Targeting PI3Kγ Research Articles

Abstract 170: Targeting of macrophage PI3Kγin prostate cancer using Eganelisib (IPI-549) reprograms immune-suppressive infiltrating macrophages to enhance anti-tumor immune responses and promote immunologically mediated tumor growth

Abstract Background: Prostate cancer (PCa) is traditionally considered an immunologically 'cold’ indication, as evidenced in part by significant infiltration of immunosuppressive tumor associated macrophages (TAMs) and few activated effector CD8+ T cells. Attempts to (re)invigorate an anti-prostate cancer immune response with immune checkpoint blockade (ICB) such as anti-CTLA-4 and anti-PD-1 have been largely ineffective clinically, despite promising results in other cancers. This is likely due to a multitude of immunosuppressive cell types and secreted factors within the tumor microenvironment (TME) that diminish T cell activation and trafficking. Reducing the immunosuppressive burden intratumorally by targeting TAMs is a clinically attractive approach and offers opportunity to sensitize PCa to immune checkpoint blockade. While PI3K signaling is ubiquitous in most cell types, specific catalytic isoforms and regulatory elements are differentially expressed depending on cell type, with PI3Kγ being significantly enriched in myeloid cells and therefore offering a unique therapeutic opportunity for specific targeting. Methods: Utilizing novel syngeneic prostate cancer tumors, PI3Kγ deletion and antagonism using Eganelisib (a small molecule inhibitor for PI3Kγ), we tested our hypothesis that targeting PI3Kγ signaling in PCa TAMs would lead to tumor growth inhibition and anti-tumor immune responses. Results: We show here that PCa tumor cells do not express the PI3Kγ isoform and, therapeutic inhibition of PI3Kγ with Eganelisib reduces the TAM suppressive phenotype. We also show PI3Kγ signaling is essential to monocyte anti-apoptotic signaling, and inhibition of PI3Kγ can enhance anti-tumor immune responses in preclinical models of PCa. Specifically, inhibition of PI3Kγ alone is sufficient to enhance anti-tumor immune responses in the less aggressive B6CaP PCa model, but insufficient in the more aggressive RM-1 PCa model. However, treatment of RM-1 tumors with Eganelisib produces improved tumor control when combined with anti-PD-1 therapy, relative to Eganelisib or anti-PD1 as a monotherapy. Conclusions: Altogether, these data highlight the integral role of PI3Kγ signaling in TAMs and the potential for therapeutic targeting with Eganelisib to sensitize PCa to ICB. Citation Format: Kenneth M. Adusei, Thomas R. Nirschl, Alex J. Lee, Fan Shen, Xiaoxu Wang, Monali Praharaj, Jade Alvarez, Iyana Gross, Kara A. Lombardo, Tamara L. Lotan, Judith A. Varner, Jelani C. Zarif. Targeting of macrophage PI3Kγin prostate cancer using Eganelisib (IPI-549) reprograms immune-suppressive infiltrating macrophages to enhance anti-tumor immune responses and promote immunologically mediated tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 170.

Defining a Targetable Leukemia Intrinsic Dependency on Noncanonical PI3Kgamma Signaling

