pan-PI3K Inhibitor Research Articles

Human Breast Cancer Cells Harboring a Gatekeeper T798M Mutation in HER2 Overexpress EGFR Ligands and Are Sensitive to Dual Inhibition of EGFR and HER2

Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M-induced resistance to identify potential strategies to overcome that resistance. HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR/HER2/HER3/PI3K axis. A low 3% allelic frequency of the T798M mutant shifted 10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT, and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin, and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting that increased EGFR ligand production was causally associated with drug resistance. Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene-amplified breast cancer cells harboring T798M mutant alleles.

A pharmacological model reveals biased dependency on PI3K isoforms for tumor cell growth

To identify the contribution of individual isoform (α, β, γ, δ) of class I PI3Ks to tumor cell growth for proper use of PI3K inhibitors in cancer therapy. A panel of human rhabdomyosarcoma Rh30 cells stably expressing myristoylation (Myr)-tagged one of class I PI3K p110 subunits was established. PI3K activity was analyzed by measuring phosphorylated Akt with Western blotting, and isoform-specific PI3K activities were validated with PI3K isoform-selective inhibitors. The growth of prostate cancer PC-3 cells and B cell type leukemia Raji cells was determined using SRB assay and CCK-8 assay, respectively. The phosphorylation of Akt in Rh30-Myr-p110α, β, γ, δ cells was preferentially inhibited by PI3K isoform-selective inhibitors A66 (PI3Kα), TGX221 (PI3Kβ), AS604850 (PI3Kγ) and CAL-101 (PI3Kδ), respectively. A newly obtained PI3K inhibitor WJD008 (10 μmol/L) completely abrogated Akt phosphorylation by all the isoforms of class I PI3Ks, thus acted as a pan-PI3K inhibitor. In prostate cancer PC-3 cells, the PI3K isoform-selective inhibitors were much less potent than WJD008 in suppression of the proliferation. In B cell type leukemia Raji cells, inhibition of PI3Kδ alone or all the isoforms of class I PI3Ks displayed similar potency against the cell proliferation, whereas selective inhibition of individual PI3Kα/β/γ isoforms resulted in negligible activity. Rh30-Myr-p110α, β, γ, δ cells are a useful cell model to identify the selectivity of PI3K inhibitors. Pan-PI3K inhibitors are suitable for treating PC-3 cells, whereas selective PI3Kδ inhibitor is sufficient to block Raji cell growth. The biased dependency on PI3K isoforms for tumor cell growth rationalizes the use of PI3K inhibitors with different selectivity for cancer therapy.

PI3K pathway dependencies in endometrioid endometrial cancer cell lines.

Endometrioid endometrial cancers (EEC) frequently harbor coexisting mutations in phosphoinositide 3-kinase (PI3K) pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to define the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cell lines rely on p110β signaling for survival. Twenty-four human EEC cell lines were characterized for their mutation profile and activation state of PI3K and mitogen-activated protein kinase (MAPK) signaling pathway proteins. Cells were treated with pan-class I PI3K, p110α, and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, mitogen-activated protein/extracellular signal-regulated kinase (MEK), and RAF inhibitors. RNA interference (RNAi) was used to assess effects of KRAS silencing in EEC cells. EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition, and only 2 of 6 KRAS-mutant cell lines showed response to MEK inhibition. KRAS RNAi silencing did not induce apoptosis in KRAS-mutant EEC cells. PTEN-mutant EEC cell lines were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only in combination with the p110α selective inhibitor A66 was a decrease in cell viability observed. Targeted pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA- and PTEN-mutant tumors, respectively, even in a subset of EECs concurrently harboring KRAS mutations. Inhibition of p110β alone may not be sufficient to sensitize PTEN-mutant EEC cells and combination with other targeted agents may be required.

Effect of pan-PI3K and HER2 blockade on antitumor activity in preclinical models of breast cancer resistance to trastuzumab therapy.

