Small Molecule Inhibitors Of PI3K Research Articles

Pictilisib-Induced Resistance Is Mediated through FOXO1-Dependent Activation of Receptor Tyrosine Kinases in Mucinous Colorectal Adenocarcinoma Cells.

The phosphatidylinositol (PI3K)/AKT/mTOR axis represents an important therapeutic target to treat human cancers. A well-described downstream target of the PI3K pathway is the forkhead box O (FOXO) transcription factor family. FOXOs have been implicated in many cellular responses, including drug-induced resistance in cancer cells. However, FOXO-dependent acute phase resistance mediated by pictilisib, a potent small molecule PI3K inhibitor (PI3Ki), has not been studied. Here, we report that pictilisib-induced adaptive resistance is regulated by the FOXO-dependent rebound activity of receptor tyrosine kinases (RTKs) in mucinous colorectal adenocarcinoma (MCA) cells. The resistance mediated by PI3K inhibition involves the nuclear localization of FOXO and the altered expression of RTKs, including ErbB2, ErbB3, EphA7, EphA10, IR, and IGF-R1 in MCA cells. Further, in the presence of FOXO siRNA, the pictilisib-induced feedback activation of RTK regulators (pERK and pAKT) was altered in MCA cells. Interestingly, the combinational treatment of pictilisib (Pi3Ki) and FOXO1i (AS1842856) synergistically reduced MCA cell viability and increased apoptosis. These results demonstrate that pictilisib used as a single agent induces acute resistance, partly through FOXO1 inhibition. Therefore, overcoming PI3Ki single-agent adaptive resistance by rational design of FOXO1 and PI3K inhibitor combinations could significantly enhance the therapeutic efficacy of PI3K-targeting drugs in MCA cells.

DHW-208, A Novel Phosphatidylinositol 3-Kinase (PI3K) Inhibitor, Has Anti-Hepatocellular Carcinoma Activity Through Promoting Apoptosis and Inhibiting Angiogenesis.

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide with high prevalence and lethality. Due to insidious onset and lack of early symptoms, most HCC patients are diagnosed at advanced stages without adequate methods but systemic therapies. PI3K/AKT/mTOR signaling pathway plays a crucial role in the progression and development of HCC. Aberrant activation of PI3K/AKT/mTOR pathway is involved in diverse biological processes, including cell proliferation, apoptosis, migration, invasion and angiogenesis. Therefore, the development of PI3K-targeted inhibitors is of great significance for the treatment of HCC. DHW-208 is a novel 4-aminoquinazoline derivative pan-PI3K inhibitor. This study aimed to assess the therapeutic efficacy of DHW-208 in HCC and investigate its underlying mechanism. DHW-208 could inhibit the proliferation, migration, invasion and angiogenesis of HCC through the PI3K/AKT/mTOR signaling pathway in vitro. Consistent with the in vitro results, in vivo studies demonstrated that DHW-208 elicits an antitumor effect by inhibiting the PI3K/AKT/mTOR-signaling pathway with a high degree of safety in HCC. Therefore, DHW-208 is a candidate compound to be developed as a small molecule PI3K inhibitor for the treatment of HCC, and our study provides a certain theoretical basis for the treatment of HCC and the development of PI3K inhibitors.

MBRS-18. TUMOR SUPPRESSOR p53 DEFINES THE THERAPEUTIC RESPONSES IN TREATMENT OF MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common primary pediatric malignant brain tumor. Current molecular analysis classifies MB into 4 groups, classic (WNT), sonic hedgehog (Shh), group 3, and group 4. Furthermore, atypical p53 signaling is associated with disease progression and confers poor prognosis. This study investigated the correlation of mutational status of p53 and iSO17q with disease progression and metastatic potential. In addition, we used small molecule inhibitors of PI3K (Buparlisib; BKM120) and HDAC (LBH-589) on a p53-mutant MB cell line to find novel therapeutic targets. Efficacy of these drugs were assessed using functional assays (cell proliferation, migration, cell cycle and drug resistance). MB tumors (n=53) were evaluated for GLI-1, GAB-1, NPR, KV1, YAP expression and mutant p53 via immunohistochemistry and correlated to patient outcomes. Results demonstrated that: 1) high expression of GAB-1 and YAP were found in the Shh group, while KV1 expression was present in all subtypes; 2) mutant p53 expression was present in various subsets of MB with no apparent correlation with metastasis or disease progression; 3) patients displaying iSO17q (determined by fluorescence in situ hybridization (FISH) technique) exhibited metastatic disease; 4) LBH-589 and BKM120 caused both time and dose-dependent inhibition of MB cell proliferation and migration; 5) combined treatment of BKM120 and LBH-589 had a synergistic effect; 6) MB cells demonstrated drug-resistance to BKM120. In conclusion, these findings underscore use of Buparlisib and LBH-589 in treatment of MB. Further, the role of mutant p53 in disease progression remains elusive, whereas presence of iSO17q defines metastatic potential.

