PI3K Catalytic Subunit Research Articles

Phosphatidylinositol-4,5-bisphosphate 3-kinase family (version 2020.2) in the IUPHAR/BPS Guide to Pharmacology Database

PI3K activation is one of the most important signal transduction pathways used to transmit signals from cell-surface receptors to regulate intracellular processes (cell growth, survival, proliferation and movement). PI3K catalytic (and regulatory) subunits play vital roles in normal cell function and in disease. Progress made in developing PI3K-targeted agents as potential therapeutics for treating cancer and other diseases is reviewed by Fruman et al. (2017) [37].

Age‐Dependent Decrease in PI3K p110 gamma

We previously demonstrated that the hypothalamic supraoptic nucleus (SON) undergoes a robust axonal sprouting response following unilateral transection of the hypothalamo‐neurohypophysial tract in a young, 35‐day‐old rat. However, no collateral sprouting occurs following axotomy in a 125‐day‐old rat. Our lab has in vitro evidence demonstrating the role of CNTF (ciliary neurotrophic factor) in promoting process outgrowth via PI3K signaling. Thus, we compared the protein levels of the PI3K catalytic and regulatory subunits in the SON between 35‐ and 125‐day rats. We found less protein of the catalytic domain, PI3K p110γ, in the 125‐day rat SON; however, we did not observe changes in the other three catalytic domains nor the regulatory domains that we analyzed. Dual fluorescent studies demonstrated that all PI3K catalytic and regulatory subunits analyzed are localized to astrocytes and neurons in the SON. These data suggest that the lack of PI3K p110γ found in the 125‐day rat SON may prevent the collateral sprouting response from occurring, but future studies need to be performed to confirm this hypothesis.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under Grant No. 1643998

Targeting PIK3CG in Combination with Paclitaxel as a Potential Therapeutic Regimen in Claudin-Low Breast Cancer.

PurposeMolecular targeting is a powerful approach for aggressive claudin-low breast cancer (CLBC). Overexpression of PI3K catalytic subunit gamma (PIK3CG) in human CLBC is offering a promising opportunity for targeted therapies. We utilized a specific inhibitor of PIK3CG combined with paclitaxel (PTX) to treat CLBC cells in vitro and in vivo.Patients and MethodsThe tumor cells growth and apoptosis in vitro were analyzed by CCK8, plate clone formation assay, tumorsphere assay, Hoechst staining and flow cytometry. The invasion and metastasis ability of tumor cells in vitro were investigated by wound healing and transwell experiments. Critical gene expression levels were checked by qRT-PCR and Western blot. Xenograft models with CLBC cell lines in SCID mice were established to investigate the effect of combined drugs in vivo.ResultsWe identified that PIK3CG was a potential therapeutic target for CLBC patients. Targeting PIK3CG potentiated CLBC cells growth inhibition in 2D and 3D cultures by PTX. Inhibition of PIK3CG activation could enhance CLBC cells apoptosis and migration suppression induced by PTX. Manipulating autophagy was a validated approach for the use of PIK3CG inhibitor. Using CLBC xenograft mice model, we found that CLBC tumors in vivo could be well treated by combined drugs of PIK3CG inhibitor and PTX.ConclusionWe demonstrated that PIK3CG was a potential target for the therapy of CLBC and inhibition of PIK3CG activation could reinforce the therapeutic effect of this aggressive disease by PTX. The combined use of PIK3CG inhibitor and PTX might be a potential regimen for treating this subtype of breast cancer.

PI3K Catalytic Subunits α and β Modulate Cell Death and IL-6 Secretion Induced by Talc Particles in Human Lung Carcinoma Cells.

Hydrated magnesium silicate (or "talc" particles) is a sclerosis agent commonly used in the management of malignant pleural effusions, a common symptom of metastatic diseases, including lung cancers. However, the direct effects of talc particles to lung carcinoma cells, which can be found in the malignant pleural effusion fluids from patients with lung cancer, are not fully understood. Here, we report a study of the signaling pathways that can modulate the cell death and IL-6 secretion induced by talc particles in human lung carcinoma cells. We found that talc-sensitive cells have higher mRNA and protein expression of PI3K catalytic subunits α and β. Further experiments confirmed that modulation (inhibition or activation) of the PI3K pathway reduces or enhances cellular sensitivity to talc particles, respectively, independent of the inflammasome. By knocking down specific PI3K isoforms, we also confirmed that both PI3Kα and -β mediate the observed talc effects. Our results suggest a novel role of the PI3K pathway in talc-induced cell death and IL-6 secretion in lung carcinoma cells. These cellular events are known to drive fibrosis, and thus further studies of the PI3K pathway may provide a better understanding of the mechanisms of talc sclerosis in the malignant pleural space.

Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer.

Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells. IMPLICATIONS: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.

Boolean model of anchorage dependence and contact inhibition points to coordinated inhibition but semi-independent induction of proliferation and migration

Epithelial cells respond to their physical neighborhood with mechano-sensitive behaviors required for development and tissue maintenance. These include anchorage dependence, matrix stiffness-dependent proliferation, contact inhibition of proliferation and migration, and collective migration that balances cell crawling with the maintenance of cell junctions. While required for development and tissue repair, these coordinated responses to the microenvironment also contribute to cancer metastasis. Predictive models of the signaling networks that coordinate these behaviors are critical in controlling cell behavior to halt disease. Here we propose a Boolean regulatory network model that synthesizes mechanosensitive signaling that links anchorage to a matrix of varying stiffness and cell density sensing to contact inhibition, proliferation, migration, and apoptosis. Our model can reproduce anchorage dependence and anoikis, detachment-induced cytokinesis errors, the effect of matrix stiffness on proliferation, and contact inhibition of proliferation and migration by two mechanisms that converge on the YAP transcription factor. In addition, we offer testable predictions related to cell cycle-dependent anoikis sensitivity, the molecular requirements for abolishing contact inhibition, and substrate stiffness dependent expression of the catalytic subunit of PI3K. Moreover, our model predicts heterogeneity in migratory vs. non-migratory phenotypes in sub-confluent monolayers, and co-inhibition but semi-independent induction of proliferation vs. migration as a function of cell density and mitogenic stimulation. Our model serves as a stepping-stone towards modeling mechanosensitive routes to the epithelial to mesenchymal transition, capturing the effects of the mesenchymal state on anoikis resistance, and understanding the balance between migration versus proliferation at each stage of the epithelial to mesenchymal transition.

Predictive and Pharmacodynamic Biomarkers of Response to the Phosphatidylinositol 3-Kinase Inhibitor Taselisib in Breast Cancer Preclinical Models.

The PI3K signaling pathway serves as a central node in regulating cell survival, proliferation, and metabolism. PIK3CA, the gene encoding the PI3K catalytic subunit p110-alpha, is commonly altered in breast cancer resulting in the constitutive activation of the PI3K pathway. Using an unbiased cell line screening approach, we tested the sensitivity of breast cancer cell lines to taselisib, a potent PI3K inhibitor, and correlated sensitivity with key biomarkers (PIK3CA, HER2, PTEN, and ESR1). We further assessed how taselisib modulates downstream signaling in the different genomic backgrounds that occur within breast cancer. We found that sensitivity to taselisib correlated with the presence of PIK3CA mutations, but was independent of HER2 status. We further showed that HER2-amplified/PIK3CA wild-type cell lines are not as sensitive to taselisib when compared with HER2-amplified/PIK3CA-mutant cell lines. In a PIK3CA-mutant/PTEN null background, PI3K downstream signaling rebounded in the presence of taselisib correlating with decreased sensitivity at later time points. Finally, we observed that PIK3CA mutations cooccurred with mutations in the estrogen receptor (ER; ESR1) in metastatic tumors from patients with ER+ breast cancer. However, the cooccurrence of an ESR1 mutation with a PIK3CA mutation did not affect response to taselisib in a single agent setting or in combination with fulvestrant. In summary, these data suggest that development of taselisib in breast cancer should occur in a PIK3CA-mutant setting with cotreatments determined by the specific subtypes under investigation.

Prostate epithelial-specific expression of activated PI3K drives stromal collagen production and accumulation.