e13570 Background: PI3K-AKT-mTOR pathway signaling is important for the oncogenic function of HER2. Activating alterations of this pathway are frequently observed in HER2-enriched breast cancer and generally herald a poor response and resistance to trastuzumab (T). Targeting the PI3K-AKT-mTOR pathway is an attractive strategy in HER2+ breast cancer that is refractory to trastuzumab. The hypothesis is that the suppression of this pathway by pan-PI3K inhibitor, GDC-0941 may lead to overcome trastuzumab-resistance. Methods: The antiproliferative and HER2-mediated cellular signaling (pAKT, pP70S6K, pS6RP, p4EBP1 and p-ERK) effects of GDC-0941 alone and in combination with T were evaluated in HER2 amplified T-sensitive (BT474), T-resistant (BT474HR), and HER2 amplified/PIK3CA mutated (HCC1954, UACC893) BT cell lines by MTT assay and Western blots. Athymic mice bearing BT474 and BT474HR xenograft tumors were treated with GDC-0941 and T (alone and in combination). Results: (1) GDC-0941 exhibited in vitro cell killing activity in MTT assay with IC50’s ranging from 0.35 µM to 1 µm and potency was augmented by the addition of T, (2) inhibition of phosphorylation of AKT(S473, T308), P70S6K, S6RP, and 4EBP1(T37/46, T70) was observed following GDC-0941 treatment, and the combination of GDC-0941 and T more effectively blocked the PI3K-AKT-mTOR pathway, (4) GDC-0941 dose-dependently blocked 3D-ON-TOP clonogenic growth of HER2+ cells. This effect was potentiated in the presence of T and (5) xenograft data show that the combination of GDC-0941 and T has strongly enhanced anti-tumor effect in both sensitive (82%) and resistant models (79%), something that cannot be achieved by either monotherapy. Conclusions: Our data suggest that 1) therapeutic targeting of the PI3K-AKT-mTOR signaling should be effective in abrogating resistance to T therapy in HER2+ BT, and 2) targeting both the HER2 and the PI3K signaling pathways is an attractive strategy to enhance the clinical activity of T therapy, as well as to prevent or delay the development of resistance.

Tumor pharmacokinetics (PK) and pharmacodynamics (PD) of SAR245409 (XL765) and SAR245408 (XL147) administered as single agents to patients with recurrent glioblastoma (GBM): An Ivy Foundation early-phase clinical trials consortium study.

2012 Background: XL765 is a potent pan PI3K inhibitor with activity against mTOR and XL147 is a potent pan-PI3K inhibitor. Inhibition of the PI3K/mTOR pathway may be beneficial in the treatment of GBM. Methods: Patients with GBM who were candidates for a surgical resection were eligible for this exploratory study. Cohorts of 6-10 patients were treated with one of three regimens: Cohort 1: XL765 50mg twice daily (BID), Cohort 2: XL147 200mg once daily (QD), Cohort 3: XL765 90mg QD for >10 days prior to undergoing tumor resection. Tumor tissue was obtained at ~12, 24 and 3 hours after the last dose, respectively. Frozen tumor tissue and blood were analyzed for drug concentration (PK) and pathway modulation was analyzed in post-dose frozen tumor tissue and compared to reference tumor samples from GBM patients not treated with XL765 or XL147. Pharmacodynamic impact (PD) on the pathway, apoptosis and proliferation was examined by immunohistochemistry (IHC) in an FFPE tumor sample from each patient and compared to an archived tumor sample from an earlier surgery. Results: Enrollment is complete with 21 patients enrolled; 6, 6 and 7 were evaluable for the PK/PD analysis in cohorts 1, 2 and 3, respectively. The toxicity profiles for both drugs were consistent with previous studies. PK analyses revealed a mean tumor to plasma ratio of 0.38 and 0.40 in cohorts 1 and 3 and 0.27 in cohort 2. PD analysis by IHC revealed reduction compared to archived tumor in pS6K1 in 4/6 and 7/7 patients in cohorts 1 and 3 and 6/6 patients in cohort 2. Reduction in Ki67 was observed in 6/6 and 5/7 patients in cohorts 1 and 3 and 4/6 patients in cohort 2. Conclusions: XL765 when given on a QD or BID schedule to patients with glioma yields moderately higher distribution of XL765 into CNS tumor compared to XL147 based on tumor to plasma ratios. Decreases in pS6K1, consistent with pathway inhibition, and decreases in Ki67, consistent with inhibition of proliferation, were observed following treatment with both XL147 and XL765. Clinical trial information: NCT01240460.

A combination of dual PI3K-mTOR inhibitor, GDC-0980, with PARP inhibitor plus carboplatin blocked tumor growth of BRCA-competent triple-negative breast cancer cells.

