P110β Inhibitor Research Articles

Functional characterization of a novel somatic oncogenic mutation of PIK3CB.

Class I phosphoinositide 3-kinase (PI3K) enzymes have attracted considerable attention as drug targets in cancer therapy over the last 20 years. The signaling pathway triggered by class I PI3Ks is dysregulated in a range of tumor types, impacting cell proliferation, survival and apoptosis. Frequent oncogenic mutations of PIK3CA have previously been discovered. In contrast, reports of PIK3CB mutations have been limited; however, in most cases, those that have been identified have been shown to be activating and oncogenic. The functional characterization of a PIK3CB catalytic domain mutant, p110βE1051K, first discovered by others in castrate-resistant prostate cancer (mCRPC), is outlined in this report; our data suggest that p110βE1051K is a gain-of-function mutation, driving PI3K signaling, tumorigenic cell growth and migration. Tumor cells expressing p110βE1051K are sensitive to p110β inhibition; its characterization as an oncogenic driver adds to the rationale for targeting p110β and indicates a continuing need to further develop specific PI3K inhibitors for clinical development in cancer therapy.

CRKL Mediates p110β-Dependent PI3K Signaling in PTEN-Deficient Cancer Cells

The p110β isoform of PI3K is preferentially activated in many tumors deficient in the phosphatase and tensin homolog (PTEN). However, the mechanism(s) linking PTEN loss to p110β activation remain(s) mysterious. Here, we identify CRKL as a member ofthe class of PI3Kβ-interacting proteins. Silencing CRKL expression in PTEN-null human cancer cells leads to a decrease in p110β-dependent PI3K signaling and cell proliferation. In contrast, CRKL depletion does not impair p110α-mediated signaling. Further study showed that CRKL binds to tyrosine-phosphorylated p130Cas in PTEN-null cancer cells. Since Src family kinases are known both to be regulated by PTEN and to phosphorylate and activate p130Cas, we tested and found that Src inhibition cooperated with p110β inhibition to suppress the growth of PTEN-null cells. These data suggest both a potential mechanism linking PTEN loss to p110β activation and the possible benefit of dual inhibition of Src and PI3K for PTEN-null tumors.

Combined Inhibition of Both p110α and p110β Isoforms of Phosphatidylinositol 3-Kinase Is Required for Sustained Therapeutic Effect in PTEN-Deficient, ER+ Breast Cancer.

Purpose: Determine the roles of the PI3K isoforms p110α and p110β in PTEN-deficient, estrogen receptor α (ER)-positive breast cancer, and the therapeutic potential of isoform-selective inhibitors.Experimental Design: Anti-estrogen-sensitive and -resistant PTEN-deficient, ER+ human breast cancer cell lines, and mice bearing anti-estrogen-resistant xenografts were treated with the anti-estrogen fulvestrant, the p110α inhibitor BYL719, the p110β inhibitor GSK2636771, or combinations. Temporal response to growth factor receptor-initiated signaling, growth, apoptosis, predictive biomarkers, and tumor volumes were measured.Results: p110β primed cells for response to growth factor stimulation. Although p110β inhibition suppressed cell and tumor growth, dual targeting of p110α/β enhanced apoptosis and provided sustained tumor response. The growth of anti-estrogen-sensitive cells was inhibited by fulvestrant, but fulvestrant inconsistently provided additional therapeutic effects beyond PI3K inhibition alone. Treatment-induced decreases in phosphorylation of AKT and Rb were predictive of therapeutic response. Short-term drug treatment induced tumor cell apoptosis and proliferative arrest to induce tumor regression, whereas long-term treatment only suppressed proliferation to provide durable regression.Conclusions: p110β is the dominant PI3K isoform in PTEN-deficient, ER+ breast cancer cells. Upon p110β inhibition, p110α did not induce significant reactivation of AKT, but combined targeting of p110α/β most effectively induced apoptosis in vitro and in vivo and provided durable tumor regression. Because apoptosis and tumor regression occurred early but not late in the treatment course, and proliferative arrest was maintained throughout treatment, p110α/β inhibitors may be considered short-term cytotoxic agents and long-term cytostatic agents. Clin Cancer Res; 23(11); 2795-805. ©2016 AACR.