Phosphatidylinositol-3-kinase (PI3K)/Akt Signaling is Functionally Essential in Myxoid Liposarcoma.

Myxoid liposarcoma (MLS) is an aggressive soft-tissue tumor characterized by a specific reciprocal t(12;16) translocation resulting in expression of the chimeric FUS-DDIT3 fusion protein, an oncogenic transcription factor. Similar to other translocation-associated sarcomas, MLS is characterized by a low frequency of somatic mutations, albeit a subset of MLS has previously been shown to be associated with activating PIK3CA mutations. This study was performed to assess the prevalence of PI3K/Akt signaling alterations in MLS and the potential of PI3K-directed therapeutic concepts. In a large cohort of MLS, key components of the PI3K/Akt signaling cascade were evaluated by next generation seqeuncing (NGS), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). In three MLS cell lines, PI3K activity was inhibited by RNAi and the small-molecule PI3K inhibitor BKM120 (buparlisib) in vitro An MLS cell line-based avian chorioallantoic membrane model was applied for in vivo confirmation. In total, 26.8% of MLS cases displayed activating alterations in PI3K/Akt signaling components, with PIK3CA gain-of-function mutations representing the most prevalent finding (14.2%). IHC suggested PI3K/Akt activation in a far larger subgroup of MLS, implying alternative mechanisms of pathway activation. PI3K-directed therapeutic interference showed that MLS cell proliferation and viability significantly depended on PI3K-mediated signals in vitro and in vivo Our preclinical study underlines the elementary role of PI3K/Akt signals in MLS tumorigenesis and provides a molecularly based rationale for a PI3K-targeted therapeutic approach which may be particularly effective in the subgroup of tumors carrying activating genetic alterations in PI3K/Akt signaling components.

The therapeutic efficacy for targeting the PI3K signaling pathway for fighting breast, prostate and multiple myeloma cancer cells

The PI3K signaling pathway is involved in the regulation of cancer cell growth, motility, survival and metabolism. This pathway is frequently active in many different types of cancer as breast, prostate and multiple myeloma. Targetable genetic aberrations in this pathway give the researchers many opportunities for the development of targeted therapies for different types of cancer. The high frequency of mutations in this pathway in multiple types of cancer has led to the development of small-molecule inhibitors of PI3K, several of which are currently in clinical trials. However, several feedback mechanisms either within the PI3K pathway or in compensatory pathways can render tumor cells resistant to therapy. Here, we give insight into the importance of the PI3K pathway as a target for cancer therapy and discuss the potential clinical efficacy of PI3K inhibitors. We mainly focused on the roles of PI3K signaling pathway in three cancer cell types including breast cancer, prostate cancer and multiple myeloma cancer.

Abstract B097: NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy

Abstract Aberrant activation of the PI3K pathway leading to increases in AKT activity is seen in many prostate cancer patients refractory to androgen blockade therapy. A number of small-molecule inhibitors of PI3K, including BKM120 (Novartis), have entered clinical trials. As with PI3K-AKT activation, highly expressed Pim kinase has been shown to drive the growth of prostate cancer. Recent studies have demonstrated that Pim kinase plays a critical role in promoting resistance of breast cancer to PI3K inhibition. Similarly, when the Pim-1 protein kinase is overexpressed, prostate cancer cell lines become resistant to PI3K inhibitor. In both tissue culture and animal models, the combination of a Pim inhibitor, PIM447, and BKM120 acted synergistically in blocking the survival and growth of prostate cancer cells. We therefore investigated the biochemical mechanism by which Pim-1 mediates cell resistance to the PI3K inhibitor. Previously, we have demonstrated that knockout of Pim kinases led to accumulation of reactive oxygen species (ROS) accompanied by a lower level of expression of ROS scavengers. To explore whether Pim-1 overexpression reverses this phenotype in prostate epithelial cancer, using lentiviral transduction, we expressed Pim-1 in mouse prostate epithelial cancer cells derived from spontaneous prostate adenocarcinomas that arose in a mouse model in which Tp53 and Rb1 are deleted in prostate epithelium. Pim-1 overexpression promoted anchorage-independent growth and formation of mouse prostate epithelial tumors in vivo. Gene expression arrays demonstrated that Pim-1 induced an Nrf2 antioxidant response element gene signature, including increases in Heme oxygenase-1 (Hmox1), NAD(P)H quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase kappa1 (Gstk1) genes. Similarly, overexpression of Pim-1 in human prostate cancer LNCaP cells results in increased expression of Nrf2 and Hmox1 protein. As expected, depletion of Nrf2 with shRNA prevented Pim-1-mediated increases in Hmox1 expression, indicating that Nrf2 activation is required for the Pim-1 induction of ROS scavengers. In line with this result, in Pim-1 overexpressing cell lines knockdown of either Hmox1 or Nrf2 restored sensitivity to BKM120 treatment. A vital role of ROS scavengers in mediating the resistance to BKM120 was further confirmed by the treatment of cells and xenografted tumors with buthionine sulfoximine (inhibitor of glutathione synthesis), which resensitized the Pim-1 overexpressing cells to BKM120 via ROS induction. Our data further demonstrated that Pim-1 overexpression decreases the ubiquitination of Nrf2 protein, thus preventing its degradation, and increasing its half-life. Conversely, inhibition of Pim kinase promoted Nrf2 ubiquitination and proteasomal degradation. Taken together, our results show that the increased expression of Pim kinases in prostate cancer blocks therapeutic efficacy of PI3K inhibitor by inducing Nrf2 and antioxidant scavengers that inhibit the induction of ROS. Citation Format: Jin H. Song, Libia A. Leuvano, Neha Singh, Andrew S. Kraft. NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B097.