There are overlapping clinical and pathologic characteristics between poor prognosis subsets of acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN), but common dependencies are ill-defined. We hypothesized that identifying novel shared targets and biomarkers could elucidate biology and nominate new therapies for both malignancies. We performed genome-wide CRISPR interference in BPDCN cells and identified 327 dependency genes, which were then analyzed informatically and functionally in AML, ALL, and BPDCN cells and patient-derived xenografts (PDXs). We defined a leukemia subset characterized by PIK3R5 activation and sensitivity to phosphoinositide 3-kinase gamma (PI3Kγ) inhibition via a noncanonical downstream pathway. The PI3Kγ complex includes an enzymatic subunit p110γ encoded by PIK3CG and a regulatory subunit p101 encoded by PIK3R5. Intriguingly, despite elevation of both p110γ and p101 proteins in this leukemia subset, only the PIK3R5 mRNA is overexpressed. To investigate this disconnection, we measured protein half-life and found that p110γ stability is positively regulated by PIK3R5/p101, associated with reduced p110γ ubiquitination. This suggested that increased expression of PIK3R5/p101 leads to posttranslational p110γ elevation by protecting it from ubiquitin proteasome-mediated degradation. To identify the upstream activator of PIK3R5, we analyzed RNA-seq data in >200 leukemia PDXs and found that AMLs with activated PIK3R5 are enriched for an innate immune response signature (IIRS). Supporting this model and indicating potential clinical relevance, analysis of TCGA AML patients revealed that the IIRS, which includes PIK3R5, was associated with M4/M5 monocytic AML and poor prognosis, independent of age, cytogenetics, or somatic mutations. Furthermore, in cells with low baseline expression, PIK3R5 was induced by the Toll-like receptor agonist resiquimod, showing that PIK3R5 is activated by inflammatory signaling and consistent with a monocytic/dendritic lineage association. By integrated analysis of ATAC-seq and ChIP-seq in PIK3R5 high vs low leukemias, as well as gene dependency scores, we identified the transcription factor PU.1 as responsible for PIK3R5 activation. Treatment with resiquimod enhanced the interaction between PU.1 and the PIK3R5 promoter, while depletion of PU.1 nullified the ability of resiquimod to activate PIK3R5. To elucidate molecular events downstream of p101/PI3Kγ, we studied consequences of pathway inactivation using PIK3R5 interference or the PI3Kγ-selective inhibitor eganelisib. Surprisingly, there was no effect of PI3Kγ inactivation on AKT phosphorylation or canonical PI3K-AKT-mTOR mRNA targets. Therefore, we performed phosphoproteomics and found that PAK1 S144, a phosphorylation site not previously associated with PI3K signaling, was suppressed by PIK3R5/PI3Kγ inhibition ( panel a). Selective inhibitors of PAK1, but not AKT, replicated the effect of eganelisib. Furthermore, introduction of constitutively active PAK1, but not inactive PAK1, eliminated sensitivity of leukemias to eganelisib, supporting the functional relationship between PAK1 phosphorylation and PI3Kγ dependency. PI3Kγ inhibitors are being tested in solid tumor trials to repolarize macrophages and potentiate immune checkpoint blockade, but their cancer cell-intrinsic role has not been explored. To assess PI3Kγ inhibition in vivo, we first performed intradermal xenografting assays using BPDCN derived from a patient's skin tumor. Eganelisib achieved comparable efficacy to cytarabine chemotherapy and the combination of eganelisib and cytarabine exerted significant synergy (combination index=0.63, p<0.0001). Next, we measured the effect of PI3Kγ inhibition on survival in disseminated PDX models. As predicted, single agent eganelisib significantly prolonged survival in AML, ALL, and BPDCN with high PIK3R5, while having no benefit in cases with low PIK3R5. To our surprise, the combination of eganelisib/cytarabine synergistically increased survival in all cases, regardless of baseline PIK3R5 ( panel b). We conclude that inflammatory signaling-activated PIK3R5 is a biomarker for sensitivity to PI3Kγ inhibition via an unappreciated PI3Kγ-PAK1 axis. Synergy with cytarabine unmasked eganelisib sensitivity in additional leukemias, expanding the therapeutic potential of targeting PI3Kγ.

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.