2613 Background: PARP is a promising target in the TNBC. The PI3K pathway, in addition to its pro-proliferative effects on tumor cells, also controls the repair of DSB (Kumar, 2010; Friedman, 2009; Juvekar, 2012; Ibrahim, 2012). We hypothesize that the growth of TNBC tumor will be blocked due to the inhibition of (1) HR / NHEJ, and (2) PI3K-mTOR pathway mediated survival signals following GDC-0980 (G), when combined with PARP inhibitor (impaired DNA-SSB-repair), and carboplatin (C) (increased DNA-DSB). Methods: We tested the in vivo efficacy of a combination of G with ABT888 (A) plus C in BRCA-competent TNBC cells, and compared the triple combination arm (A+C+G) with a combination arm of A+C+ pan PI3K inhibitor GDC-0941 in mice xenograft (MDA-MB231) model. Mechanistically, we tested, (1) cell survival/ proliferation (5-7 BRCA-wt and mutant cell lines) using MTT assay, CelltiterGLO, and cell cycle analyses, (2) anchorage independent and dependent clonogenic growth (3D ON-TOP and soft-agar assay), (3) apoptosis, and (4) cell signaling marker(s). Results: (1) G alone decreased the tumor growth by 40-50%. (2) The G + A + C combination blocked the growth of established tumors by 90% as compared to control(s). (3) Interestingly, G + A + C combination had markedly higher percentage of inhibition of tumor growth than the inhibition observed in the A+C+GDC-0941 arm (36%). (4) G treatment time-dependently increased cl-caspase 3, 9, and cl-PARP and increased AnnexinV positivity both time (24 and 48 hrs) and dose dependently (50 and 200 nM) in cells. (5) G when combined with A+C was most effective in inducing apoptosis in PIK3CA-mutated BT20 cells. (6) The triple combination (50 nM G+10 μM A + 10 μM C) inhibited clonogenic growth of MDA-MB231, MDA-MB468 and BT20 cells. (7)Treatment of G decreased downstream effectors of PI3K-mTOR pathway, pAKTS473, pP70S6K and pS6RP. Conclusions: Tumor growth of BRCA-competent TNBC cells is blocked by a combination treatment of G with A+C. The profound anti-tumor effect of this triple combination can be explained by the anti-proliferative and pro-apoptotic actions of the drugs. The combination of G with A+C merits further investigation in TNBC.

Ph III randomized studies of the oral pan-PI3K inhibitor buparlisib (BKM120) with fulvestrant in postmenopausal women with HR+/HER2– locally advanced or metastatic breast cancer (BC) after aromatase inhibitor (AI; BELLE-2) or AI and mTOR inhibitor (BELLE-3) treatment.

TPS650^ Background: The PI3K/Akt/mTOR pathway is commonly dysregulated in BC and linked to resistance to endocrine therapy, including AIs. Buparlisib is an oral inhibitor of class I PI3K isoforms (α, β, γ, δ), showing near complete tumor regression in an in vivo model of ER+ BC in combination with fulvestrant. Preliminary clinical activity in BC pts has been observed with buparlisib as a single agent and in combination with letrozole (Mayer et al. ASCO 2012). Methods: BELLE-2 (NCT01610284) and BELLE-3 (NCT01633060) are 2 Ph III randomized, double-blind, placebo-controlled trials of oral buparlisib (100 mg/d) in combination with fulvestrant (500 mg) given on D1 and 15 of Cycle 1 and D1 of each 28-day cycle thereafter in postmenopausal women with HR+/HER2– locally advanced/metastatic BC. Additional eligibility criteria: disease progression on/after AI treatment for BELLE-2; prior treatment with AI and progression on mTOR inhibitor in combination with endocrine therapy (as last therapy prior to study entry) for BELLE-3; ≤1 previous line of chemotherapy for advanced disease; available archival tumor tissue for PI3K-related biomarker analysis. Randomization to either arm (1:1 for BELLE-2 and 2:1 for BELLE-3) is stratified according to PI3K pathway activation status (activated vs non-activated vs unknown) and visceral disease status (present vsabsent). Study treatment is given until disease progression or discontinuation for any reason. Co-primary endpoints in both trials: PFS in full and PI3K pathway-activated populations based on local investigator assessment (RECIST 1.1). Key secondary endpoints: overall survival (OS) in full and PI3K pathway-activated populations; other secondary endpoints: PFS and OS in PI3K non-activated/unknown population, overall response rate, clinical benefit rate, safety (CTCAE 4.03), PK, and patient-reported outcomes (EORTC QLQ-C30 and QLQ-BR23). Estimated enrollment is 842 pts for BELLE-2 and 615 for BELLE-3. As of January 2013, 96 pts were randomized to BELLE-2 and 1 pt to BELLE-3. Recruitment is ongoing globally. Clinical trial information: NCT01610284 and NCT01633060.

PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-l-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.

PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1/PD-L1 in airway epithelium (P3199)

Abstract B7-H1/PD-L1 is implicated in the escape of viruses from the host defense systems, leading to persistence of infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. We previously reported that stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC), upregulated the expression of B7-H1 via activation of NF-κB. The mechanism of upregulation was further investigated with regard to involvement of phosphatidylinositol 3-kinases. Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by a chemical inhibitor or a small interfering RNA for PI3Kδ. The expression of p110δ was detected by Western blot and suppressed by PI3Kδ siRNA. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. The transcriptional activity of NF-κB is regulated by histone deacetylase. The activity of histone deacetylase was suppressed by poly IC, which was protected by a PI3Kδ inhibitor. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 in airway mucosa and a causal link between oxidative stress and epigenetic regulation in virus-associated B7-H1 upregulation.

CpG-ODN Attenuates Pathological Cardiac Hypertrophy and Heart Failure by Activation of PI3Kα-Akt Signaling

Phosphoinositide-3-kinase α (PI3Kα) represents a potential novel drug target for pathological cardiac hypertrophy (PCH) and heart failure. Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are classic agonists of Toll-like receptor 9 (TLR9), which typically activates PI3K-Akt signaling in immune cells; however, the role of the nucleotide TLR9 agonists in cardiac myocytes is largely unknown. Here we report that CpG-ODN C274 could both attenuate PCH and improve cardiac dysfunction by activating PI3Kα-Akt signaling cascade. In vitro studies indicated that C274 could blunt reactivation of fetal cardiac genes and cell enlargement induced by a hypertrophic agent, isoproterenol. The anti-hypertrophic effect of C274 was suppressed by a pan-PI3K inhibitor, LY294002, or a small interfering RNA targeting PI3Kα. In vivo studies demonstrated that PCH, as marked by increased heart weight (HW) and cardiac ANF mRNA, was normalized by pre-administration with C274. In addition, Doppler echocardiography detected cardiac ventricular dilation, and contractile dysfunction in isoproterenol-treated animals, consistent with massive replacement fibrosis, reflecting cardiac cell death. As expected, pre-treatment of mice with C274 could prevent cardiac dysfunction associated with diminished cardiac cell death and fibrosis. In conclusion, CpG-ODNs are novel cardioprotective agents possessing antihypertrophic and anti-cell death activity afforded by engagement of the PI3Kα-Akt signaling. CpG-ODNs may have clinical use curbing the progression of PCH and preventing heart failure.

Abstract 2713: The Pten and p53 tumor suppressors cooperate to constrain the PI3K/mTOR pathway activation.

Abstract Loss of Pten expression and mutational inactivation of p53 are frequent molecular events in liver cancer associated with advanced disease stage and poor prognosis. The tumor suppressor PTEN is a phosphoinositide phosphatase that antagonizes the class IA PI3K-engaged oncogenic signaling by dephosphorylating PtdIns(3,4,5)P(3); however, the specific roles of class IA PI3K isoforms, p110a and p110b, have not been assessed in genetically engineered mouse models of liver cancer with Pten inactivation. Furthermore, effective treatment strategies for Pten and p53 deficient liver malignancy remain to be defined. In this study, we showed that combined deletion of Pten and p53 in liver led to rapid development of hepatocellular carcinoma and cholangiocellular carcinoma, the two most common types of liver cancer, with complete penetrance. While a single ablation of p110a or p110b markedly reduced the tumor burden, inactivation of both p110a and p110b completely blocked the liver tumorigenesis. Although liver tumors driven by inactivation of Pten and p53 showed a good response to GDC-0941, a pan-PI3K inhibitor, the combined treatment with both GDC-0941 and Rad001, an mTOR inhibitor or single-agent BEZ235, a dual pan-PI3K and mTOR inhibitor, led to marked tumor regression. These studies suggest that Pten and p53 cooperate to constrain the PI3K/mTOR pathway activation and inhibitors targeting both PI3K and mTOR may effectively treat Pten and p53 deficient liver cancers. Citation Format: Pixu Liu, Hailing Cheng, Lynn Symonds, Erbo Xu, Sauveur-Michel Maira, Emmanuelle di Tomaso, Thomas Roberts, Jean Zhao. The Pten and p53 tumor suppressors cooperate to constrain the PI3K/mTOR pathway activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2713. doi:10.1158/1538-7445.AM2013-2713

Abstract 4472: PI3K pathway dependencies in endometrioid endometrial cancer.