Role of the E3 ubiquitin ligase RNF157 as a novel downstream effector linking PI3K and MAPK signaling pathways to the cell cycle

The interconnected PI3K and MAPK signaling pathways are commonly perturbed in cancer. Dual inhibition of these pathways by the small-molecule PI3K inhibitor pictilisib (GDC-0941) and the MEK inhibitor cobimetinib (GDC-0973) suppresses cell proliferation and induces cell death better than either single agent in several preclinical models. Using mass spectrometry-based phosphoproteomics, we have identified the RING finger E3 ubiquitin ligase RNF157 as a target at the intersection of PI3K and MAPK signaling. We demonstrate that RNF157 phosphorylation downstream of the PI3K and MAPK pathways influences the ubiquitination and stability of RNF157 during the cell cycle in an anaphase-promoting complex/cyclosome–CDH1-dependent manner. Deletion of these phosphorylation-targeted residues on RNF157 disrupts binding to CDH1 and protects RNF157 from ubiquitination and degradation. Expression of the cyclin-dependent kinase 2 (CDK2), itself a downstream target of PI3K/MAPK signaling, leads to increased phosphorylation of RNF157 on the same residues modulated by PI3K and MAPK signaling. Inhibition of PI3K and MEK in combination or of CDK2 by their respective small-molecule inhibitors reduces RNF157 phosphorylation at these residues and attenuates RNF157 interaction with CDH1 and its subsequent degradation. Knockdown of endogenous RNF157 in melanoma cells leads to late S phase and G2/M arrest and induces apoptosis, the latter further potentiated by concurrent PI3K/MEK inhibition, consistent with a role for RNF157 in the cell cycle. We propose that RNF157 serves as a novel node integrating oncogenic signaling pathways with the cell cycle machinery and promoting optimal cell cycle progression in transformed cells.

Hypercapnia Inhibits Host Defense Against Influenza A Virus by Potentiating Virus‐induced Activation of the PI3K/Akt Pathway

RationaleHypercapnia, the elevation of CO2 in blood and tissue, commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary bacterial and viral infections, including influenza. We previously reported that hypercapnia downregulates several innate immune genes and increases the mortality of bacterial pneumonia and influenza A virus (IAV) infection in mice. High CO2 also increases viral nucleoprotein (NP) expression in the mouse lung, primarily in alveolar macrophages (MØs). In the current study, we explored the effect of hypercapnia on the PI3K/Akt pathway during IAV infection in MØs. In addition, using pharmacologic PI3K and Akt inhibitors we studied the role of hypercapnia‐induced activation of Akt on the expression of antiviral proteins, including MX dynamin like GTPase 1 (MX1), 2′‐5′‐oligoadenylate synthetase 1 (OAS1) and viperin; viral nonstructural 1 protein 1 (NS1) and NP.MethodsHuman alveolar and PMA‐differentiated THP‐1 MØs as well as murine alveolar and bone marrow derived MØs were cultured in 5% CO2 (PCO2 36 mmHg; normocapnia) or 15% CO2 (PCO2 108 mmHg; hypercapnia) and infected with IAV. Akt activation was assessed by immunoblot and immunofluorescence microscopy (IF). MX1, OAS1 and viperin mRNA and protein levels were determined using qPCR and immunoblot or IF, respectively. IAV replication was assessed using MDCK plaque assay, and NS1 and NP expression were determined by immunoblot or IF. For in vivo studies, mice were pre‐exposed to normoxic hypercapnia (10% CO2/21% O2) for 3 days, or air as control, then infected with IAV. Mice were sacrificed 4 days after infection for determination of antiviral and viral proteins expression in lung tissue by immunoblot and IF.ResultsHypercapnia potentiated IAV‐induced Akt activation in MØs in vivo and in vitro, an effect blocked by the PI3K inhibitor LY294002 and the Akt inhibitor MK2206. In addition, elevated CO2 decreased MX1, OAS1 and viperin mRNA and protein expression and increased viral replication and NS1 and NP proteins in IAV infected human and mouse MØs. Hypercapnia also increased viral protein expression in alveolar MØs, and decreased antiviral proteins and augmented viral titers in the lungs of IAV‐infected mice. We also found that the PI3K and Akt inhibitors blocked the hypercapnia‐induced suppression of antiviral proteins and the increase on viral proteins and IAV replication in vitro.ConclusionHypercapnia decreases antiviral proteins and increases viral NS1 and NP proteins and virus replication in MØs following IAV infection in vivo and in vitro. The effect of elevated CO2 on the macrophage antiviral response and virus production can be blocked by small molecule inhibitors of PI3K and Akt, suggesting a link between hypercapnia‐induced activation of the PI3K/Akt pathway and an impaired antiviral host response. Therefore, PI3K/Akt inhibitors may hold promise as pharmacotherapy to improve antiviral host defense in patients with advanced lung disease and hypercapnia.Support or Funding InformationR01HL107629, R56HL131745, P01AG049665