We genetically engineered expression of an activated form of P110 alpha, the catalytic subunit of PI3K, in mouse prostate epithelium to create a mouse model of direct PI3K activation (Pbsn-cre4Prb;PI3KGOF/+ ). We hypothesized that direct activation would cause rapid neoplasia and cancer progression. Pbsn-cre4Prb;PI3KGOF/+ mice developed widespread prostate intraepithelial hyperplasia, but stromal invasion was limited and overall progression was slower than anticipated. However, the model produced profound and progressive stromal remodeling prior to explicit epithelial neoplasia. Increased stromal cellularity and inflammatory infiltrate were evident as early as 4 months of age and progressively increased through 12 months of age, the terminal endpoint of this study. Prostatic collagen density and phosphorylated SMAD2-positive prostatic stromal cells were expansive and accumulated with age, consistent with pro-fibrotic TGF-β pathway activation. Few reported mouse models accumulate prostate-specific collagen to the degree observed in Pbsn-cre4Prb;PI3KGOF/+ . Our results indicate a signaling process beginning with prostatic epithelial PI3K and TGF-β signaling that drives prostatic stromal hypertrophy and collagen accumulation. These mice afford a unique opportunity to explore molecular mechanisms of prostatic collagen accumulation that is relevant to cancer progression, metastasis, inflammation and urinary dysfunction. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Patient-derived organoid models of metastatic colorectal cancer to study response to PIK3CA inhibitors

Abstract Background More than 70% of patients diagnosed with colorectal cancer with liver metastasis have unresectable disease, so the available treatment is systemic chemotherapy that has limited efficacy and causes harmful side-effects. Thus novel and more effective drugs are needed to improve patients’ survival and quality of life. Recent studies have identified several genomic alterations in CRLM that can theoretically be targeted by novel cancer-specific drugs. Among those is the gain-of-function mutations in PIK3CA – the gene encoding the p110a catalytic subunit of PI3K – that are present in 18% of metastatic colorectal tumours. Following the recent FDA-approval of Alpelisib (a PI3Ka-specific inhibitor) HR-positive breast cancer, there is increasing interest to test this drug efficacy to treat CRLM patients with PIK3CA mutations. Although widely used to test cancer cell response, immortalized cell lines fail to represent tumour genomic alterations and heterogeneity. Our goal is to develop patient-derived primary cell models that can be used to reliably test patients’ drug response. Methods Fresh colorectal cancer liver metastasis surgical specimens were dissociated and digested into single cells and seeded in matrigel with appropriate culture media. Originated organoid cultures were then submitted to a minimum of 5 passages followed by a freeze-thaw cycle before deemed established culture. To validate organoid tumour origin, we then performed whole-genome sequencing of paired organoid and respective fresh tumour. Results The established patient-derived organoids from colorectal liver metastasis add an overall mutation match of > 80% when compared to with the original tumour. We have found so far two CRLM organoid lines with mutations in the helical domain of PIK3CA (E545A and E542K). Conclusions We have demonstrated patient-derived organoids recapitulate the genome of their tumour of origin. Libraries of these primary cell models should in the near future be used to more accurately determine drug effectiveness prior to clinical trials. Ongoing testing of Alpelisib alone or in combination with other PI3K pathway target genes will determine whether these patients could benefit from these drugs. Legal entity responsible for the study Barros-Silva, J.D. Funding Cancer Research UK. Disclosure All authors have declared no conflicts of interest.

Abstract 3438: PI3Kα/δ inhibitor, copanlisib is highly effective in ER-positive breast cancer