Abstract Background: Endometrioid endometrial cancers (EECs) frequently harbor coexisting mutations in PI3K pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to determine the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cells rely on p110β signaling for survival. Methods: Twenty-four human EEC cell lines were characterized for their mutation profile using the Sequenom OncoCarta Panel v1.0 and Sanger sequencing for PTEN, PIK3CA, and PIK3R1 transcripts, and their protein and phospho-protein expression levels of PI3K and MAPK signaling pathway components. The cell line panel was treated with pan-class I PI3K, p110α and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, MEK and RAF inhibitors, and the correlations between protein expression, gene mutations, and sensitivity to targeted therapeutics determined. RNA interference was employed to assess effects of KRAS silencing on EEC cells. Results: The prevalence of PI3K pathway mutations in the EEC cell line panel studied mirrored that reported for primary EECs. PI3K pathway activation as determined by levels of AKTSer473 phosphorylation was significantly associated with PTEN but not PIK3CA or KRAS mutation status. EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor Temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition. Only 2/6 KRAS-mutant EEC cell lines showed response to MEK inhibition, and KRAS siRNA silencing did not induce apoptosis in KRAS-mutant EEC cells. EEC cell lines harboring PTEN mutations were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only combination with the p110α selective inhibitor A66 resulted in a decrease in cell viability. Conclusions: Our findings provide evidence to suggest that selective pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA- and PTEN-mutant cancers, respectively, and that these PI3K inhibitors may even be effective in a subset of EECs concurrently harboring KRAS mutations. Our results further demonstrate that p110β inhibition alone is not sufficient to sensitize PTEN-mutant EEC cells, suggesting that combination with other targeted agents may be required to increase efficacy. Citation Format: Britta Weigelt, Patricia H. Warne, Maryou B. Lambros, Jorge S. Reis-Filho, Julian Downward. PI3K pathway dependencies in endometrioid endometrial cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4472. doi:10.1158/1538-7445.AM2013-4472

Abstract 4471: Combined blockade of PI3K/AKT and EGFR/HER3 enhances antitumor activity in triple negative breast cancer.

Abstract Background: Up to 60% of triple negative breast cancers (TNBCs) express high levels of EGFR. Moreover, TNBCs are associated with increased frequency of phosphatase and tension homologue (PTEN) loss of function, leading to hyperactivation of the phosphoinositide 3-kinase (PI3K)/AKT pathway. This provides the rationale for using PI3K/AKT inhibitors in this subset of patients. However, compensatory expression of receptor tyrosine kinases (RTKs) such as HER3 may limit efficacy of PI3K/AKT inhibitors. Therefore, we hypothesize that combined targeting of both EGFR and HER3 and the PI3K/AKT pathway will result in superior antitumor activity compared to single agent in TNBC. Materials and Methods: Several TNBC cell lines were treated with MEHD7945A, a dual-action antibody that targets both EGFR and HER3, AKT inhibitor GDC-0068, and pan-PI3K inhibitor GDC-0941. Cell viability was measured by CellTiter-Glo and Crystal Violet. Both cell line- and patient-derived xenograft models of TNBC were treated with MEHD7945A, GDC-0068, GDC-0941, or the combination of MEHD7945A with either GDC-0068 or GDC-0941. Tumor size and histology were examined. Protein expression was measured by Western blot, Mass Spectometry, CEER and immunohistochemistry. Results: GDC-0068 and GDC-0941 treatment resulted in variable inhibition of cell viability, with IC50s ranging from 170 nM to >1 μM across all TNBC cell lines. In cells stimulated with either EGF or Heregulin MEHD7945A prevented EGFR/HER3 receptors phosphorylation and improved the antiproliferative activity of the PI3K/AKT inhibitors. To test the activity of these compounds in vivo we used three different models of TNBC, two cell line (MDA-MB-468 and HCC70)-based and a patient-derived xenograft. MEHD7945, GDC-0941 and GDC-0068 showed variable delay in tumor growth whereas combination of MEHD7945A with either GDC-0068 or GDC-0941 was always superior to single agent treatment. Both combinations either prevented tumor growth or led to tumor shrinkage with complete responses achieved in 1/2 mice in each cohorts. Of note, all the treatments (up to 9 weeks of therapeutic exposure) were well tolerated. Analysis of treated tumors reveals potent inhibition of the PI3K/AKT pathway, with decreased levels phospho-PRAS40 and phospho-S6. Moreover, we confirmed that MEHD7945A effectively prevented EGFR and HER3 phosphorylation consequent to PI3K inhibition. Conclusions: Combined therapy with MEHD7945A and either GDC-0068 or GDC-0941 was superior to monotherapy in preclinical models of TNBC. We are currently exploring the levels of expression/activation of EGFR and HER3 following PI3K/Akt blockade in samples from patients treated with GDC-0068. These findings provide the rationale for combined targeting of PI3K/AKT and EGFR/HER3 in triple negative breast cancers. Citation Format: Maurizio Scaltriti, Jessica Tao, Dejan Juric, Neil Auricchio, Pau Castel, Natasha Morse, Phillip Kim, Sharat Singh, Saswati Hazra, Todd Hembrough, Jon Burrows, Jose Baselga. Combined blockade of PI3K/AKT and EGFR/HER3 enhances antitumor activity in triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4471. doi:10.1158/1538-7445.AM2013-4471

Abstract 3258: Discovery and characterization of SAR260301, a novel PI3Kβ-selective inhibitor in clinical development for the treatment of PTEN-deficient tumors .