Counteracting PD-L1/PD-L2 Immune Suppression in Multiple Myeloma through Kinase Inhibition

Introduction: Multiple myeloma (MM) is clonal plasma cell malignancy that remains incurable. Checkpoint inhibitors represent a revolutionary form of cancer therapy that empowers the immune system to defeat cancer. Programmed death-1 (PD-1) is an inhibitory receptor expressed on immune cells, particularly cytotoxic T cells, that interacts with two ligands, PD-ligand 1 (PD-L1) and PD-L2 expressed on tumor cells. PD-L1 and PD-L2 engage PD-1 on the T cell surface to negatively modulate the magnitude of T-cell-mediated responses. Such negative feedback is critically important in maintaining homeostasis of the immune response to prevent autoimmunity during infection or inflammation in normal tissue. However, in cancers, it presents a major problem in blocking cellular antitumor responses. PD-L1/PD-L2 ligation with PD-1 provides a mechanism of immune escape for tumor cells by turning off the cytotoxic T cells. Currently available monoclonal antibodies (mAbs) that disrupt the PD-1/PD-L1 interaction have exhibited remarkable responses in selected patients and tumor types. However, mAbs demonstrate many drawbacks that include a lack of tumor cell specificity, low response rates in unselected patient populations and induction of de novoautoimmune disease that excludes many patients from therapy. We hypothesize that small molecule checkpoint inhibitors can provide greater specificity, shortened half-lives to diminish autoimmune or other adverse events, increased oral bioavailability, enhanced bio-efficacy, and higher stability at ambient temperature facilitating purification during production. Here, we identified novel, small molecule PI3K inhibitors that reduce PD-L1/PD-L2 levels on MM cells and enhance the antimyeloma activity of autologous T cells.Methods: To determine the effect of proteasome inhibitors on PD-L1/PD-L2 surface expression, RPMI8226 cells were incubated with bortezomib (BTZ), carfilzomib (CFZ) or ixazomib (IXZ) (1nM) for 36 h. Cells were then incubated in PBS/10% normal goat serum followed by antibodies (1/100) for 30 min at 4ºC and then treated with Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2. RPMI8226 cells were also incubated with the small molecule PI3K inhibitor DT97 (500nM) alone or combined with BTZ, CFZ, or IXZ. Cells were similarly stained using Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2 and a BD LSRFortessa™ cell analyzer was used for multicolor flow cytometry to acquire >10,000 events/sample. To determine the effect of DT97 on T cell-mediated cytolysis, bone marrow biopsy was performed on an MM patient and CD138+ cells were isolated by Miltenyi positive selection and CD4+ T cells by MojoSort™ CD4+ Human T Cell isolation. CD138+ cells were incubated with CD4+ T cells, proteasome inhibitors, or DT97 as indicated for 16 h at 37ºC. CD138+cells were then affinity-isolated, incubated with a FITC-conjugated anti-Annexin-V antibody and quantitated using a BD LSRFortessa™ cell analyzer.Results: Treatment of MM cells with BTZ, CFZ, or IXZ significantly increased the surface expression of PD-L1 and PD-L2 (Fig. 1). In contrast, treatment with the PI3K inhibitor DT97 suppressed the expression of PD-L1 and PD-L2 on MM cells (Fig. 2). Importantly, DT97 co-treatment with either BTZ, CFZ, or IXZ suppressed the induction of PD-L1 and PD-L2 seen after treatment with proteasome inhibitors alone. Treatment with the proteasome inhibitors alone or combined with T cells did not promote significant killing of MM cells (Fig. 3). However, DT97 treatment significantly enhanced autologous T cell-mediated MM death and the effect was further enhanced by addition of proteasome inhibitors.Conclusions: The results presented here establish the proof-of-principle that small molecule PI3K inhibitors reduce PD-L1/PD-L2 levels on tumor cells and enhance the anti-myeloma effect of autologous T cells. Small molecules checkpoint inhibitors represent a safe therapeutic alternative that can avoid the problems associated with antibodies, while retaining their functionality. Taken together, our studies indicate the promise of small molecules checkpoint inhibitors and support further translational and clinical development as a transformative form of cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Systematic Functional Characterization of Resistance to PI3K Inhibition in Breast Cancer.