Attenuation of Ventilation-Enhanced Epithelial–Mesenchymal Transition through the Phosphoinositide 3-Kinase-γ in a Murine Bleomycin-Induced Acute Lung Injury Model

Mechanical ventilation (MV) used in patients with acute lung injury (ALI) induces lung inflammation and causes fibroblast proliferation and excessive collagen deposition-a process termed epithelial-mesenchymal transition (EMT). Phosphoinositide 3-kinase-γ (PI3K-γ) is crucial in modulating EMT during the reparative phase of ALI; however, the mechanisms regulating the interactions among MV, EMT, and PI3K-γ remain unclear. We hypothesized that MV with or without bleomycin treatment would increase EMT through the PI3K-γ pathway. C57BL/6 mice, either wild-type or PI3K-γ-deficient, were exposed to 6 or 30 mL/kg MV for 5 h after receiving 5 mg/kg AS605240 intraperitoneally 5 days after bleomycin administration. We found that, after bleomycin exposure in wild-type mice, high-tidal-volume MV induced substantial increases in inflammatory cytokine production, oxidative loads, Masson's trichrome staining level, positive staining of α-smooth muscle actin, PI3K-γ expression, and bronchial epithelial apoptosis (p < 0.05). Decreased respiratory function, antioxidants, and staining of the epithelial marker Zonula occludens-1 were also observed (p < 0.05). MV-augmented bleomycin-induced pulmonary fibrogenesis and epithelial apoptosis were attenuated in PI3K-γ-deficient mice, and we found pharmacological inhibition of PI3K-γ activity through AS605240 (p < 0.05). Our data suggest that MV augmented EMT after bleomycin-induced ALI, partially through the PI3K-γ pathway. Therapy targeting PI3K-γ may ameliorate MV-associated EMT.

Baicalein Potentiated M1 Macrophage Polarization in Cancer Through Targeting PI3Kγ/ NF-κB Signaling

Baicalein is one of the bioactive compounds extracted from Scutellaria baicalensis. Recent studies indicated the antitumor effects of baicalein, however, the underlying mechanisms are needed to be further determined. In this study, we found that baicalein could inhibit the tumor growth in mice models of breast cancer and melanoma and worked as an immunomodulator to promote the infiltration of tumor-associated macrophages (TAMs) and skew the TAMs towards the M1-like phenotype. Baicalein also induced M1-like phenotype polarization in THP-1-derived macrophages. Meanwhile, the expression of pro-inflammatory factors associated with M1 macrophages, including TNF-α, IL-1β, CXCL9 and CXCL10, were increased after baicalein treatment. Mechanistically, the RNA-seq data suggested that baicalein potentiated the M1 macrophage polarization via the NF-κB/TNF-α signaling pathway. ELISA and confocal microscopy assay confirmed that baicalein significantly induced the production of TNF-α and the activation of NF-κB, while TNF-α neutralization inhibited baicalein-induced macrophage polarization toward M1, and NF-κB P65 knock-down suppressed baicalein-induced TNF-α production in THP-1-derived macrophages. Phosphoinositide 3-kinase (PI3k) γ has been reported as a key molecule in macrophage polarization, and inhibition of PI3Kγ activates the NF-κB-related inflammatory signals. Our pharmacological network analysis predicted that PI3Kγ might be one of the major targets of baicalein. The results from the docking program and surface plasmon resonance (SPR) confirmed that baicalein displayed good binding activity to PI3Kγ. We further found that baicalein not only exhibited a direct inhibitory effect on the protein kinase activity of PI3Kγ, but also reduced the mRNA and protein expression of PI3Kγ, indicating that baicalein might be a novel PI3Kγ inhibitor. In summary, baicalein mediated the TAMs skewing to M1-TAMs, and then retarded tumor growth. These effects, at least in part, were linked to the PI3Kγ/NF-κB signaling.