Role of PI3K p110β in the differentiation of human embryonic stem cells into islet-like cells

To investigate the effects of the PI3K inhibitors on the differentiation of insulin-producing cells derived from human embryonic stem cells. Here, we report that human embryonic stem cells induced by phosphatidylinositol-3-kinase (PI3K) p110β inhibitors could produce more mature islet-like cells. Findings were validated by immunofluorescence analysis, quantitative real-time PCR, insulin secretion in vitro and cell transplantation for the diabetic SCID mice. Immunofluorescence analysis revealed that unihormonal insulin-positive cells were predominant in cultures with rare polyhormonal cells. Real-time PCR data showed that islet-like cells expressed key markers of pancreatic endocrine hormones and mature pancreatic β cells including MAFA. Furthermore, this study showed that the expression of most pancreatic endocrine hormones was similar between groups treated with the LY294002 (nonselective PI3K inhibitor) and TGX-221 (PI3K isoform selective inhibitors of class 1β) derivatives. However, the level of insulin mRNA in TGX-221-treated cells was significantly higher than that in LY294002-treated cells. In addition, islet-like cells displayed glucose-stimulated insulin secretion in vitro. After transplantation, islet-like cells improved glycaemic control and ameliorated the survival outcome in diabetic mice. This study demonstrated an important role for PI3K p110β in regulating the differentiation and maturation of islet-like cells derived from human embryonic stem cells.

Endometrial cancer cells exhibit high expression of p110β and its selective inhibition induces variable responses on PI3K signaling, cell survival and proliferation.

PTEN loss and constitutive activation of the class I phosphoinositide 3-kinase (PI3K) pathway are key drivers of endometrial tumorigenesis. In some cancer types, PTEN-deficient tumors are reliant on class I PI3K p110β (encoded by PIK3CB) activity but little is known about this contribution in endometrial tumorigenesis. In this study, we find that p110β is overexpressed in a panel of 7 endometrial cancer cell lines compared to non-transformed cells. Furthermore, in 234 clinically annotated patient samples, PIK3CB mRNA levels increase significantly in the early phase of tumorigenesis from precursors to low grade primary malignant lesions whereas PIK3CA levels are higher in non-endometrioid compared to endometrioid primary tumors. While high levels of either PIK3CA or PIK3CB associate with poor prognosis, only elevated PIK3CB mRNA levels correlate with a high cell cycle signature score in clinical samples. In cancer cell lines, p110α inhibition reduces cell viability by inducing cell death in PIK3CA mutant cells while p110β inhibition delayed proliferation in PTEN-deficient cells, but not in WT cells. Taken together, our findings suggest that PIK3CB/p110β contributes to some of the pleiotropic functions of PI3K in endometrial cancer, particularly in the early steps by contributing to cell proliferation.

Skeletal muscle PI3K p110β regulates expression of AMP-activated protein kinase

Skeletal muscle metabolic homeostasis is maintained through numerous biochemical and physiological processes. Two principal molecular regulators of skeletal muscle metabolism include AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K); however, PI3K exists as multiple isoforms, and specific metabolic actions of each isoform have not yet been fully elucidated in skeletal muscle. Given this lack of knowledge, we performed a series of experiments to define the extent to which PI3K p110β mediated expression and (or) activation of AMPK in skeletal muscle. To determine the effect of p110β inhibition on AMPK expression and phosphorylation in cultured cells, C2C12 myoblasts were treated with a pharmacological inhibitor of p110β (TGX-221), siRNA against p110β, or overexpression of kinase-dead p110β. Expression and phosphorylation of AMPK were unaffected in myoblasts treated with TGX-221 or expressing kinase-dead p110β. However, expressions of total and phosphorylated AMPK at T172 were reduced in myoblasts treated with p110β siRNA. When normalized to expression of total AMPK, phosphorylation of AMPK S485/491 was elevated in p110β-deficient myoblasts. Similar results were observed in tibialis anterior muscle from mice with conditional deletion of p110β (p110β-mKO mice). Analysis of AMPK transcript expression revealed decreased expression of Prkaa2 in p110β-deficient myoblasts and in p110β-mKO muscle. Loss of p110β had no effect on oligomycin-stimulated phosphorylation of AMPK or phosphorylated Acetyl-CoA carboxylase (ACC), although oligomycin-induced AMPK and ACC phosphorylation were increased in p110β-deficient myoblasts compared to oligomycin-stimulated control myoblasts when normalized to levels of total AMPK or ACC. Overall, these results suggest that p110β positively regulates expression of AMPK in cultured myoblasts and in skeletal muscle in vivo; moreover, despite the reduced abundance of AMPK in p110β-deficient myoblasts, loss of p110β does not appear to impair AMPK activation following stimulus. These findings thus reveal a novel role for p110β in mediating skeletal muscle metabolic signaling.