PIM kinase overexpression confers resistance to small-molecule PI3K inhibitors. Combined inhibition of PIM and PI3K may therefore be warranted in a subset of breast cancers. Cancer Discov; 6(10); 1134-47. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069.

Abstract 4634: Novel dual HDAC & PI3K inhibitor, CUDC-907, for MYC-driven malignancies

Abstract MYC family genes are among the most frequently deregulated oncogenic drivers in human cancer. Pharmacologic inhibition of HDAC activity and blockade of the PI3K pathway have both been shown to suppress MYC-induced transcription. HDAC activity is critical for MYC gene regulation, as MYC represses transcription of target genes through recruitment of HDACs. HDAC inhibitors have been shown to restore expression of genes suppressed by MYC family members and to induce rapid downregulation of expression of MYC itself. The PI3K pathway plays a central role in regulating MYC at the post-transcriptional level. Activation of PI3K signaling leads to activation of AKT, which phosphorylates and inhibits GSK3β. As GSK3β normally phosphorylates MYC which facilitates the degradation of MYC, activation of PI3K signaling leads to increased stability of MYC, whereas PI3K inhibitors decrease MYC stability. A recent study has demonstrated addiction to MYC signaling and hypersensitivity to PI3K inhibition in PTEN-deficient diffuse large B-cell (DLBCL) cell lines, suggesting that MYC-driven cancers may be particularly sensitive to PI3K inhibition. As HDACs and PI3K regulate MYC protein levels and functions through nonoverlapping mechanisms, simultaneous HDAC and PI3K inhibition may further enhance MYC suppression. CUDC-907 is an orally bioavailable, small-molecule dual HDAC and PI3K inhibitor that primarily inhibits class I and II HDACs and the PI3Kα, β, and δ isoforms. CUDC-907 shows greater anti-tumor activity in vitro than single-target HDAC or PI3K inhibitors, especially in MYC-dependent cell types, such as DLBCL and NUT midline carcinoma (NMC). In preclinical testing, CUDC-907 treatment leads to a dose-dependent decrease in MYC protein levels, and is also more potent in decreasing MYC than the HDAC inhibitor panobinostat and the pan-PI3K inhibitor pictilisib alone or in combination. Significant antitumor effects have been consistently observed in MYC-driven DLBCL xenograft and genetically engineered mouse models exposed to CUDC907. In particular, certain MYC translocation (Daudi), double-hit (concurrent MYC and BLC2 translocation, WSUDLCL2 and DOHH2), double-expresser (expression of MYC and BCL2 proteins, U2932) xenograft models, and the Eμ-Myc transgenic mouse model achieve tumor growth inhibition of 100%, 69%, 56%, 97% and 72%, respectively. These findings raise the possibility that hematologic and solid tumors driven by aberrant overexpression of MYC family genes (e.g., MYC-altered DLBCL and NMC) might be more responsive to simultaneous HDAC and PI3K inhibition with CUDC-907 than they are to single-target therapy. Clinical Phase 1 studies are currently testing CUDC-907 in patients with relapsed/refractory (R/R) DLBCL and advanced MYC-aberrant solid tumors. Preliminary data are encouraging and support the planned Phase 2 study in R/R MYC-altered DLBCL, as well as further testing in other MYC-driven malignancies. Citation Format: Kaiming Sun, Ruzanna Atoyan, Mylissa A. Borek, Steven Dellarocca, Maria E. Samson, Anna W. Ma, Guangxin Xu, Troy Patterson, David P. Tuck, Jaye L. Viner, Ali Fattaey, Jing Wang. Novel dual HDAC & PI3K inhibitor, CUDC-907, for MYC-driven malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4634.

Molecular Pathways: Targeting the PI3K Pathway in Cancer-BET Inhibitors to the Rescue.

The PI3K signaling pathway is a complex and tightly regulated network that is critical for many physiologic processes, such as cell growth, proliferation, metabolism, and survival. Aberrant activation of this pathway can occur through mutation of almost any of its major nodes and has been implicated in a number of human diseases, including cancer. The high frequency of mutations in this pathway in multiple types of cancer has led to the development of small-molecule inhibitors of PI3K, several of which are currently in clinical trials. However, several feedback mechanisms either within the PI3K pathway or in compensatory pathways can render tumor cells resistant to therapy. Recently, targeting proteins of the bromodomain and extraterminal (BET) family of epigenetic readers of histone acetylation has been shown to effectively block adaptive signaling response of cancer cells to inhibitors of the PI3K pathway, which at least in some cases can restore sensitivity. BET inhibitors also enforce blockade of the MAPK, JAK/STAT, and ER pathways, suggesting they may be a rational combinatorial partner for divergent oncogenic signals that are subject to homeostatic regulation. Here, we review the PI3K pathway as a target for cancer therapy and discuss the potential use of BET inhibition to enhance the clinical efficacy of PI3K inhibitors. Clin Cancer Res; 22(11); 2605-10. ©2016 AACR.