Abstract IA26: Reprogramming tumor-associated macrophages by targeting PI3K-gamma with IPI-549

Abstract The PI3 kinases (PI3K) belong to a family of signal-transducing enzymes that mediate key cellular functions in cancer and immunity. The PI3K-gamma (γ) isoform is highly expressed in myeloid cells/macrophages and plays an important role in M2 macrophage polarization (Kaneda, Nature 2016). PI3K-γ knockout mice also demonstrated the role for PI3K-γ in tumor growth and immune tolerance (Gunderson, Cancer Discovery 2015; Kaneda, 2016). IPI-549 is an oral, potent, and selective inhibitor of PI3K-γ that shows single-agent antitumor activity in multiple murine tumor models (Kaneda, 2016) and enhanced antitumor activity and improved survival when combined with immune-checkpoint blockade (ICB) (De Henau, Nature 2016). This antitumor activity is dependent on the presence of both immune-suppressive tumor-associated CD11b+ myeloid cells and CD8+ cytotoxic T cells. IPI-549 can reduce the suppression of T-cell activity by both murine and human myeloid-derived suppressor cells in vitro and in vivo. IPI 549 treatment of tumor-bearing mice leads to a shift in tumor-associated myeloid cells from an immunosuppressive M2 phenotype to a proinflammatory M1 phenotype and an increased frequency of circulating tumor-specific T cells and tumor-infiltrating CD8+ T cells. In addition, preclinical tumor model systems show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells (De Henau, Nature 2016). Treatment of ICB-resistant 4T1 and B16GMCSF tumor-bearing mice with IPI-549 and anti-PD1 for 14 days led to a significant antitumor activity and an increased CD8+/T-reg cell ratio, confirming that selective pharmacologic targeting PI3K-γ by IPI-549 restores sensitivity to ICB. We are conducting a Ph 1/1b study IPI-549-01 (ClinicalTrials.gov NCT02637531) evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunomodulatory activity of IPI-549 to determine its recommended phase 2 dose and preliminary clinical activity, as both a monotherapy and in combination with nivolumab, in patients with advanced solid tumors. Citation Format: Jeffery L. Kutok. Reprogramming tumor-associated macrophages by targeting PI3K-gamma with IPI-549 [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA26.

Lipid accumulation in macrophages confers protumorigenic polarization and immunity in gastric cancer.

Heterotypic interactions between tumor cells and macrophages can enable tumor progression and hold potential for the development of therapeutic interventions. However, the communication between tumors and macrophages and its mechanism are poorly understood. Here, we find that tumor‐associated macrophages (TAM) from tumor‐bearing mice have high amounts of lipid as compared to macrophages from tumor‐free mice. TAM also present high lipid content in clinical human gastric cancer patients. Functionally, TAM with high lipid levels are characterized by polarized M2‐like profiling, and exhibit decreased phagocytic potency and upregulated programmed death ligand 1 (PD‐L1) expression, blocking anti–tumor T cell responses to support their immunosuppressive function. Mechanistically, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identifies the specific PI3K pathway enriched within lipid‐laid TAM. Lipid accumulation in TAM is mainly caused by increased uptake of extracellular lipids from tumor cells, which leads to the upregulated expression of gamma isoform of phosphoinositide 3‐kinase (PI3K‐γ) polarizing TAM to M2‐like profiling. Correspondingly, a preclinical gastric cancer model is used to show pharmacological targeting of PI3K‐γ in high‐lipid TAM with a selective inhibitor, IPI549. IPI549 restores the functional activity of macrophages and substantially enhances the phagocytosis activity and promotes cytotoxic‐T‐cell‐mediated tumor regression. Collectively, this symbiotic tumor‐macrophage interplay provides a potential therapeutic target for gastric cancer patients through targeting PI3K‐γ in lipid‐laden TAM.