Abstract The PI3K/mTOR pathway is involved in promoting tumor cell proliferation, survival and metastasis. The development of new anticancer therapies targeting different components of this pathway has been motivated by the identification of somatic PIK3CA missense mutations as well as the high frequency of loss of negative regulatory proteins such as the tumor suppressor PTEN. In this last case, the abnormal activation of downstream effectors is mediated by PI3Kβ. PTEN deficiency has also been reported to be involved in the resistance to a variety of anticancer therapies. The preceding findings support investigation into selective modulators of the lipid kinase activity of p110β as agents for the treatment of PTEN-deficient tumors, with potentially reduced on-target adverse events described for pan-PI3K inhibitors currently undergoing clinical trials. SAR260301 is a selective PI3Kβ lipid kinase inhibitor that has demonstrated higher potency in vitro on p110β (IC50= 52 nM) than on p110δ and p110α isoforms and no or weak activity against p110γ, mTOR and a wide range of protein kinases (>400 enzymes). The potency and selectivity against the intended target was further confirmed in mechanistic cellular models (IC50 = 32 nM, p110β; 26- and 88-fold selective over p110δ and p110α, respectively). Moreover, SAR260301 also modulates pAkt levels exclusively in PTEN-deficient tumor cell lines with IC50 values ranging from 39 to 310 nM and from 20 to 209 nM for pAkt-S473 and pAkt-T308, respectively. When administered orally, the compound shows dose and time-dependent inhibition of the PI3K pathway in human PTEN-deficient tumor xenografts in mice. In these models, target engagement correlates with compound exposure and is associated with good tolerability. The MAPK pathway also plays an important role in cancer and is often constitutively and simultaneously activated with the PI3K pathway. Furthermore, PTEN loss has been shown to correlate with resistance to MAPK inhibitors. Preclinical data evidencing the ability of SAR260301 to modulate pAkt levels in genetic contexts where PTEN deficiency co-exists with MAPK pathway activation, as well as the effects of combining SAR260301 with MAPK inhibitors will also be presented. The foregoing studies support ongoing Phase I clinical investigation of SAR260301 in patients with PTEN-deficient malignancies. Citation Format: Angela Virone-Oddos, Hélène Bonnevaux, Olivier Lemaitre, Loic Vincent, Franck Halley, Brigitte Demers, Véronique Charrier, Olivier Courtin, Stéphane Guérif, Laurent Besret, Pierre-Yves Abecassis, Sylvaine Cartot-Cotton, Gary Emmons, Alan Roberts, David Compton, Bin Wu, Laurent Schio, Christoph Lengauer, Carlos Garcia-Echeverria. Discovery and characterization of SAR260301, a novel PI3Kβ-selective inhibitor in clinical development for the treatment of PTEN-deficient tumors . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3258. doi:10.1158/1538-7445.AM2013-3258

The Tandem PH Domain-Containing Protein 2 (TAPP2) Regulates Chemokine-Induced Cytoskeletal Reorganization and Malignant B Cell Migration