Combination Nanopreparations of a Novel Proapoptotic Drug - NCL-240, TRAIL and siRNA.

To develop a multifunctional nanoparticle system carrying a combination of pro-apoptotic drug, NCL-240, TRAIL [tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand] and anti-survivin siRNA and to test the combination preparation for anti-cancer effects in different cancer cells. Polyethylene glycol-phosphoethanolamine (PEG-PE) - based polymeric micelles were prepared carrying NCL-240. These micelles were used in combination with TRAIL-conjugated micelles and anti-survivin siRNA-S-S-PE containing micelles. All the micelles were characterized for size, zeta potential, and drug encapsulation efficiency. Different cancer cells were used to study the cytotoxicity potential of the individual as well as the combination formulations. Other cell based assays included cellular association studies of transferrin-targeted NCL-240 micelles and study of cellular survivin protein downregulation by anti-survivin siRNA-S-S-PE containing micelles. NCL-240 micelles and the combination NCL-240/TRAIL micelles significantly increased cytotoxicity in the resistant strains of SKOV-3, MCF-7 and A549 as compared to free drugs or single drug formulations. The NCL-240/TRAIL micelles were also more effective in NCI/ADR-RES cancer cell spheroids. Anti-survivin siRNA micelles alone displayed a dose-dependent reduction in survivin protein levels in A2780 cells. Treatment with NCL-240/TRAIL after pre-incubation with anti-survivin siRNA inhibited cancer cell proliferation. Additionally, a single multifunctional system composed of NCL-240/TRAIL/siRNA PM also had significant cytotoxic effects in vitro in multiple cell lines. These results demonstrate the efficacy of a combination of small-molecule PI3K inhibitors, TRAIL, and siRNA delivered by micellar preparations in multiple cancer cell lines.

Abstract IA06: PI 3-kinase and Akt at the interface of signaling and metabolism

Abstract The PI 3-Kinase and Akt signaling pathway plays a critical role in modulating all phenotypes associated with human malignancies, including cell survival, proliferation and metabolism. Recent studies point to a major role for Akt signaling in modulating changes in metabolic pathways, although the specific mechanisms are unknown. Using unbiased metabolic profiling, we have uncovered a role for oncogenic PI 3-K and Akt in sensing redox changes in cancer cells, specifically by up-regulating glutathione levels and enhancing glutathione biosynthetic flux. Mechanistically, this is mediated by mTORC1-mediated up-regulation of the oxidative pentose phosphate pathway and Nrf2 activation. These findings indicate a post-translational mechanism by which oncogenic Akt signaling directly activates Nrf2 in cancer. Enhanced glutathione biosynthesis provides several advantages to cancer cells, including oxidative stress resistance and anchorage-independent growth, and thus constitute a metabolic dependency that can be targeted as a therapeutic strategy within breast cancer cells driven by Akt hyperactivation. In a separate study, we show that the chemotherapy agent doxorubicin triggers rapid and sustained activation of Akt signaling in breast cancer lines. Small molecule inhibitors of PI3K or Akt block Akt signaling downstream of DNA damage and sensitize cells to the cytotoxicity of doxorubicin. We have identified MERIT40, a component of the BRCA1-A DNA damage repair complex, as a novel Akt substrate that is phosphorylated in doxorubicin-treated cells. MERIT40 phosphorylation is required for formation and stabilization of the multicomponent BRCA1-A complex in response to DNA damage and this contributes to the resolution of DNA damage. We propose that disruption of MERIT40 phosphorylation contributes to the ability of PI3K/Akt inhibitors to sensitize breast cancer cells to chemotherapy through disruption of BRCA1 A complex integrity which is required for DNA damage repair and cell survival in the DNA damage response. Citation Format: Alex Toker. PI 3-kinase and Akt at the interface of signaling and metabolism. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA06.

Abstract A22: Role of cap-dependent translation in response to upstream kinase-targeted therapy in colorectal cancer

Abstract Mutational activation of the PI3K/AKT and RAS/RAF/MEK/ERK pathways is associated with colorectal cancer (CRC) progression and metastasis. Several small-molecule inhibitors of PI3K, AKT, RAF and MEK have been tested in the clinic for the treatment of CRC but have shown only limited activity as a single agent. We find that a potential mechanism of AKT or ERK-independence is associated with redundant activation of eIF4E-initiated cap-dependent translation mediated by convergent regulation of the translation repressor 4E-BP1 by the AKT and ERK pathways. Combined inhibition of both pathways exhibits synergistic anti-CRC effects, both in vitro and in vivo, by effectively inhibiting cap-dependent translation via maximal activation of 4E-BP1 function. Specifically, we identify that survivin is a key translationally-regulated target of both pathways through convergence on the mTORC1/4E-BP1/eIF4E axis, and continuous translation of survivin by AKT and ERK signaling is crucial for CRC progression to metastasis. These findings reveal that the cap-dependent translation functions as a critical regulatory node that integrates oncogenic AKT and ERK signals for metastatic progression of CRC. Thus, targeting cap-dependent translation that could simultaneously block upstream oncogenic signals and their downstream targets may provide a promising therapeutic strategy for advanced CRC. Citation Format: Qing Ye, Weijia Cai, B. Mark Evers, David Solit, Neal Rosen, Qing-Bai She. Role of cap-dependent translation in response to upstream kinase-targeted therapy in colorectal cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A22.