Abstract Purpose: Over 60% of ER+ breast cancer (BC) cases are associated with activation of the PI3K-AKT-mTORC1/C2 pathway. PIK3CA, the catalytic subunit of PI3K, is by far the most frequently mutated gene in luminal ER+ BC patients. It has long been known that hormone receptor and oncogene cooperation plays a key role in driving tumor development and that most hormone therapy-resistant BC depends on the PI3K pathway. Here, we present the results of a preclinical study of the PI3K α/δ (dominant) inhibitor copanlisib alone and in combination with letrozole in ER+ BC cell lines. Method: Anti-proliferative, apoptotic, cell cycle, and intracellular signaling effects of copanlisib alone and in combination with letrozole were evaluated in a panel of ER +/PIK3CA mutated, and ER+/PTEN mutated breast cancer cell lines. Results: 1) Copanlisib inhibited PI3K, mTOR and their downstream signaling molecules in ER+/PIK3CA or ER+/PTEN mutated BC cell lines; 2) interestingly, copanlisib also inhibited RAS-MAPK signaling in the earlier time points in ER+/PIK3CA mutated cells (T47D & MCF7) but not in ER+/PTEN mutated cells (CAMA1); 3) copanlisib caused a strong differential growth inhibition in ER+ BC cell lines as shown by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom. Inhibition was greater when copanlisib was combined with letrozole; 4) administration of copanlisib induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner; 5) unlike everolimus, copanlisib blocked HIF1α accumulation in hypoxic conditions and this blocking effect was reversed by prior treatment with the proteasome inhibitor carfilzomib, suggesting copalnisib enhanced proteasome-mediated HIF1α degradation; and 6) copanlisib also attenuated ER+ cell migration, an important phenotypic feature for metastasis, along with integrin-mediated RAC1 activation. Conclusions: Copanlisib is highly effective (blocks proliferation, induces apoptosis, and inhibits PI3K and its downstream signaling targets) in ER+ BC cell lines (both PIK3CA and PTEN mutated). The addition of copanlisib to aromatase inhibitor might represent an improved treatment strategy for patients with hormone-resistant/relapse ER+ BC. Citation Format: Pradip De, Jennifer C. Aske, Casey Williams, Nandini Dey, Brian Leyland-Jones. PI3Kα/δ inhibitor, copanlisib is highly effective in ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3438.

Abstract 1787: Selective activation of a PI3K catalytic isoform by G protein-coupled receptors in glioblastoma

Abstract Objective: To determine how PI3K catalytic isoforms become dysregulated in glioblastoma. Background: Recurrence in glioblastoma (GBM) is common and even more resistant to treatment than initial GBM. The Sheng Lab has previously identified that PIK3CB expression levels, but not other PI3K subtypes, are higher in recurrent GBM and are associated with GBM recurrence in newly diagnosed individuals. PI3KCB encodes for PI3K catalytic subunit p110β, and PI3K/AKT pathway activation is a well-known promoter of cell survival. Understanding mechanisms of PIK3CB activation in GBM may identify new therapeutic targets and improve GBM survival. Current knowledge of PI3K isoform activity in GBM is insufficient and contradictory. Given previous studies, it was hypothesized that G-Coupled Protein Receptor (GPCR) promoters preferentially activate PI3K/AKT in PIK3CBhigh GBM cells. Further, that knockdown of PIK3CB, but not other PI3K isoforms, inhibits PI3K/AKT reactivation in PIK3CBhigh cell lines. Design/Methods: U87MG cells, an established p110βhigh GBM cell line, were starved overnight in serum free media, and then treated with growth factors, promoters or inhibitors of several previously hypothesized pathways. Cell lysates were collected and PI3K/AKT pathway activity was measured with immunoblotting. The ratio of pAKT/AKT/ACTB quantified PI3K/AKT pathway activity. Results: Overnight serum starvation depleted pAKT, and incubation with 1% FBS restored pAKT signaling in a time dependent manner. Neither EGF nor FGF-2 activated PI3K/AKT signaling. Gefitinib, an EGFR inhibitor, failed to inhibit reactivation when cells were co-treated with 1% FBS. However, GPCR stimulation by Lysophosphatidic acid (LPA) increased pAKT/AKT ratios in a dose dependent manner. More significantly, Gallein, a GPCR inhibitor, suppressed PI3K/AKT signaling when cells were co-treated with 1% FBS. Selective PI3K inhibitors showed little PI3K/AKT reactivation at multiple doses. Conclusions: These results suggest that GPCR signaling is responsible for PI3K/AKT pathway activation in p110βhigh cells. Citation Format: Abigail R. Winn, Zhi Sheng. Selective activation of a PI3K catalytic isoform by G protein-coupled receptors in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1787.

The H1047R PIK3CA oncogene induces a senescence-like state, pleiotropy and acute HSP90 dependency in HER2+ mammary epithelial cells.