P110β Inhibition Reduces Histone H3K4 Di-Methylation in Prostate Cancer.

Epigenetic alteration plays a major role in the development and progression of human cancers, including prostate cancer. Histones are the key factors in modulating gene accessibility to transcription factors and post-translational modification of the histone N-terminal tail including methylation is associated with either transcriptional activation (H3K4me2) or repression (H3K9me3). Furthermore, phosphoinositide 3-kinase (PI3 K) signaling and the androgen receptor (AR) are the key determinants in prostate cancer development and progression. We recently showed that prostate-targeted nano-micelles loaded with PI3 K/p110beta specific inhibitor TGX221 blocked prostate cancer growth in vitro and in vivo. Our objective of this study was to determine the role of PI3 K signaling in histone methylation in prostate cancer, with emphasis on histone H3K4 methylation. PI3 K non-specific inhibitor LY294002 and p110beta-specific inhibitor TGX221 were used to block PI3 K/p110beta signaling. The global levels of H3K4 and H3K9 methylation in prostate cancer cells and tissue specimens were evaluated by Western blot assay and immunohistochemical staining. A synthetic androgen R1881 was used to stimulate AR activity in prostate cancer cells. A castration-resistant prostate cancer (CRPC) specific human tissue microarray (TMA) was used to assess the global levels of H3K4me2 methylation by immunostaining approach. Our data revealed that H3K4me2 levels were significantly elevated after androgen stimulation. With RNA silencing and pharmacology approaches, we further defined that inhibition of PI3 K/p110beta activity through gene-specific knocking down and small chemical inhibitor TGX221 abolished androgen-stimulated H3K4me2 methylation. Consistently, prostate cancer-targeted delivery of TGX221 in vivo dramatically reduced the global levels of H3K4me2 as assessed by immunohistochemical staining on tissue section of mouse xenografts from CRPC cell lines 22RV1 and C4-2. Finally, immunostaining data revealed a strong H3K4me2 immunosignal in CRPC tissues compared to primary tumors and benign prostate tissues. Taken together, our results suggest that PI3 K/p110beta-dependent signaling is involved in androgen-stimulated H3K4me2 methylation in prostate cancer, which might be used as a novel biomarker for disease prognosis and targeted therapy. Prostate 77:299-308, 2017. © 2016 Wiley Periodicals, Inc.

PI3K p110β isoform synergizes with JNK in the regulation of glioblastoma cell proliferation and migration through Akt and FAK inhibition.

BackgroundGlioblastoma multiforme is the most aggressive malignant primary brain tumor, characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma. Both PI3K/Akt and JNK pathways are essential to glioblastoma cell survival, migration and invasion. Due to their hyperactivation in glioblastoma cells, PI3K and JNK are promising targets for glioblastoma treatment.MethodsTo investigate the combination effects of class IA PI3K catalytic isoforms (p110α, p110β and p110δ) and JNK inhibition on tumor cell growth and motility, glioblastoma cells and xenografts in nude mice were treated with isoform-selective PI3K inhibitors in combination with JNK inhibitor.ResultsWe showed that combined inhibition of these PI3K isoforms and JNK exerted divergent effects on the proliferation, migration and invasion of glioblastoma cells in vitro. Pharmacological inhibition of p110β or p110δ, but not p110α, displayed synergistic inhibitory effect with JNK inhibition on glioblastoma cell proliferation and migration through decreasing phosphorylation of Akt, FAK and zyxin, leading to blockade of lamellipodia and membrane ruffles formation. No synergistic effect on invasion was observed in all the combination treatment. In vivo, combination of p110β and JNK inhibitors significantly reduced xenograft tumor growth compared with single inhibitor alone.ConclusionConcurrent inhibition of p110β and JNK exhibited synergistic effects on suppressing glioblastoma cell proliferation and migration in vitro and xenograft tumor growth in vivo. Our data suggest that combined inhibition of PI3K p110β isoform and JNK may serve as a potent and promising therapeutic approach for glioblastoma multiforme.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0356-5) contains supplementary material, which is available to authorized users.

PI3K-p110α mediates resistance to HER2-targeted therapy in HER2+, PTEN-deficient breast cancers.