Decatenation checkpoint-defective melanomas are dependent on PI3K for survival.

Melanoma cell lines are commonly defective for the G2-phase cell cycle checkpoint that responds to incomplete catenation of the replicated chromosomes. Here, we demonstrate that melanomas defective for this checkpoint response are less sensitive to genotoxic stress, suggesting that the defective cell lines compensated for the checkpoint loss by increasing their ability to cope with DNA damage. We performed an siRNA kinome screen to identify kinases responsible and identified PI3K pathway components. Checkpoint-defective cell lines were three-fold more sensitive to small molecule inhibitors of PI3K. The PI3K inhibitor PF-05212384 promoted apoptosis in the checkpoint-defective lines, and the increased sensitivity to PI3K inhibition correlated with increased levels of activated Akt. This work demonstrates that increased PI3K pathway activation is a necessary adaption for the continued viability of melanomas with a defective decatenation checkpoint.

Dual mTORC1 and mTORC2 inhibitor Palomid 529 penetrates the Blood–Brain Barrier without restriction by ABCB1 and ABCG2

Palomid 529, a novel dual mTORC1/2 inhibitor has displayed interesting activities in experimental models and is a candidate for clinical evaluation. We have assessed the interaction of Palomid 529 with ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-gp/P-glycoprotein) and ABCG2 (BCRP/Breast Cancer Resistant Protein) by in vitro transwell assays, and their effects on the brain penetration using drug disposition analysis of i.v. and oral Palomid 529 in wild-type (WT) and Abcb1 and/or Abcg2 knockout (KO) mice. Palomid 529 lacked affinity for these transporters in vitro, in contrast to GDC-0941, a small molecule PI3K inhibitor, which we used as control substance for in vitro transport. The plasma AUCi.v. of micronized and DMSO formulated Palomid 529 was similar in WT and KO mice. Importantly, the brain and brain tumor concentration of Palomid 529 at a high dose (54 mg/kg) was also similar in both strains, whereas a less than 1.4-fold difference (p < 0.05) was found at the low (5.4 mg/kg) dose. Because of poor solubility, the oral bioavailability of micronized Palomid 529 was only 5%. Olive oil or spray-dried formulation greatly improved the bioavailability up to 50%. Finally, Palomid 529 effectively inhibits the orthotopic U87 glioblastoma growth. In summary, Palomid 529 is the first mTOR targeting drug lacking affinity for ABCB1/ABCG2 and having good brain penetration. This warrants further evaluation of Palomid 529 for treatment of high-grade gliomas and other intracranial malignancies.

Initial testing (stage 1) of the phosphatidylinositol 3′ kinase inhibitor, SAR245408 (XL147) by the pediatric preclinical testing program

Activation of the PI3 kinase pathway occurs frequently in many adult cancers and is implicated in tumor cell proliferation, survival, and resistance to chemotherapy and radiotherapy. However, less is known regarding the relevance of this pathway in pediatric cancers. Here we have evaluated SAR245408, a novel small molecule PI3K inhibitor, against childhood cancer cell lines and xenografts. SAR245408 was tested against the PPTP in vitro cell line panel at concentrations from 10 to 100 µM and against the PPTP in vivo xenograft panels at a dose of 100 mg/kg administered orally daily × 14. In vitro SAR245408 demonstrated cytotoxic activity, with a median relative IC50 value of 10.9 µM (range 2.7-24.5 µM). SAR245408 was well tolerated in vivo, and all 44 tested xenograft models were evaluable for efficacy. SAR245408 induced significant differences in EFS distribution compared to control in 29 of 37 (79%) of solid tumor xenografts and in two of seven (29%) ALL xenografts. SAR245408 induced tumor growth inhibition meeting criteria for intermediate EFS T/C activity (EFS T/C > 2) in 4 of 37 (11%) solid tumor xenografts. Intermediate EFS T/C activity was also observed for two of seven (29%) evaluable ALL xenografts. Objective responses were not observed for solid tumor or for ALL xenografts. Under the conditions evaluated in this study, SAR245408 achieved modest single-agent activity against most PPTP preclinical models. Further exploration of SAR245408 in combination with standard agents or with other signaling inhibitors could be considered.