In pre-clinical models, co-existence of Human Epidermal Growth Factor Receptor-2 (HER2)-amplification and PI3K catalytic subunit (PIK3CA) mutations results in aggressive, anti-HER2 therapy-resistant breast tumors. This is not always reflected in clinical setting. We speculated that the complex interaction between the HER2 and PIK3CA oncogenes is responsible for such inconsistency. We performed series of biochemical, molecular and cellular assays on genetically engineered isogenic mammary epithelial cell lines and breast cancer cells expressing both oncogenes. In vitro observations were validated in xenografts models. We showed that H1047R, one of the most common PIK3CA mutations, is responsible for endowing a senescence-like state in mammary epithelial cells overexpressing HER2. Instead of imposing a permanent growth arrest characteristic of oncogene-induced senescence, the proteome secreted by the mutant cells promotes stem cell enrichment, angiogenesis, epithelial-to-mesenchymal transition, altered immune surveillance and acute vulnerability toward HSP90 inhibition. We inferred that the pleiotropism, as observed here, conferred by the mutated oncogene, depending on the host microenvironment, contributes to conflicting pre-clinical and clinical characteristics of HER2+, mutated PIK3CA-bearing tumor cells. We also came up with a plausible model for evolution of breast tumors from mammary epithelial cells harboring these two molecular lesions.

Somatic loss of PIK3R1 may sensitize breast cancer to inhibitors of the MAPK pathway.

The PI3K pathway, which includes the PI3K catalytic subunits p110α (PIK3CA) and the PI3K regulatory subunit p85α (PIK3R1), is the most frequently altered pathway in cancer. We encountered a breast cancer patient whose tumor contained a somatic alteration in PIK3R1. Some commercial sequencing platforms suggest that somatic mutations in PIK3R1 may sensitize cancers to drugs that inhibit the mammalian target of rapamycin (mTOR). However, a review of the preclinical and clinical literature did not find evidence substantiating that hypothesis. The purpose of this study was to knock out PIK3R1 in order to determine the optimal therapeutic approach for breast cancers lacking p85α. We created an isogenic cellular system by knocking out both alleles of the PIK3R1 gene in the non-tumorigenic human breast cell line MCF-10A. Knockout cells were compared with wild-type cells by measuring growth, cellular signaling, and response to drugs. We observed hyperphosphorylation of MEK in these knockouts, which sensitized PIK3R1-null cells to a MEK inhibitor, trametinib. However, they were not sensitized to the mTOR inhibitor, everolimus. Our findings suggest that breast cancers with loss of p85α may not respond to mTOR inhibition, but may be sensitive to MEK inhibition.

Functional role of SGK3 in PI3K/Pten driven liver tumor development

BackgroundHepatocellular carcinoma (HCC) is a leading cause of cancer related deaths worldwide. The PI3K cascade is one of the major signaling pathways underlying HCC development and progression. Activating mutations of PI3K catalytic subunit alpha (PIK3CA) and/or loss of Pten often occur in human HCCs. Serum and glucocorticoid kinase 3 (SGK3) belongs to the SGK family of AGK kinases and functions in parallel to AKT downstream of PI3K. Previous studies have shown that SGK3 may be the major kinase responsible for the oncogenic potential of PIK3CA helical domain mutants, such as PIK3CA(E545K), but not kinase domain mutants, such as PIK3CA(H1047R).MethodsWe investigated the functional contribution of SGK3 in mediating activated PIK3CA mutant or loss of Pten induced HCC development using Sgk3 knockout mice.ResultsWe found that ablation of Sgk3 does not affect PIK3CA(H1047R) or PIK3CA(E545K) induced lipogenesis in the liver. Using PIK3CA(H1047R)/c-Met, PIK3CA(E545K)/c-Met, and sgPten/c-Met murine HCC models, we also demonstrated that deletion of Sgk3 moderately delays PIK3CA(E545K)/c-Met driven HCC, while not affecting PIK3CA(H1047R)/c-Met or sgPten/c-Met HCC formation in mice. Similarly, in human HCC cell lines, silencing of SGK3 reduced PIK3CA(E545K) -but not PIK3CA(H1047R)- induced accelerated tumor cell proliferation.ConclusionAltogether, our data suggest that SGK3 plays a role in transducing helical domain mutant PIK3CA signaling during liver tumor development.