The intracellular signaling processes controlling malignant B cell migration and tissue localization remain largely undefined. Tandem PH domain-containing proteins TAPP1 and TAPP2 are adaptor proteins that specifically bind to phosphatidylinositol-3,4-bisphosphate, or PI(3,4)P2, a product of phosphoinositide 3-kinases (PI3K). While PI3K enzymes have a number of functions in cell biology, including cell migration, the functions of PI(3,4)P2 and its binding proteins are not well understood. Previously we found that TAPP2 is highly expressed in primary leukemic B cells that have strong migratory capacity. Here we find that SDF-1-dependent migration of human malignant B cells requires both PI3K signaling and TAPP2. Migration in a transwell assay is significantly impaired by pan-PI3K and isoform-selective PI3K inhibitors, or by TAPP2 shRNA knockdown (KD). Strikingly, TAPP2 KD in combination with PI3K inhibitor treatment nearly abolished the migration response, suggesting that TAPP2 may contribute some functions independent of the PI3K pathway. In microfluidic chamber cell tracking assays, TAPP2 KD cells show reduction in percentage of migrating cells, migration velocity and directionality. TAPP2 KD led to alterations in chemokine-induced rearrangement of the actin cytoskeleton and failure to form polarized morphology. TAPP2 co-localized with the stable F-actin-binding protein utrophin, with both molecules reciprocally localizing against F-actin accumulated at the leading edge upon SDF-1 stimulation. In TAPP2 KD cells, Rac was over-activated and localized to multiple membrane protrusions, suggesting that TAPP2 may act in concert with utrophin and stable F-actin to spatially restrict Rac activation and reduce formation of multiple membrane protrusions. TAPP2 function in cell migration is also apparent in the more complex context of B cell migration into stromal cell layers – a process that is only partially dependent on PI3K and SDF-1. In summary, this study identified TAPP2 as a novel regulator of malignant B cell migration and a potential therapeutic intervention target.

Trastuzumab-Resistant Cells Rely on a HER2-PI3K-FoxO-Survivin Axis and Are Sensitive to PI3K Inhibitors

The antibody trastuzumab is approved for treatment of patients with HER2 (ERBB2)-overexpressing breast cancer. A significant fraction of these tumors are either intrinsically resistant or acquire resistance rendering the drug ineffective. The development of resistance has been attributed to failure of the antibody to inhibit phosphoinositide 3-kinase (PI3K), which is activated by the HER2 network. Herein, we examined the effects of PI3K blockade in trastuzumab-resistant breast cancer cell lines. Treatment with the pan-PI3K inhibitor XL147 and trastuzumab reduced proliferation and pAKT levels, triggering apoptosis of trastuzumab-resistant cells. Compared with XL147 alone, the combination exhibited a superior antitumor effect against trastuzumab-resistant tumor xenografts. Furthermore, treatment with XL147 and trastuzumab reduced the cancer stem-cell (CSC) fraction within trastuzumab-resistant cells both in vitro and in vivo. These effects were associated with FoxO-mediated inhibition of transcription of the antiapoptosis gene survivin (BIRC5) and the CSC-associated cytokine interleukin-8. RNA interference-mediated or pharmacologic inhibition of survivin restored sensitivity to trastuzumab in resistant cells. In a cohort of patients with HER2-overexpressing breast cancer treated with trastuzumab, higher pretreatment tumor levels of survivin RNA correlated with poor response to therapy. Together, our results suggest that survivin blockade is required for therapeutic responses to trastuzumab and that by combining trastuzumab and PI3K inhibitors, CSCs can be reduced within HER2(+) tumors, potentially preventing acquired resistance to anti-HER2 therapy.

An integrin-targeted, pan-isoform, phosphoinositide-3 kinase inhibitor, SF1126, has activity against multiple myeloma in vivo

Multiple reports point to an important role for the phosphoinositide-3 kinase (PI3K) and AKT signaling pathways in tumor survival and chemoresistance in multiple myeloma (MM). The goals of our study were: (1) to generate the preclinical results necessary to justify a Phase I clinical trial of SF1126 in hematopoietic malignancies including MM and (2) to begin combining pan-PI3K inhibitors with other agents to augment antitumor activity of this class of agent in preparation for combination therapy in Phase I/II trials. We determined the in vitro activity of SF1126 with 16 human MM cell lines. In vivo tumor growth suppression was determined with human myeloma (MM.1R) xenografts in athymic mice. In addition, we provide evidence that SF1126 has pharmacodynamic activity in the treatment of patients with MM. SF1126 was cytotoxic to all tested MM lines, and potency was augmented by the addition of bortezomib. SF1126 affected MM.1R cell line signaling in vitro, inhibiting phospho-AKT, phospho-ERK, and the hypoxic stabilization of HIF1α. Tumor growth was 94 % inhibited, with a marked decrease in both cellular proliferation (PCNA immunostaining) and angiogenesis (tumor microvessel density via CD31 immunostaining). Our clinical results demonstrate pharmacodynamic knockdown of p-AKT in primary patient-derived MM tumor cells in vivo. Our results establish three important points: (1) SF1126, a pan-PI3K inhibitor has potent antitumor activity against MM in vitro and in vivo, (2) SF1126 displays augmented antimyeloma activity when combined with proteasome inhibitor, bortezomib/Velcade(®), and (3) SF1126 blocks the IGF-1-induced activation of AKT in primary MM tumor cells isolated from SF1126-treated patients The results support the ongoing early Phase I clinical trial in MM and suggest a future Phase I trial in combination with bortezomib in hematopoietic malignancies.