Abstract 901: Mitotic kinase inhibitors synergize with PI3K and Akt inhibitors in a novel model of anaplastic thyroid carcinomas

Abstract Anaplastic thyroid carcinoma (ATC) is the most aggressive form of thyroid cancer, and often derives from pre-existing well-differentiated tumors. Despite a relatively low prevalence, it accounts for a disproportionate number of thyroid cancer-related deaths, due to its resistance to any therapeutic approach. We have generated a mouse model in which the simultaneous selective activation of the PI3K pathway and inactivation of p53 in the thyroid epithelial cells leads to the development of ATCs. These tumors display all the features of their human counterpart, including pleomorphism, epithelial-mesenchymal transition, aneuploidy, local invasion and distant metastases. Expression profiling of the murine ATCs reveals a significant overlap with genes found deregulated in human ATC, including genes involved in mitosis control, such as Aurora kinase A (AURKA), its co-activator TPX2, and Polo like kinase-1 (PLK1). These proteins are crucial driving forces for normal mitotic spindle formation, centrosome maturation and separation, and their overexpression has been demonstrated in a wide range of tumors. MLN8237 (AURKA inhibitor), TAK960 and GSK461364 (PLK1 inhibitors) are a promising new generation of anti-mitotic agents. We show that these molecules inhibit cell proliferation and induce cell death in mouse ATC-derived cell lines and in the human ATC cell lines CAL62 and C643. A dose-dependent decrease in the degree of chromosome alignment and spindle bipolarity during mitosis is observed after the treatments, together with a change in mitotic index indicated by the decrease of the mitotic marker pHisH3. FACS analysis reveals a G2/M phase arrest followed by apoptosis. Furthermore, small molecule inhibitors of PI3K or AKT potently synergize with these anti-mitotic compounds. Taken together these data suggest that AURKA and PLK1 inhibitors are promising therapeutic agents against undifferentiated thyroid carcinoma cells, and that concurrent inhibition of key driver pathways is a viable option to increase efficacy in this otherwise invariably lethal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 901. doi:1538-7445.AM2012-901

Abstract PL2-1: Drug Development — Targeting HER2 beyond Trastuzumab

Abstract The use of trastuzumab in the treatment of early and advanced stage HER2 overexpressing (HER2+) breast cancers represents a significant advance in the treatment of breast cancer. In the advanced stage setting, the development of therapeutic resistance, either primary or secondary/acquired, is a clinical problem limiting the clinical efficacy of trastuzumab. This discussion will include alternative approaches to the treatment of HER2+ breast cancers, and strategies to enhance the clinical efficacy of trastuzumab by overcoming the development of therapeutic resistance. Elucidating mechanisms of resistance provides an opportunity to develop combination strategies based on scientific rationale to overcome or prevent its development. For example, the IGF1/IGF-1R signaling axis, which is frequently co-expressed in HER2+ breast cancers promotes trastuzumab resistance in preclinical models. IGF-1R inhibitors overcomes trastuzumab resistance in preclinical models, providing a strong rationale for evaluating the combination in clinical trials. Similarly, the pro-survival PI3K-Akt signaling network is frequently deregulated in breast cancers as a consequence of PI3KCA gain-of-function mutations or loss of the tumor suppressor PTEN. In preclinical models, deregulated PI3K signaling mediates therapeutic resistance to trastuzumab, and small molecule PI3K inhibitors have been shown to overcome trastuzumab resistance. Other mechanisms of resistance to trastuzumab include the presence of heregulin (HRG), a soluble growth factor ligand to HER3 and HER4, which promotes the formation of HER2/HER3 heterodimers, a complex that potently activates PI3K signaling. Other HER2 antibodies e.g. pertuzumab, are more effective at blocking the effects of HRG on HER2/HER3 dimerization. Another mechanism of resistance is truncation of HER2, resulting in a receptor that lacks the extracellular domain that contains the trastuzumab binding site. Strategies to prevent HER2 truncation have been examined preclinically. HER2 signaling has also been targeted using small molecules that compete with ATP for binding at the catalytic kinase domain of HER2. Lapatinib, an oral, reversible inhibitor of HER2 and EGFR tyrosine kinases is approved in combination with capecitabine for treating advanced stage HER2+ breast cancers that progressed on prior trastuzumab-based therapies. The dilemma is that the majority of women treated with lapatinib develop therapeutic resistance within 12 months of initiating therapy. Mechanisms of therapeutic resistance to lapatinib have been identified including (i) de-repression of estrogen receptor (ER) signaling, and (ii) activation of the pro-survival mediator NF-kB; the role of PTEN loss and the presence of PI3K activating mutations in the development of lapatinib resistance remains controversial. This insight has led to rational combinations to overcome or perhaps even delay the development of lapatinib resistance e.g. approval of letrozole plus lapatinib in HER2+/ER+ breast cancers. And finally, other HER2 targeted approaches will be discussed, including hsp90 antagonists, HER2 antibody conjugates (e.g. T-DM1), HER2 vaccines, and combinations of antibody-based and HER2 kinase inhibitors. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PL2-1.