PIK3CA Amplification Associates with Aggressive Phenotype but Not Markers of AKT-MTOR Signaling in Endometrial Carcinoma

Amplification of PIK3CA, encoding the PI3K catalytic subunit alpha, is common in uterine corpus endometrial carcinoma (UCEC) and linked to an aggressive phenotype. However, it is unclear whether PIK3CA amplification acts via PI3K activation. We investigated the association between PIK3CA amplification, markers of PI3K activity, and prognosis in a large cohort of UCEC specimens. UCECs from 591 clinically annotated patients including 83 tumors with matching metastasis (n = 188) were analyzed by FISH to determine PIK3CA copy-number status. These data were integrated with mRNA and protein expression and clinicopathologic data. Results were verified in The Cancer Genome Atlas dataset. PIK3CA amplifications were associated with disease-specific mortality and with other markers of aggressive disease. PIK3CA amplifications were also associated with other amplifications characteristic of the serous-like somatic copy-number alteration (SCNA)-high subgroup of UCEC. Tumors with PIK3CA amplification also demonstrated an increase in phospho-p70S6K but had decreased levels of activated phospho-AKT1-3 as assessed by Reverse Phase Protein Arrays and an mRNA signature of MTOR inhibition. PIK3CA amplification is a strong prognostic marker and a potential marker for the aggressive SCNA-high subgroup of UCEC. Although PIK3CA amplification associates with some surrogate measures of increased PI3K activity, markers for AKT1-3 and MTOR signaling are decreased, suggesting that this signaling is not a predominant pathway to promote cancer growth of aggressive serous-like UCEC. Moreover, these associations may reflect features of the SCNA-high subgroup of UCEC rather than effects of PIK3CA amplification itself.

Application of Hyperpolarized 13C Magnetic Resonance Imaging to Detect Target Inhibition of NFkB Activation in Preclinical Patient-Derived Models of CNS Lymphoma

To gain insights into the tumor microenvironment in primary and secondary CNS lymphomas, we applied LC/MS and GC/MS for differential metabolomic profiling of the cerebrospinal fluid (CSF) of CNS lymphoma patients compared to control subjects. Among 145 analytes identified, the majority were involved in energy metabolism; one of the most significantly upregulated metabolites in CNS lymphoma was lactate (1.8 fold, p<0.001). Subsequently we determined that baseline elevated CSF lactate, quantified by a Beckman Coulter Unicell Clinical Chemistry analyzer, correlated with short survival in three phase I investigations involving immunotherapy in relapsed CNS lymphoma (p<0.0001). (Blood Advances 2018). Given this, we hypothesize that tumor-associated lactate significantly contributes to the pathogenesis of CNS lymphoma as a potential mediator of tumor invasion as well as immunosuppression, and can serve as a novel biomarker in CNS lymphoma. To pursue this, we are testing the hypothesis that metabolic imaging techniques including proton magnetic resonance spectroscopy (MRS) and hyperpolarized (HP) 13C MRS can facilitate prognosis and response assessment to targeted therapies.Thus far we have applied these metabolic imaging approaches to preclinical murine models involving diffusely invasive, intracranial, patient-derived xenografts of CNS DLBCL in RAG-/- mice, to detect tumor-associated lactate production generated by infiltrating lymphoma. We demonstrated that each of these MRS approaches detect highly invasive lymphoma that is undetectable by conventional gadolinium-enhanced T1, T2 sequences, or diffusion-weighted imaging. Because of its ability to detect real-time changes in metabolic pathways, we focused on the application of HP13C pyruvate metabolic imaging as a non-invasive imaging tool for NF-kB pathway inhibition in CNS lymphoma using these models. We evaluated the metabolic response to AZ1495, a novel, CNS penetrant, orally-bioavailable inhibitor of IRAK4 kinase, comparing MYD88 wild type vs. MYD88 L265P mutant tumor models. Using a 14.1T imaging system for MR acquisition, we demonstrated similar tumor-associated production of HP 13C lactate in both MYD88wt and MY88 mutant tumors at 3 weeks post-implantation. We determined that while AZ1495 did not significantly impact lactate production in MYD88wt lymphoma, this agent significantly down-regulated tumor-associated HP pyruvate to lactate conversion (>47%) within 2 days in MYD88 mutant CNS lymphoma (p<0.02). (Figure 1). In parallel, we determined that AZ1495 potently antagonized phosphorylation of p65 REL-A selectively in intracranial xenografts harboring L265P MYD88 mutation. Transcriptional profiling by RNA-Seq demonstrated > 2-fold down-modulation of NF-kB gene expression at 4h of AZ1495 therapy, including transcripts encoding LDH-A as well as the catalytic subunit of PI3K, suggesting interaction with the B cell receptor pathway. Combination AZ1495 plus ibrutinib starting d+5 was synergistic in survival prolongation compared to AZ1495 monotherapy (p<0.003), ibrutinib monotherapy (p<0.005), or control gavage (p<0.002) in a MYD88 L265P mutant, clinically refractory patient-derived intracranial DLBCL xenograft model. Taken together, these data demonstrate the ability of HP13C MRI metabolic imaging to identify highly infiltrative CNS lymphoma, not detectable by conventional MR sequences, as well as its potential to provide an early, non-invasive pharmacodynamic biomarker of response in an NFkB pathway-specific manner, and to facilitate precision medicine in CNS lymphoma. Supported by the National Cancer Institute, the Leukemia and Lymphoma Society and by the Sandler Program for Breakthrough Biomedical Research. [Display omitted] DisclosuresGao:Glaxo Smith Kline: Employment. Drew:AstraZeneca: Employment. Degorce:Astra Zeneca: Employment. Mayo:Astra Zeneca: Employment. Dillman:Astra Zeneca: Employment. Anjum:Astra Zeneca: Employment. Bloecher:Astra Zeneca: Employment. Rubenstein:Celgene: Research Funding; Genentech: Research Funding.