Human epidermal growth factor receptor-2 (HER2) amplification/overexpression (HER2+) frequently co-occurs with PI3K pathway activation in breast tumors. PI3K signaling is most often activated by PIK3CA mutation or PTEN loss, which frequently results in sensitivity to p110α or p110β inhibitors, respectively. To examine the p110 isoform dependence in HER2+, PTEN-deficient tumors, we generated genetic mouse models of breast tumors driven by concurrent Her2 activation and Pten loss coupled with deletion of p110α or p110β. Ablation of p110α, but not p110β, significantly impaired the development of Her2+/Pten-null tumors in mice. We further show that p110α primarily mediates oncogenic signaling in HER2+/PTEN-deficient human cancers while p110β conditionally mediates PI3K/AKT signaling only upon HER2 inhibition. Combined HER2 and p110α inhibition effectively reduced PI3K/AKT signaling and growth of cancer cells both in vitro and in vivo. Addition of the p110β inhibitor to dual HER2 and p110α inhibition induced tumor regression in a xenograft model of HER2+/PTEN-deficient human cancers. Together, our data suggest that combined inhibition of HER2 and p110α/β may serve as a potent and durable therapeutic regimen for the treatment of HER2+, PTEN-deficient breast tumors.

A PI3K p110β-Rac signalling loop mediates Pten-loss-induced perturbation of haematopoiesis and leukaemogenesis.

The tumour suppressor PTEN, which antagonizes PI3K signalling, is frequently inactivated in haematologic malignancies. In mice, deletion of PTEN in haematopoietic stem cells (HSCs) causes perturbed haematopoiesis, myeloproliferative neoplasia (MPN) and leukaemia. Although the roles of the PI3K isoforms have been studied in PTEN-deficient tumours, their individual roles in PTEN-deficient HSCs are unknown. Here we show that when we delete PTEN in HSCs using the Mx1–Cre system, p110β ablation prevents MPN, improves HSC function and suppresses leukaemia initiation. Pharmacologic inhibition of p110β in PTEN-deficient mice recapitulates these genetic findings, but suggests involvement of both Akt-dependent and -independent pathways. Further investigation reveals that a p110β–Rac signalling loop plays a critical role in PTEN-deficient HSCs. Together, these data suggest that myeloid neoplasia driven by PTEN loss is dependent on p110β via p110β–Rac-positive-feedback loop, and that disruption of this loop may offer a new and effective therapeutic strategy for PTEN-deficient leukaemia.

Novel Role for p110β PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells.

The organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110β and AR have previously been reported to functionally interact.

Oncogenic activation of the PI3-kinase p110β isoform via the tumor-derived PIK3Cβ(D1067V) kinase domain mutation.

Activation of the phosphoinositide 3-kinase (PI3K) pathway occurs widely in human cancers. Although somatic mutations in the PI3K pathway genes PIK3CA and PTEN are known to drive PI3K pathway activation and cancer growth, the significance of somatic mutations in other PI3K pathway genes is less clear. Here, we establish the signaling and oncogenic properties of a recurrent somatic mutation in the PI3K p110β isoform that resides within its kinase domain (PIK3Cβ(D1067V)). We initially observed PIK3Cβ(D1067V) by exome sequencing analysis of an EGFR-mutant non-small cell lung cancer (NSCLC) tumor biopsy from a patient with acquired erlotinib resistance. On the basis of this finding, we hypothesized that PIK3Cβ(D1067V) might function as a novel tumor-promoting genetic alteration, and potentially an oncogene, in certain cancers. Consistent with this hypothesis, analysis of additional tumor exome data sets revealed the presence of PIK3Cβ(D1067V) at low frequency in other patient tumor samples (including renal cell carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, melanoma, thyroid carcinoma and endometrial carcinoma). Functional studies revealed that PIK3Cβ(D1067V) promoted PI3K pathway signaling, enhanced cell growth in vitro, and was sufficient for tumor formation in vivo. Pharmacologic inhibition of PIK3Cβ with TGX-221 (isoform-selective p110β inhibitor) specifically suppressed growth in patient-derived renal-cell carcinoma cells with endogenous PIK3Cβ(D1067V) and in NIH-3T3 and human EGFR-mutant lung adenocarcinoma cells engineered to express this mutant PI3K. In the EGFR-mutant lung adenocarcinoma cells, expression of PIK3Cβ(D1067V) also promoted erlotinib resistance. Our data establish a novel oncogenic form of PI3K, revealing the signaling and oncogenic properties of PIK3Cβ(D1067V) and its potential therapeutic relevance in cancer. Our findings provide new insight into the genetic mechanisms underlying PI3K pathway activation in human tumors and indicate that PIK3Cβ(D1067V) is a rational therapeutic target in certain cancers.

