, PI3Kα H1047R-mutant T47D model. LOXO-783 also demonstrated an additive effect in combination with these endocrine therapies in vivo. Similar results were observed in a T47D model engineered to express ESR1 D538G, as well as in an HR+, HER2- PI3Kα double in-cis mutant model (H1047R/D350G) also harboring ESR1 D538G and derived from a patient who had progressed on prior letrozole plus taselisib. Moreover, LOXO-783 plus abemaciclib demonstrated an additive effect in vitro (CI at 50% inhibition = 0.61), and in T47D xenograft and PDX models in vivo. Combinations of LOXO-783 with abemaciclib plus imlunestrant resulted in a mean tumor regression of –48.1%; LOXO-783 with abemaciclib plus FUL showed mean tumor regression of –43.9% in T47D xenografts. Similar efficacy was not observed in the absence of LOXO-783 (mean tumor regression was –7.3% with abemaciclib plus imlunestrant, and 3.2% with abemaciclib plus FUL). We observed comparable results in PDX models. These data collectively demonstrate the additive effect of LOXO-783 with SOC treatments. Extending these studies to additional treatment settings, LOXO-783 was similarly efficacious as a single agent in abemaciclib-resistant and abemaciclib/FUL double-resistant models, and was additive in combination with paclitaxel in a triple negative breast cancer model in vitro and in vivo. Conclusions LOXO-783 shows additive effects when combined with SOC in breast cancers harboring the PI3Kα H1047R-mutation (as single or double in-cis mutations) in both HR+ and triple negative settings. LOXO-783 is also efficacious in ESR1 mutant as well as in abemaciclib and abemaciclib/FUL double-resistant models. A phase 1 trial of LOXO-783 alone or in combination with anticancer therapies is ongoing (PIKASSO-01; NCT05307705). Citation Format: Loredana Puca, Michele S. Dowless, Carmen M. Perez-Ferreiro, Maria Jesus Ortiz-Ruiz, Gregory P. Donoho, Andrew Capen, Lysiane Huber, Sarah M. Bogner, Dongling Fei, Jason R. Manro, Chun Ping Yu, Wei Guo Xu, Rui Wang, Shuang Chen, Mark A. Hicks, Parisa Zolfaghari, Andrew Faber, Raymond Gilmour, Monica D. Ramstetter, Matthew T. Chang, Maria Jose Lallena, Xuequian Gong, David M. Hyman, Lillian M. Smyth, Barbara J. Brandhuber, Barry S. Taylor, Anke Klippel. LOXO-783: A potent, highly mutant selective and brain-penetrant allosteric PI3Kα H1047R inhibitor in combination with standard of care (SOC) treatments in preclinical PI3Kα H1047R-mutant breast cancer models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-02.

Abstract OT3-22-01: First-in-human global multi-center study of RLY-2608, a pan mutant and isoform selective PI3Kα inhibitor, as a single agent in advanced solid tumor patients and in combination with fulvestrant in patients with advanced breast cancer

Abstract Background: Targeting constitutively active mutant kinases with selective small molecule inhibitors is a key therapeutic pillar of precision oncology. Phosphatidylinositol-4,5bisphosphate-3 kinase, catalytic subunit alpha (PIK3CA) mutations leading to oncogenic activation of PI3Kα represent the largest opportunity for this approach in solid tumors. However, there is no selective inhibitor that targets mutant PI3Kα in the clinic. Toxicity related to non-selective inhibition of WT PI3Kα (hyperglycemia) and other PI3K isoforms limits the tolerability, dosing and efficacy of the orthosteric inhibitor, alpelisib, the only approved solid tumor PI3K inhibitor. RLY-2608, a novel oral allosteric PI3Kα inhibitor, is uniquely designed to overcome these limitations via mutant- and isoform-selective PI3Kα inhibition for greater target coverage, improved tolerability and antitumor activity. We initiated a first-in-human (FIH), study to evaluate the clinical activity of RLY-2608 as a single agent in advanced solid tumor patients (pts) with PI3KCA mutations and in combination with fulvestrant in pts with PIK3CA mutant, HR+, HER2- metastatic breast cancer (MBC). Methods: This is a global, multi-center, dose escalation/expansion study (NCT05216432) of RLY2608 as a single agent in adults who have advanced solid tumors and are refractory, intolerant, or declined standard therapy and RLY-2608 in combination with fulvestrant in previously treated pts with HR+/HER2- MBC. Eligibility criteria include presence of PI3KCA mutation (blood or tumor) per local assessment, ECOG performance status 0-1, measurable or evaluable disease per RECIST 1.1 and no prior PI3K inhibitor (except combination group 2). RLY-2608 is administered on a continuous schedule with 4-week cycles. Adverse events (AEs) per CTCAE v5, PK, biomarkers (mutant ctDNAs and insulin pathway markers) and anti-tumor activity are assessed serially. Dose escalation employs a Bayesian Optimal Interval design to identify MTD and RP2D. Following dose escalation, pts will be treated with RLY-2608 at the MTD/RP2D in a monotherapy dose expansion with 5 groups (N=75, 15 each): 1. Clear cell ovarian carcinoma 2. Head and neck squamous cell carcinoma 3. Cervical cancer 4. Other solid tumors 5. PI3KCA double mutations. In addition, two expansion cohorts will enroll patients with HR+/HER2- MBC treated with RLY-2608 and fulvestrant combination (N = 30, 15 each): 1. No prior PI3K therapy 2. Intolerant to PI3K inhibitors. The primary endpoints are MTD/RP2D and AE profile for single agent and combination; key secondary endpoints are PI3KCA genotype in blood and tumor, PK, biomarkers, and overall response rate. US enrollment began December 2021 and ex-USA startup is under way. Citation Format: Andreas Varkaris, Erika Hamilton, Jason Henry, Alexander I. Spira, Alison M. Schram, Julia E. McGuinness, Gege Tan, Xiaoyan Li, Tamieka Hunter, Ramin Samadani, Alison Timm, Djuro Karanovic, Vivek Subbiah, Cesar A. Perez. First-in-human global multi-center study of RLY-2608, a pan mutant and isoform selective PI3Kα inhibitor, as a single agent in advanced solid tumor patients and in combination with fulvestrant in patients with advanced breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-22-01.