Wogonin Attenuates Isoprenaline-Induced Myocardial Hypertrophy in Mice by Suppressing the PI3K/Akt Pathway.

Many studies have focused on identifying therapeutic targets of myocardial hypertrophy for the treatment of correlative cardiac events. Wogonin is a natural flavonoid compound that displays a potent anti-hypertrophic effect. Knowledge of its pharmacological mechanisms might reveal an effective way to search for therapeutic targets. Myocardial hypertrophy was replicated by the subcutaneous implantation of an isoprenaline mini-pump in mice or isoprenaline treatment of H9C2 cells. Pathologic changes in cardiac structure were assessed by echocardiographic and histological examinations. The signaling transduction in hypertrophy-promoting pathways and the genes involved were detected by western blot and RT-qPCR. Wogonin significantly attenuated isoprenaline-induced myocardial hypertrophy in vivo and in vitro by suppressing phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) hypertrophy-promoting pathway. Wogonin promoted the ubiquitination and degradation of PI3K catalytic subunit alpha (Pik3ca), the catalytic subunit of PI3K, which was upregulated by isoprenaline treatment. Wogonin also increased the expression of neural precursor cells expressing developmentally down-regulated gene 4-like (Nedd4l), the ubiquitin E3 ligase of Pik3ca. Therefore, wogonin targets Nedd4l to induce the degradation of Pik3ca, which reverses the over-activation of the PI3K/Akt pathway and consequently relieves the isoprenaline-induced myocardial hypertrophy.

Genetic Defects in Phosphoinositide 3-Kinase δ Influence CD8+ T Cell Survival, Differentiation, and Function.

Activated phosphoinositide 3-kinase delta syndrome (APDS), also known as p110 delta-activating mutation causing senescent T cells, lymphadenopathy and immunodeficiency (PASLI), is an autosomal dominant primary human immunodeficiency (PID) caused by heterozygous gain-of-function mutations in PIK3CD, which encodes the p110δ catalytic subunit of PI3K. This recently described PID is characterized by diverse and heterogeneous clinical manifestations that include recurrent respiratory infections, lymphoproliferation, progressive lymphopenia, and defective antibody responses. A major clinical manifestation observed in the NIH cohort of patients with PIK3CD mutations is chronic Epstein–Barr virus (EBV) and/or cytomegalovirus viremia. Despite uncontrolled EBV infection, many APDS/PASLI patients had normal or higher frequencies of EBV-specific CD8+ T cells. In this review, we discuss data pertaining to CD8+ T cell function in APDS/PASLI, including increased cell death, expression of exhaustion markers, and altered killing of autologous EBV-infected B cells, and how these and other data on PI3K provide insight into potential cellular defects that prevent clearance of chronic infections.