Abstract P5-20-02: PTEN status dictates the roles of PI3K isoforms p110α and p110β in modulation of AKT/mTOR and response to growth factor signaling in ER+ breast cancer

Abstract Class 1A phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, survival, and metabolism. PI3Ks are heterodimeric lipid kinases composed of a p85 regulatory subunit and a p110 catalytic subunit (p110α, p110β, or p110δ). p110α and p110β play distinct roles in PI3K signaling in carcinoma cells. p110α is frequently activated by growth factor receptor kinase signaling. In contrast, p110β was shown to play a role in insulin metabolic action, G protein-coupled receptor (GPCR) and Rac1 signal transduction, and oncogenic transformation. Cancer cells deficient in PTEN, the tumor suppressor phosphatase that antagonizes PI3K signaling, are often sensitized to pharmacological inhibitors of p110β. As a result, early clinical studies with p110β inhibitors are often restricted to patients with PTEN-deficient cancers. However, analysis of data from the Genomics of Drug Sensitivity in Cancer database revealed that genetic lesions in PTEN or PIK3CA (encodes p110α) were significantly and independently associated with increased sensitivity to the p110β inhibitor AZD6284. Among 668 cancer cell lines evaluated, 61 lines had AZD6482 IC50 values ≤2 mM, but only 25 lines harbored an alteration in PTEN and/or PIK3CA. Thus, a significant fraction of cancer cell lines (and tumors) without PTEN/PIK3CA alterations are likely to be sensitive to p110β inhibition. To explore the role of p110β in PI3K signaling in ER+ PTEN-deficient breast cancer, we treated cells with the p110β inhibitor GSK2636771 and the p110α inhibitor BYL-719, alone or in combination, and assessed effects on steady state and growth factor-induced activation of AKT and MEK/ERK activation, and cell growth. p110β inhibition reduced the viability of PTEN-deficient cells; however, combined inhibition of p110α and p110β was more effective at reducing AKT and ERK phosphorylation and increasing apoptosis in ER+ PTEN-deficient cells. Furthermore, anti-estrogen treatment potentiated the anti-proliferative effects of PI3K inhibition. p110β inhibition reduced insulin-like growth factor 1 (IGF-1)-induced pAKT levels, and delayed AKT phosphorylation in both PTEN-deficient and PTEN-wild-type cells. In contrast, p110β inhibition sensitized both PTEN-wild-type and PTEN-deficient cells to heregulin stimulation, and promoted PI3K (p85)/HER3 interaction. These results indicate that p110β inhibition desensitizes cells to IGF-1 stimulation, hypersensitizes cells to heregulin, and modulates downstream AKT and MEK/ERK activation in response to growth factor receptor activation. Our findings suggest that the anti-tumor efficacy of p110β inhibitors may be related to growth factor dependence and PTEN status. Citation Format: Lloye M Dillon, Stephanie J Bouley, Todd W Miller. PTEN status dictates the roles of PI3K isoforms p110α and p110β in modulation of AKT/mTOR and response to growth factor signaling in ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-20-02.

Role of phosphoinositide 3-OH kinase p110β in skeletal myogenesis.

Phosphoinositide 3-OH kinase (PI3K) regulates a number of developmental and physiologic processes in skeletal muscle; however, the contributions of individual PI3K p110 catalytic subunits to these processes are not well-defined. To address this question, we investigated the role of the 110-kDa PI3K catalytic subunit β (p110β) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110β delayed differentiation. We next generated mice with conditional deletion of p110β in skeletal muscle (p110β muscle knockout [p110β-mKO] mice). While young p110β-mKO mice possessed a lower quadriceps mass and exhibited less strength than control littermates, no differences in muscle mass or strength were observed between genotypes in old mice. However, old p110β-mKO mice were less glucose tolerant than old control mice. Overexpression of p110β accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110β had the opposite effect. p110β overexpression was unable to promote myoblast differentiation under conditions of p110α inhibition, but expression of p110α was able to promote differentiation under conditions of p110β inhibition. These findings reveal a role for p110β during myogenesis and demonstrate that long-term reduction of skeletal muscle p110β impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice.