Therapeutic effect of gedatolisib, a pan-PI3K/mTOR inhibitor, on prostate cancer models with PI3K or PTEN mutational status.

149 Background: Metastatic castration-resistant prostate cancer (mCRPrC) is characterized by a loss of androgen receptor (AR) sensitivity and oncogenic activation of the PI3K, AKT, and mTOR (PAM) pathway. Loss of the PI3K regulator, PTEN, is frequent during prostate cancer (PrC) initiation, progression, and therapeutic resistance. Co-targeting PAM/AR pathways is a promising PrC treatment strategy but hampered by complex PAM-AR pathway crosstalk driving either reciprocal negative feedback inhibition or feedback relief. Most PAM inhibitors (PAM-i) selectively spare (or weakly inhibit) one or more key PAM pathway components, potentiating drug resistance depending on the PAM mutation status of patients. AKT-i demonstrated limited efficacy in PTEN-deficient PrC and even less efficacy in PTEN-wild type (wt) in recent clinical studies, suggesting that more comprehensive inhibition of the PI3K isoforms and mTOR may be required. We posited that gedatolisib, a well-tolerated and potent inhibitor of all Class I PI3K isoforms, mTORC1, and mTORC2, would be effective in both PTEN-wt and PTEN-deficient PrC. Methods: A panel of PrC cell lines with different PI3K or PTEN status ( wt: 22RV1, MDA-PCa-2b, DU145; null: LNCaP, PC3, C4-2) were assayed under different conditions for their sensitivity to gedatolisib and other PAM-I ( PI3K: alpelisib, copanlisib; AKT: capivasertib, ipatasertib; mTOR: everolimus; pan-PI3K/mTOR: samotolisib, gedatolisib). A combination of cell viability, growth rate, cytotoxicity and phospho-FACS analytical assays were used. Xenograft studies evaluating gedatolisib with a subset of these models were also performed in castrated mice. Results: We confirmed that AKT-i is more effective in PTEN-null than PTEN-wt PrC. In contrast, the two pan-PI3K/mTOR-i evaluated were effective independent of PI3K or PTEN status. Gedatolisib exhibited superior potency and efficacy compared to each of the other PAM-i for cell proliferation, death, or PAM pathway activation. In vivo, gedatolisib substantively inhibited tumor growth regardless of PTEN or PI3K status. Conclusions: We demonstrated that gedatolisib exerts superior activity regardless of PI3K or PTEN status in all PrC cell lines evaluated compared to the other PAM pathway inhibitors evaluated. Our results indicate potent and simultaneous blockade of all Class I PI3K isoforms, mTORC1, and mTORC2 could circumvent PTEN dependent resistance. Gedatolisib as a single agent and in combination with other therapies reported promising preliminary efficacy and safety in various solid tumor types. In light of the PrC findings reported here, development of gedatolisib may be warranted in PrC patients regardless of their PTEN or PI3K status.

Phosphoinositol 3-kinase-driven NET formation involves different isoforms and signaling partners depending on the stimulus.

Neutrophil extracellular traps (NETs) serve to immobilize and kill pathogens, but also can contribute to the progression of several inflammatory and auto-immune diseases, as well as cancer. Whence the importance of elucidating the mechanisms underlying NET formation. In this regard, the PI3K signaling pathway has been shown to be crucial; yet little is known about which of its components are involved. Here, we identified the PI3K isoforms and associated signaling partners that are mobilized in response to different classes of physiological NET inducers (inflammatory cytokines, growth factors, chemoattractants). NET generation was assessed by microscopy and signalling molecule activation by immunoblot using phospho-antibodies. Across the various stimuli, PI3Kα and PI3Kγ isoforms clearly contributed to NET induction, while the participation of other isoforms was stimulus-dependent. Some PI3K isoforms were also found to signal through Akt, the canonical downstream effector of PI3K, while others did not. Downstream of PI3K, mTOR and PLCγ2 were used by all stimuli to control NET generation. Conversely, the involvement of other kinases depended on the stimulus - both TNFα and GM-CSF relied on PDK1 and Akt; and both TNFα and fMLP additionally used S6K. We further established that all PI3K isoforms and downstream effectors act belatedly in NET generation, as reported previously for PI3K. Finally, we revisited the PI3K-PDK1-Akt signaling hierarchy in human neutrophils and again found stimulus-dependent differences. Our data uncover unsuspected complexity and redundancy in the signaling machinery controlling NET formation through the all-important PI3K pathway. Conserved signaling molecules represent therapeutic targets for pathologies involving NETs and in this regard, the existence of drugs currently used in the clinic or undergoing clinical trials (which target PI3K isoforms, mTOR or Akt), underscores the translational potential of our findings.