Abstract 5788: Preclinical evaluation of HZB0071, a small molecule inhibitor of AKT

Abstract Objective: AKT is a component of the PI3K/AKT/mTOR (PAM) pathway, which plays an important part in cell proliferation, growth and survival, activated in many cancers and has been validated as a therapeutic target in the clinic. Inhibition of AKT activity can block the PAM pathway and proliferation of various types of tumor cells with loss of tumor suppressor PTEN, mutational activation of p110a catalytic subunit of PI3K and amplification of the gene encoding either AKT or PI3K. The purpose of this study is to investigate the in vitro and in vivo antitumor activity of HZB0071, a small molecule AKT kinase inhibitor, in preclinical models of solid cancer with AKT overexpression. Method: Kinase inhibiting activity of HZB0071 was determined with AKT1, 2, 3 kinase assays. Cellular anti-proliferative activity was evaluated with AKT overexpressed LNcap cell line and DU145 cell line without AKT overexpression. The antitumor activity of HZB0071 was evaluate in vivo and the synergistic effect combined with Paclitaxel in AKT highly expressed patient-derived mouse xenograft (PDX) models of Gastric Cancer ST-02-0013 and cell-derived mouse xenograft (CDX) models of Breast Cancer BT474. Result: HZB0071 displayed potent kinase inhibiting activity for AKT1, 2, 3 with IC50 1.3±0.41 nM, 50.3±13.4 nM and 90.4±5.84 nM, respectively. HZB0071 inhibited cell proliferation in AKT overexpressed LNcap cells with IC50 67±7 nM. In contrast, HZB0071 showed much weaker anti-proliferative activity in DU145 cells with IC50 &amp;gt;10,000 nM. HZB0071 showed antitumor efficacy in AKT highly expressed gastric cancer PDX model ST-02-0013 (TGI = 62% @30 mpk, QD as single agent; TGI = 61% @15 mpk + Paclitaxel @10 mpk, QD, in combination with Paclitaxel), as well as in the breast cancer BT474 CDX model (TGI = 88% @30 mpk + Paclitaxel @15 mpk, QD). Conclusion: We have identified a novel potent AKT inhibitor HZB0071. Preclinical studies shows antitumor efficacy of HZB0071 in AKT overexpressed solid cancer models. HZB0071 represents a promising clinical candidate for treating solid cancers with high AKT expression. Citation Format: Gang Li, Lihong Hu, Weifeng Mao, Dongfang Li, Charles Z. Ding, Shuhui Chen. Preclinical evaluation of HZB0071, a small molecule inhibitor of AKT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5788.

PI3K Positively Regulates YAP and TAZ in Mammary Tumorigenesis Through Multiple Signaling Pathways.

Breast cancer is a leading cause of death in women worldwide. Active mutations of PI3K catalytic subunit PIK3CA (e.g., H1047R) and amplification of its homolog PIK3CB are observed in a large number of breast cancers. In recent years, aberrant activation of Transcriptional coactivator with PDZ binding motif (TAZ) and its paralog Yes-associated protein (YAP) have also been found to be important for breast cancer development and progression. However, whether PI3K interacts with YAP/TAZ during mammary tumorigenesis is unknown. Through a systematic gain-of-function screen for kinases involved in mammary tumorigenesis, we identified PIK3CB as a transformation-inducing kinase in breast cells. We further determined that PIK3CB positively regulates YAP and TAZ to promote transformation and inhibit mammary cell death in vitro PIK3CB coexpression with TAZ, rather than PIK3CB or TAZ alone, in human MCF10A nontumorigenic mammary cells is sufficient for tumor formation in mice in vivo Interestingly, we also determined that PIK3CA-H1047R enhances YAP and TAZ activity in mammary tumorigenesis in vitro Mechanistically, the regulation of YAP/TAZ by both PIK3CA and PIK3CB occurs through multiple signaling pathways including LATS-dependent and LATS-independent pathways. Therefore, in this study, we determine that PI3K and YAP/TAZ interact to promote breast cancer cell transformation.Implications: This study provides the first evidence that the Hippo pathway effectors TAZ and YAP are critical mediators of PI3K-induced mammary tumorigenesis and synergistically function together with PI3K in transformation of mammary cells. These findings may provide a novel rationale for targeting YAP/TAZ alone or in combination with PI3K inhibitors for breast cancer therapy in the future. Mol Cancer Res; 16(6); 1046-58. ©2018 AACR.