Measurement of PIP3 Levels Reveals an Unexpected Role for p110β in Early Adaptive Responses to p110α-Specific Inhibitors in Luminal Breast Cancer

BYL719, which selectively inhibits the alpha isoform of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110a), is currently in clinical trials for the treatment of solid tumors, especially luminal breast cancers with PIK3CA mutations and/or HER2 amplification. This study reveals that, even among these sensitive cancers, the initial efficacy of p110α inhibition is mitigated by rapid re-accumulation of the PI3K product PIP3 produced by the p110β isoform. Importantly, the reactivation of PI3K mediated by p110β does not invariably restore AKT phosphorylation, demonstrating the limitations of using phospho-AKT as a surrogate to measure PI3K activation. Consistently, we show that the addition of the p110β inhibitor to BYL719 prevents the PIP3 rebound and induces greater antitumor efficacy in HER2-amplified and PIK3CA mutant cancers.

Abstract 3342: The p110β isoform of PI3K modulates response to growth factor receptor signaling in breast cancer cells irrespective of PTEN status

Abstract The class 1a phosphatidylinositol 3-kinases (PI3Ks) regulate cell growth, survival, and metabolism. These PI3Ks are heterodimeric lipid kinases composed of a p85 regulatory subunit and a p110 catalytic subunit (p110α, p110β, or p110δ). p110α and p110β play distinct roles in PI3K signaling in carcinoma cells. p110α is activated by growth factor receptor kinase signaling. In contrast, p110β was shown to play a role in insulin metabolic action, G protein-coupled receptor (GPCR) signaling, and oncogenic transformation. Cancer cells deficient in PTEN, the tumor suppressor phosphatase that antagonizes PI3K signaling, have been shown to be sensitized to pharmacological inhibitors of p110β. As a result, early clinical studies with p110β inhibitors are restricted to patients with PTEN-deficient cancers. However, analysis of data from the Genomics of Drug Sensitivity in Cancer database revealed that genetic lesions in PTEN or PIK3CA (encodes p110α) were significantly and independently associated with increased sensitivity to the p110β inhibitor AZD6284. Among 668 cancer cell lines, 96, 57, and 5 lines harbored alterations in PTEN, PIK3CA, or both, respectively. Among 61 cell lines with IC50 values ≤2 μM, only 25 harbored an alteration in PTEN and/or PIK3CA. Thus, a significant fraction of cancer cell lines (and tumors) without PTEN alterations are likely to be sensitive to p110β inhibition. To explore the role of p110β in PI3K signaling in ER+ breast cancer, we treated cells with the p110β inhibitor GSK2636771 and monitored effects on growth factor-induced activation of AKT and MEK/ERK. We found that p110β inhibition reduced basal pAKT and pERK levels in PTEN-deficient cells. Furthermore, p110β inhibition reduced insulin-like growth factor 1 (IGF-1)-induced pAKT levels and delayed the onset of AKT phosphorylation in both PTEN-deficient and PTEN-wild-type cells. In contrast, p110β inhibition sensitized both PTEN-wildtype and PTEN-deficient cells to heregulin stimulation. These results indicate that p110β desensitizes cells to IGF-1 stimulation and hyper-sensitizes them to heregulin stimulation and suggests that p110β regulates downstream AKT and MEK/ERK activation in response to growth factor receptor activation. Our findings advocate for the testing of p110β inhibitors in cell lines (and tumors) selected based on growth factor dependence and PTEN status. Citation Format: Stephanie J. Bouley, Lloye M. Dillon, Todd W. Miller. The p110β isoform of PI3K modulates response to growth factor receptor signaling in breast cancer cells irrespective of PTEN status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3342. doi:10.1158/1538-7445.AM2014-3342

Abstract LB-327: Loss of PTEN leads to clinical resistance to the PI3Kα inhibitor BYL719 and provides evidence of convergent evolution under selective therapeutic pressure