Inhibition of Phosphatidylinositol 3-Kinase γ by IPI-549 Attenuates Abdominal Aortic Aneurysm Formation in Mice

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway plays a pivotal role in abdominal aortic aneurysm (AAA). However, systemic inhibition of this pathway causes serious side effects, thus limiting the clinical use of pan-PI3K inhibitors. In this study, it was hypothesised that the γ subunit of PI3K plays an important role in the PI3K/AKT signalling pathway during AAA, and that specifically targeting PI3Kγ may prevent this process. Aortic specimens were collected from AAA patients and organ donors. Furthermore, a classical AAA model in male C57BL/6 mice was created via an intra-aortic porcine pancreatic elastase (PPE) infusion and aortas were collected. A specific PI3Kγ inhibitor, IPI-549, was administered to mice orally. The protein expression level of PI3Kγ was examined by immunohistochemistry and western blotting. The aortic leukocytes were examined by immunohistochemistry and flow cytometry. PI3Kγ protein levels were elevated in the aortas of AAA patients and PPE infused mice. Three color immunofluorescence staining revealed the predominant area of PI3Kγ by T cells and macrophages in aneurysmal aortas. IPI-549 treatment significantly prevented AAA formation in mice. Aortic macrophages, T cells and neo-angiogenesis were significantly reduced in mice treated with IPI-549 compared with vehicle treated PPE infused mice. Flow cytometry analysis also revealed that CD45+ leukocytes and CD45+ F4/80+ macrophages in IPI-549 treated mouse aortas decreased dramatically. Additionally, IPI-549 treatment inhibited the phosphorylation of AKT in experimental aneurysmal lesions. Specific inhibition of PI3Kγ limits AAA formation. Targeting PI3Kγ prevents inflammatory cell infiltration through inhibition of AKT phosphorylation in AAA.

In-silico & In-vitro Identification of Structure-Activity Relationship Pattern of Serpentine & Gallic Acid Targeting PI3Kγ as Potential Anticancer Target.

Natural products showed anticancer activity and often induce apoptosis or autophagy in cancer cells through the PI3K/Akt/mTOR signaling pathways. The potential of natural products as PI3Ks inhibitors has been reported, which suggest PI3Ks a promising anticancer target. Phosphoinositide 3-kinase is a family of related intracellular signal transducer enzymes or lipid kinases that regulate different cellular processes involved in cancer. To identify the molecular reason behind the similar target based activity of selected shikimate pathway metabolites on PI3Kγ, a detail structure-activity relationship study was performed. In the studied work, anticancer potential of plant molecules gallic acid and serpentine was evaluated against PI3Kγ isoform and compared with wortmannin, a steroid metabolite of the fungi and a non-specific covalent known inhibitor of PI3Ks by using in-silico QSAR, docking, ADMET, chemical isolation from plant, NMR and in-vitro activity. A predictive QSAR model was developed by applying multiple linear regression which revealed identification of key structural properties regulating the inhibitory activity of serpentine and gallic acid on PI3Kγ. The model exhibited acceptable statistical parameters such as r2 0.76, r2CV 0.72, and q2 0.55. Structural elucidation was done through NMR studies. Predicted activities were further evaluated through in-vitro testing of gallic acid and serpentine targeting PI3Kγ. The identified chemical features modulating the activity were amide, amine, and secondary amine groups counts, highest occupied molecule orbital (HOMO) energy and valence connectivity index (order 2). In-silico ADME and toxicity risk assessment was done for pharmacokinetic and bioavailability compliance evaluation.

Targeting PI3Kγ scaffold function to activate airway CFTR, limit lung inflammation and promote bronchorelaxation in cystic fibrosis

Targeting PI3Kγ scaffold function to activate airway CFTR, limit lung inflammation and promote bronchorelaxation in cystic fibrosis

Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells.