Wnt-β-Catenin Pathway Regulates Vascular Mimicry in Triple Negative Breast Cancer

The Wnt-beta-catenin pathway (WP) regulates different aspects of cell fate, migration and polarity. Inappropriate deregulation of this pathway leads to oncogenic and metastatic changes. The Triple Negative (TN) subset of BC (the absence of hormone receptors and absence of amplification/overexpression of HER2 receptors) exhibits aggressive clinical behavior and has poor clinical outcome. We have recently identified WP as one of the key signature pathways in TNBC associated with metastasis [1,2]. Here we report for the first time that WP controls metastasis-associated phenotype of Vascular Mimicry (VM) in TNBC. We have established 2D and 3D models to study VM in this subset of BC and characterized different TNBC cell lines in terms of VM and identified the role of WP in the regulation of VM. To establish the role of WP in the regulation of VM in TNBC, we have used (1) a pharmacological inhibitor of beta-catenin which is a functional readout of WP and (2) signaling modulators of WP. Since PI3K-pathway is an upstream regulator of WP, we have also used inhibitors of PI3K-pathway to test our hypothesis. Mechanistically (1) sulindac sulfide (pharmacological inhibitor of beta-catenin) and XAV939 (modulator of WP) mediated decrease in beta-catenin, (2) XAV939 mediated increase in axin and (3) LY294002 or SF1126 (pan PI3K inhibitors) mediated decrease in pGSK3beta caused an abrogation of VM in various TNBC cell lines including BT20, HCC1937, SUM149, MDA-MB231 and MDA-MB468. Finally we obtained genetic proof-of-concept using beta-catenin SiRNA. SiRNAmediated downregulation of WP abrogated VM demonstrating the involvement of WP in VM. Functionally, our data show that VM in brain specific metastatic TNBC cells is mediated via activation of WP.

L-Aminoacyl-triazine Derivatives Are Isoform-Selective PI3Kβ Inhibitors That Target Nonconserved Asp862 of PI3Kβ

A series of aminoacyl-triazine derivatives based upon the pan-PI3K inhibitor ZSTK474 were identified as potent and isoform selective inhibitors of PI3Kβ. The compounds showed selectivity based upon stereochemistry with L-amino acyl derivatives preferring PI3Kβ while their D-congeners favoured PI3Kδ. The mechanistic basis of this inhibition was studied using site-directed mutants. One Asp residue, D862 was identified as a critical participant in binding to the PI3Kβ-selective inhibitors distinguishing this class from other reported PI3Kβ-selective inhibitors. The compounds show strong inhibition of cellular Akt phosphorylation and growth of PTEN-deficient MD-MBA-468 cells.

The autophagic inhibitor 3‐methyladenine potently stimulates PKA‐dependent lipolysis in adipocytes

BACKGROUND AND PURPOSE The class III PI3K inhibitor, 3-methyladenine (3-MA), is commonly used to selectively block autophagy. Recent findings suggest a strong relationship between autophagy and lipid turnover. Here, we explore the effect of 3-MA on adipocyte lipolysis. EXPERIMENTAL APPROACH Assays were performed in 3T3-L1 cells. Cells were treated with 3-MA and wortmannin, a pan PI3K and autophagy inhibitor. Pharmacological and genetic manipulation of endogenous autophagic and lipolytic pathways was used to ascertain the contribution of 3-MA to the observed effects on lipolysis. KEY RESULTS 3T3-L1 cells that were exposed to 3-MA showed a consistent increase in lipolysis, approximately 50% over basal levels. The effect of 3-MA was not secondary to autophagic inhibition as treatment of 3T3-L1 cells with wortmannin yielded no such changes. Dosing and time course experiments showed that 3-MA's ability to activate lipolysis was more sensitive than its inhibitory effect on autophagy. Knockdown of adipose triglyceride lipase (ATGL) negated the stimulatory effect of 3-MA by >90%, indicating that 3-MA enhanced ATGL-dependent hydrolysis of triacylglycerols. Additionally, the lipolytic effect of 3-MA was dependent on the activation of PKA and 3-MA induced a rapid and potent elevation of intracellular cAMP levels in adipocytes. CONCLUSIONS AND IMPLICATIONS Cumulatively, we show that 3-MA potently modulated a cellular mechanism and its underlying signalling pathways not associated with autophagy. Furthermore, we describe a novel stimulatory effect on a major signalling pathway. Our findings provide valuable information to studies employing 3-MA as a specific inhibitor for PI3K and autophagy.