Abstract Activating mutations of PIK3CA, the gene encoding the p110α subunit of PI3K, are frequent in breast cancer and selective inhibitors of this enzyme have shown promising clinical activity in breast tumors harboring these mutations. We studied the case of a patient with metastatic breast cancer harboring a PIK3CA mutation that was treated in a clinical trial with BYL719, a highly selective PI3Kα inhibitor. The treatment resulted in a robust partial response that lasted 7 months followed by rapid progression and death. A rapid autopsy was performed with collection of tissue samples from 16 different metastatic sites. We compared by whole genome and exome sequencing the original primary tumor, a rapidly progressing lung metastasis and a periaortic lesion that was still responding to BYL719 at time of death. Besides several common alterations, PTEN loss and a missense mutation were detected only in the lung metastasis. Using targeted exome sequencing we analyzed all the other available samples. Strikingly, we observed a consistent loss in PTEN (via different mechanisms such as deletion, splice site mutation and frameshift mutations) in all the lesions refractory to BYL719 but not in the responding ones. In every case, the loss of PTEN was also documented by lack of protein expression by immunohistochemistry (IHC). Finally, we were able to build a dendrogram showing the phylogenetic evolution of the lesions and the evolutionary convergence of the PTEN alterations. To validate PTEN loss as a possible mechanism of acquired resistance to selective PI3Kα inhibition, we generated doxycycline-inducible PTEN shRNA stable clones starting from three different BYL719-sensitive cell lines. In all the studied models, induction of PTEN shRNA resulted in resistance to BYL719. Since PTEN deficient genetic models have been shown to rely on the β subunit of the PI3K holoenzyme, we tested whether the concomitant inhibition of both p110α and p110β was sufficient to revert the resistant phenotype. BKM120 (a pan-PI3K inhibitor) or the addition of AZD6482 (p110β inhibitor) to BYL719 re-sensitized the cells to BYL719. To expand our findings in vivo, we generated a patient-derived xenograft (PDX) model from a PTEN-null non-responding lesion (lung). Consistently, this PDX model was refractory to the antitumor activity of BYL719 but conserved sensitivity to BKM120 or the combination of AZD6482 and BYL719. In both cases, IHC analysis revealed a decrease in PI3K/AKT downstream effectors pPRAS40 (246) and pS6 (240/4) staining with BKM120 or AZD6482+BYL719, but not with BYL719 alone. Preliminary analyses of other samples collected from patients treated with BYL719 suggest that PTEN loss is a relatively frequent event upon therapy progression. Taken together, the different mechanisms that inactivate PTEN in the tumor treated with BYL719 can be explained by convergent phenotypic evolution in a heterogeneous tumor and highlight the importance of PTEN and PI3Kβ in acquired resistance to PI3Kα inhibitors. Citation Format: Pau Castel, Dejan Juric, Helen Won, Benjamin Ainscough, Haley Ellis, Saya Ebbesen, Malachi Griffith, Obi Griffith, Iyer Gopakumar, Dennis Sgroi, Steven Isakoff, Elaine Mardis, David Solit, Scott Lowe, Cornelia Quadt, Malte Peters, Michael Berger, Maurizio Scaltriti, José Baselga. Loss of PTEN leads to clinical resistance to the PI3Kα inhibitor BYL719 and provides evidence of convergent evolution under selective therapeutic pressure. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-327. doi:10.1158/1538-7445.AM2014-LB-327

Abstract 4775: PI3K-p110β is required for leukemic transformation and HSC depletion in the absence of Pten

Abstract The tumor suppressor PTEN, which antagonizes PI3K signaling, plays critical roles in cell cycle, cell migration and stem cell self-renewal. PTEN is frequently mutated or inactivated in T-cell acute lymphoblastic leukemia (T-ALL) and acute myelogenous leukemia (AML). In mice, HSC-specific deletion of Pten leads to myeloproliferative phenotype, transplantable leukemia, and increased cycling of hematopoietic stem cells (HSCs), leading to their depletion. Rapamycin, an mTOR inhibitor, rescues this phenotype. Thus, it is likely that PI3K itself plays an important role in Pten-deficient HSCs and leukemic cells, but the specific contributions of the PI3K isoforms are poorly understood. We demonstrate that genetic ablation or pharmacological inhibition of PI3K-p110β prevents myeloid neoplasia and prolongs survival. Importantly, p110β ablation rescues HSCs in the bone marrow (BM), and reinstates long-term hematopoietic reconstitution. Moreover, Pten deficiency leads to aberrant expression of Cxcl12 in the BM and spleen, enabling the generation of an alternative splenic niche. Inactivation of p110β rescues retention of HSCs, and perturbs the splenic niche by interfering with the Cxcl12-CXCR4 axis. This suggests that selective inhibition of p110β offers an alternative therapeutic strategy in PTEN deficient leukemias while restoring untransformed HSCs to their proper niche. Citation Format: Haluk Yuzugullu, Lukas Baitsch, Allison Steiner, Linda K. Clayton, Kira Gritsman, Jean J. Zhao. PI3K-p110β is required for leukemic transformation and HSC depletion in the absence of Pten. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4775. doi:10.1158/1538-7445.AM2014-4775

Phosphoinositide 3-kinases p110α and p110β have differential roles in insulin-like growth factor-1-mediated Akt phosphorylation and platelet priming.