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

Abstract 554: Checkpoint blockade therapy is improved by altering the immune suppressive microenvironment with IPI-549, a potent and selective inhibitor of PI3K-gamma, in preclinical models

Abstract The phosphoinositide-3-kinase (PI3K) lipid kinases transduce signals in response to various stimuli in different cell types. PI3K-γ is predominantly expressed in leukocytes and not expressed in most epithelial tumors or sarcomas. Genetic studies highlight an important role for PI3K-γ in myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Schmid et al. Canc Cell 2011). Targeting PI3K-γ could therefore alter the immune tumor microenvironment, enabling the immune system to attack tumor cells more effectively. We are developing IPI-549, an investigational small molecule inhibitor of PI3K-γ, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. IPI-549 is a potent and selective inhibitor of PI3K-γ with favorable pharmacological properties. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization, but did not affect M1 polarization. Oral administration of IPI-549 to tumor-bearing mice resulted in significant tumor growth inhibition in multiple syngeneic solid tumor models at PI3K-γ selective doses. Analysis of the tumor-associated immune cells demonstrated that IPI-549 treatment results in decreased immune suppressive myeloid cells and increased CD8+ T cells, suggesting enhanced anti-tumor immunity. To address the requirement for targeting myeloid cells by IPI-549, CD11b+ cells were depleted from a transplanted whole tumor Lewis Lung Carcinoma model and the effect of IPI-549 on limiting tumor growth was abrogated. In addition, a myeloid-infiltrated B16-GMCSF model, but not the isogenic B16 model without GMCSF, was responsive to IPI-549. Studies in immune-deficient mice or CD8 T-cell depleted tumor bearing mice demonstrated the T-cell dependence of IPI-549-mediated tumor growth inhibition. IPI-549 treatment also led to a significant reduction in lung metastases in the 4T1 and B16-GMCSF models. Importantly, in vivo studies with IPI-549 in combination with the immune checkpoint inhibitors anti-PD-1, anti-PDL-1 and anti-CTLA-4 showed increased tumor growth inhibition in multiple models compared to monotherapies alone. These data can inform combinations for future clinical trials. Our studies support a role for PI3K-γ in immune suppressive myeloid cells in the tumor microenvironment and provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit tumor growth in preclinical models. A Phase 1 study evaluating IPI-549 as an orally administered therapeutic, as a single agent and in combination with an anti-PD-1 antibody therapy, in patients with selected solid tumors is expected to begin in early 2016. Citation Format: Olivier De Henau, Taha Merghoub, David Winkler, Sujata Sharma, Melissa Pink, Jeremy Tchaicha, Matthew Rausch, Jennifer Proctor, Nicole Kosmider, John Soglia, Vito Palombella, Jeffery L. Kutok, Jedd D. Wolchok, Karen McGovern. Checkpoint blockade therapy is improved by altering the immune suppressive microenvironment with IPI-549, a potent and selective inhibitor of PI3K-gamma, in preclinical models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 554.

Abstract B029: The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Abstract Introduction: The phosphoinositide-3-kinase (PI3K) lipid kinases are a family of kinase isoforms that transduce signals in response to various stimuli in different cell types. The PI3K-γ isoform is expressed in immune cells and has limited, if any, expression in epithelial cancer cells. Genetic deletion and kinase-dead knock-in studies highlight a key role for PI3K-γ in the development and function of myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Joshi Mol Canc Res 2014; Schmid Canc Cell 2011). Targeting PI3K-γ in these tumor-associated myeloid cells could therefore inhibit the immune suppressive tumor microenvironment, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results: Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD value of 0.29 nM for PI3K-γ with &amp;gt;58-fold weaker binding affinity for the other Class I PI3K isoforms. Enzymatic assays utilizing physiological ATP concentrations (3 mM) confirmed the selectivity of IPI-549 for PI3K-γ (&amp;gt;200-fold) over other Class I PI3K isoforms. Cellular assays designed to assess individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; &amp;gt;140-fold selectivity). Further selectivity screening revealed that IPI-549 is selective for PI3K-γ over other protein and lipid kinases, receptors, ion channels, and transporters. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization in response to IL-4 and MCSF1, but did not inhibit ConA-induced T-cell activation. These data indicate the potential for IPI-549 to block immune suppressive macrophage development but not T-cell activity. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of the PI3K-γ isoform relative to the other Class I PI3K isoforms. In an in vivo PI3K-γ-dependent neutrophil migration murine model, IPI-549 blocked neutrophil migration in a dose dependent manner. To evaluate the effect of PI3K-γ inhibition on tumor growth in an immunocompetent animal, IPI-549 was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple syngeneic models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects immune suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell activity. Studies in nude or CD8 T-cell depleted mice demonstrated the T-cell dependence of IPI-549 mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors showed increased tumor growth inhibition compared to either monotherapy. Conclusions: IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. IND-enabling studies with IPI-549 are ongoing to support its initial clinical exploration in the setting of solid tumors. Citation Format: Jeffery Kutok, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Karen McGovern, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Katherine Walsh, Kerry White, David Winkler, Vito Palombella. The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B029.

Abstract A192: The potent and selective phosphoinositide-3-kinase-γ inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Abstract Introduction The phosphoinositide-3-kinase (PI3K) lipid kinases transduce signals in response to various stimuli in different cell types. PI3K-γ is expressed in immune cells and has limited expression in epithelial cancer cells. Genetic inactivation of PI3K-γ highlights its role in the development and function of myeloid-derived cells that constitute a key component of the suppressive tumor microenvironment (Schmid 2011). Targeting PI3K-γ in tumor-associated myeloid cells could potentially relieve tumor immune tolerance, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD of 0.29 nM for PI3K-γ and enzymatic assays confirmed the selectivity of IPI-549 for PI3K-γ (&amp;gt;200-fold) over the other Class I PI3K isoforms. Comparison of IPI-549 to previously reported PI3K-γ inhibitors in this enzymatic confirmed its unique potency and selectivity for PI3K-γ. Cellular assays for individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; &amp;gt;140-fold selectivity). In kinase screens, IPI-549 is selective for PI3K-γ over other kinases, receptors, ion channels, and transporters. In vitro assays demonstrated that IPI-549 blocked immune suppressive M2 murine macrophage polarization in response to IL-4 and MCSF1. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of PI3K-γ relative to other Class I PI3K isoforms. To characterize IPI-549's ability to inhibit PI3K-γ in vivo, mice with air pouches treated with IPI-549 showed dose responsive inhibition of PI3K-γ-dependent neutrophil migration. The effect of IPI-549 on tumor growth was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell numbers and activity, as assessed by marker studies. Nude or CD8 T-cell depleted mice studies demonstrated a T-cell dependence of IPI-549-mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors or following chemotherapy showed increased tumor growth inhibition compared to monotherapies. Conclusions IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. Citation Format: Karen McGovern, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Kerry White, David Winkler, Vito Palombella, Jeffery Kutok. The potent and selective phosphoinositide-3-kinase-γ inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A192.

Manic fringe promotes a claudin-low breast cancer phenotype through notch-mediated PIK3CG induction.

Claudin-low breast cancer (CLBC) is a poor prognosis disease biologically characterized by stemness and mesenchymal features. These tumors disproportionately affect younger patients and women with African ancestry, causing significant morbidity and mortality, and no effective targeted therapy exists at present. CLBC is thought to originate from mammary stem cells, but little is known on how or why these tumors express a stable epithelial-to-mesenchymal transition phenotype, or what are the driving forces of this disease. Here, we report that Manic Fringe (Mfng), which encodes an O-fucosylpeptide 3-β-N-acetylglucosaminyltransferase known to modify EGF repeats in the Notch extracellular domain, is highly expressed in CLBC and functions as an oncogene in this context. We show that Mfng modulates Notch activation in human and mouse CLBC cell lines, as well as in mouse mammary gland. Mfng silencing in CLBC cell lines reduced cell migration, tumorsphere formation, and in vivo tumorigenicity associated with a decrease in the stem-like cell population. Mfng deletion in the Lfng(flox/flox);MMTV-Cre mouse model, in which one-third of mammary tumors resemble human CLBC, caused a tumor subtype shift away from CLBC. We identified the phosphoinositide kinase Pik3cg as a direct transcriptional target of Mfng-facilitated RBPJκ-dependent Notch signaling. Indeed, pharmacologic inhibition of PI3Kγ in CLBC cell lines blocked migration and tumorsphere formation. Taken together, our results define Mfng as an oncogene acting through Notch-mediated induction of Pik3cg. Furthermore, they suggest that targeting PI3Kγ may prove beneficial for the treatment of CLBC subtype.