Platelet hyperactivity is a contributing factor in the pathogenesis of cardiovascular disease and can be induced by elevated levels of circulating growth factors, such as insulin-like growth factor-1 (IGF-1). IGF-1 is a primer that cannot stimulate platelet activation by itself, but in combination with physiological stimuli can potentiate platelet functional responses via a phosphoinositide 3-kinase-dependent mechanism. In this study, we explored the role of the phosphoinositide 3-kinase p110α isoform in IGF-1-mediated enhancement of platelet function. Using a platelet-specific p110α knockout murine model, we demonstrate that genetic deletion, similar to pharmacological inactivation of p110α, did not affect proteinase-activated receptor 4 signaling to Akt/protein kinase B but significantly reduced IGF-1-mediated Akt phosphorylation. The p110β inhibitor TGX-221 abolished IGF-1-induced Akt phosphorylation in p110α-deficient platelets, demonstrating that both p110α and p110β contribute to IGF-1-mediated Akt phosphorylation. Genetic deletion of p110α had no effect on IGF-1-mediated increases in thrombus formation on collagen and enhancement of proteinase-activated receptor 4-mediated integrin activation and α-granule secretion. In contrast, pharmacological inhibition of p110α blocked IGF-1-mediated potentiation of integrin activation and α-granule secretion. Functional enhancement by IGF-1 in p110α knockout samples was lost after TGX-221 treatment, suggesting that p110β drives priming in the absence of the p110α isoform. Together, these results demonstrate that both p110α and p110β are involved in Akt signaling by IGF-1, but that it is the p110α isoform that is responsible for IGF-1-mediated potentiation of platelet function.

MP49-14 SMYD3 IS RESPONSIBLE FOR ANDROGEN-INDUCED H3K4 METHYLATION IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2014MP49-14 SMYD3 IS RESPONSIBLE FOR ANDROGEN-INDUCED H3K4 METHYLATION IN PROSTATE CANCER CELLS Zachary Hamilton, Ruibao Chen, Xiangxing Kuang, Yiling Huang, J. Brantley Thrasher, and Benyi Li Zachary HamiltonZachary Hamilton More articles by this author , Ruibao ChenRuibao Chen More articles by this author , Xiangxing KuangXiangxing Kuang More articles by this author , Yiling HuangYiling Huang More articles by this author , J. Brantley ThrasherJ. Brantley Thrasher More articles by this author , and Benyi LiBenyi Li More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1115AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Epigenetic alteration plays a major role in prostate cancer development and progression. Histones are the key factors in modulating gene accessibility to transcription factors. Post-translational modification of the histone N-terminal tail includes methylation, acetylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation and deamination. Of these, histone methylation is associated with either transcriptional activation (H3K4, H3K36 and H3K79) or repression (H3K9, H3K27 and H4K20). Furthermore, androgens and the androgen receptor (AR) are key factors for prostate cancer development and progression. SMYD3 is a histone methyltransferase that plays a role in transcriptional regulation. Previously, we have shown that p110β is critical for androgen-induced gene expression and cell proliferation in prostate cancer. The objective of this study was to investigate the involvement of p110β in histone methylation and prostate cancer cell proliferation. Methods In this study, we examined the effect of androgen stimulation on the global levels of H3K4a methylation in prostate cancer cells. Histone methylation was assessed in western blot assays using methylation site-specific antibodies. The involvement of p110β and SMYD3 were determined with gene specific small interference RNAs and chemical inhibitor TGX221. The direct interaction of SMYD3 with AR-downstream genes was evaluated with chromatin immunoprecipitation assay. The global levels of H3K4 methylation were assessed by immunohistochemical staining on tissue section. Results Our data revealed that H3K4me2 was significantly elevated after androgen stimulation, which was due to increased methylation but not reduced demethylation. Using the small interfering RNA approach, we further defined that out of eight H3K4 methyltransferases, SMYD3 was responsible for androgen-stimulated increase of H3K4me2 level. Meanwhile, inhibition of PI3K/p110β activity through p110β inhibitor TGX221 abolished androgen-stimulated H3K4me2 modification. Finally, prostate cancer cell-targeted delivery of p110β inhibitor TGX221 in vivo suppressed xenograft tumor growth, which is associated with reduced H3K4me2 methylation. Conclusions Taken together, this data suggests that androgen stimulates SMYD3-dependent H3K4me2 modification, which is regulated by the PI3K/p110β pathway in prostate cancer cells. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e507 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Zachary Hamilton More articles by this author Ruibao Chen More articles by this author Xiangxing Kuang More articles by this author Yiling Huang More articles by this author J. Brantley Thrasher More articles by this author Benyi Li More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...