Targeting PI3Kγ activity decreases vascular trauma-induced intimal hyperplasia through modulation of the Th1 response.

Interventional strategies to treat atherosclerosis, such as transluminal angioplasty and stent implantation, often cause vascular injury. This leads to intimal hyperplasia (IH) formation that induces inflammatory and fibroproliferative processes and ultimately restenosis. We show that phosphoinositide 3-kinase γ (PI3Kγ) is a key player in IH formation and is a valid therapeutic target in its prevention/treatment. PI3Kγ-deficient mice and mice expressing catalytically inactive PI3Kγ (PI3Kγ KD) showed reduced arterial occlusion and accumulation of monocytes and T cells around sites of vascular lesion. The transfer of PI3Kγ KD CD4(+) T cells into Rag2-deficient mice greatly reduced vascular occlusion compared with WT cells, clearly demonstrating the involvement of PI3Kγ in CD4(+) T cells during IH formation. In addition we found that IH is associated with increased levels of Th1 and Th17 cytokines. A specific decrease in the Th1 response was observed in the absence of PI3Kγ activity, leading to decreased CXCL10 and RANTES production by smooth muscle cells. Finally, we show that treatment with the PI3Kγ inhibitor AS-605240 is sufficient to decrease IH in both mouse and rat models, reinforcing the therapeutic potential of PI3Kγ inhibition. Altogether, these findings demonstrate a new role for PI3Kγ activity in Th1-controlled IH development.

Phosphoinositide 3-kinase gamma in T cell biology and disease therapy

Phosphoinositide 3-kinase gamma (PI3Kγ) kinase activity is important for its signaling functions in T cell development, activation, differentiation, and trafficking. Protection of PI3Kγ knockout mice from disease in multiple autoimmune models suggests that targeting PI3Kγ alone, or in combination with PI3Kδ, could be a promising approach to disease therapy.

Binding Selectivity Studies of Phosphoinositide 3-Kinases Using Free Energy Calculations

Phosphoinositide 3-kinases (PI3Ks) and their phosphatidylinositol 3,4,5-triphosphate (PIP(3)) products regulate a variety of cellular processes. Of these, PI3Kα is an attractive target for anticancer drug design. Mutations in the PI3Kα kinase domain alter the mobility of the activation loop resulting in gain of function. We employed molecular dynamics (MD) simulations-based energetic analysis using molecular mechanics/generalized born surface area (MM/GBSA) for PI3Kα and -γ. MD simulations were carried out for PI3K models based on the RESP (restrained electrostatic potential) and quantum mechanics (QM)-polarized ligand docking (QPLD)-derived partial charges. Computational alanine scanning was also used to evaluate the contributions of key binding residues to ligand binding. Our results show that both RESP and QPLD charge models of PI3Kα and PI3Kγ provide similar performance in MD simulations. For example, the predicted RESP and QPLD free energies of -9.5 and -9.3 kcal/mol for LY294002 binding to PI3Kγ and -10.9 and -11.7 kcal/mol for wortmannin binding to PI3Kα are in good agreement with experimental values. A significant loss in binding free energy was observed when hydrophobic residues were mutated to alanine, suggesting that specific hydrophobic interactions are important to optimal ligand binding. MM/GBSA calculations suggested that residues Ser774, Gln859, and Ile932 of PI3Kα might be used to design H1047R mutant-specific ligands, whereas Lys890 of PI3Kγ can be used for ligand design targeting PI3Kγ.