Phosphoinositol 3-kinase Inhibitor Research Articles

Impact of PIK3CA gain and PTEN loss on mantle cell lymphoma biology and sensitivity to targeted therapies

AbstractBesides many other mutations in known cancer driver genes, mantle cell lymphoma (MCL) is characterized by recurrent genetic alterations of important regulators of the phosphoinositol-3-kinase (PI3K) cascade including PIK3CA gains and PTEN losses. To evaluate the biological and functional consequences of these aberrations in MCL, we have introduced transgenic expression of PIK3CA (PIK3CA UP) and performed knockout/knockdown of PTEN gene (PTEN KO/KD) in 5 MCL cell lines. The modified cell lines were tested for associated phenotypes including dependence on upstream B-cell receptor (BCR) signaling (by an additional BCR knockout). PIK3CA overexpression decreased the dependence of the tested MCL on prosurvival signaling from BCR, decreased levels of oxidative phosphorylation, and increased resistance to 2-deoxy-glucose, a glycolysis inhibitor. Unchanged protein kinase B (AKT) phosphorylation status and unchanged sensitivity to a battery of PI3K inhibitors suggested that PIK3CA gain might affect MCL cells in AKT-independent manner. PTEN KO was associated with a more distinct phenotype: AKT hyperphosphorylation and overactivation, increased resistance to multiple inhibitors (most of the tested PI3K inhibitors, Bruton tyrosine kinase inhibitor ibrutinib, and BCL2 inhibitor venetoclax), increased glycolytic rates with resistance to 2-deoxy-glucose, and significantly decreased dependence on prosurvival BCR signaling. Our results suggest that the frequent aberrations of the PI3K pathway may rewire associated signaling with lower dependence on BCR signaling, better metabolic and hypoxic adaptation, and targeted therapy resistance in MCL.

The PIK3CA H1047R Mutation Confers Resistance to BRAF and MEK Inhibitors in A375 Melanoma Cells through the Cross-Activation of MAPK and PI3K-Akt Pathways.

The targeting of the Mitogen-Activated Protein Kinase (MAPK) signalling pathway in melanoma improves the prognosis of patients harbouring the V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) mutation. However, a fraction of these patients may experience tumour progression due to resistance to targeted therapy. Mutations affecting the Phosphoinositol-3-Kinase (PI3K)–Akt pathway may favour the onset of drug resistance, suggesting the existence of a crosstalk between the MAPK and PI3K–Akt pathways. We hypothesized that the inhibition of both pathways may be a therapeutic option in resistant melanoma. However, conflicting data have been generated in this context. In this study, three different A375 cell melanoma models either overexpressing or not expressing the wild-type or mutated form of the PhosphatidylInositol-4,5-bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) gene were used to clarify the therapeutic response of melanoma to BRAF, Mitogen-Activated Protein Kinase Kinase 1 (MEK), and PI3K inhibitors in the presence of the PIK3CA H1047R mutation. Our data strongly support the notion that the crosstalk between the MAPK and PI3K–Akt pathways is one of the main mechanisms associated with melanoma development and progression and that the combination of MAPK and PI3K inhibitors may sensitize melanoma cells to therapy.

PI3K activation allows immune evasion by promoting an inhibitory myeloid tumor microenvironment

BackgroundOncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood.MethodsWe use an in...

A Phase II Clinical Trial of the Pan PI3K Inhibitor, Buparlisib, for the Treatment of Relapsed and Refractory Chronic Lymphocytic Leukemia: Canadian Cancer Trials Group IND.216

Introduction: Inhibition of the phospho-inositol 3 kinase (PI3K) delta isoform is effective in the treatment of relapsed and refractory chronic lymphocytic leukemia (CLL) (Pongas G, et al. Semin Oncol 2016; 43: 647). The efficacy of this class of drugs is limited by the potency of the inhibitor and its toxicity profile. Buparlisib is an orally available pan-Class I PI3K inhibitor which has greater potency than idelalisib in vitro . Low P70S6k and raptor levels, both of which are downstream regulators of the PI3K/mTOR pathway, correlate with sensitivity to PI3K inhibitor activity in CLL lymphocytes in vitro (Amrein L, et al. Int J Cancer 2013; 133: 247-52). The recommended phase II dose of buparlisib is 100 mg daily po and the main toxicities in Phase I were hyperglycemia, hypertension and mood disturbance (Bendell JC, et al. J Clin Oncol. 2012;30:282-90). We conducted a single arm phase II trial of buparlisib in patients with relapsed and refractory CLL (NCT02340780) and attempted to correlate response with P70S6k and raptor levels.Methods: Patients with CLL requiring re-treatment after at least one prior line of therapy were eligible. Buparlisib 100 mg daily was administered continuously on 28-day cycles with dose reductions to 80 mg and 60 mg in the event of toxicity. Mood questionaires (PHQ-9 and GAD-7) were administered at study entry and with each treatment cycle. The primary endpoint was overall response rate, defined according to the Revised International Workship on CLL (Hallek 2008) incorporating updated recommendations for novel targeted agents (Cheson 2012). Additional endpoints included safety, duration of response and progression free survival. Levels of P70S6k and raptor were measured in isolated CLL cells prior to therapy with buparlisib and compared with mean values as per Amrein et al. Int J Cancer 2013;133: 247-52).Results: Between April 2015 and January 2017, fourteen patients were enrolled; Rai stage high (n=7), intermediate (n=6) and low (n=1). Of these, 13 were eligible for toxicity (1 ineligible due to inadequate washout period) and 11 for response assessment (2 did not complete 1 cycle of therapy). The median age was 74 years (61-87), male:female 8:5, ECOG 0-1 and median number of prior therapies 1 (range 1-3). No patients received prior therapy with idelalisib or other PI3K inhibitor; 3 received prior ibrutinib, including the ineligible patient. Six of 13 patients had a partial response (46.2%, 95%, CI: 19.2,74.9%), with a median duration of response of 14.4 months. Five patients with a best response of stable disease all experienced tumor shrinkage (figure). There were no complete responses. Patients received a median of 5 cycles of therapy (range 1 to 20), the dose intensity was 485.6 mg per week and 27.3% received > 90% of planned doses. Ten patients have stopped protocol therapy, 1 for progressive disease and 9 for therapy-related adverse events: hyperglycemia (n=4), mood disorder (n=3), fatigue and generalized muscle weakness (n=1), elevated AST (n=1), hypertension (n=1) and thrombocytopenia (n=1). Mood disorders improved after stopping therapy in all 3 patients. The most common related adverse events (>15%) were diarrhea, nausea, fatigue, anorexia and dysguesia; all were <grade 3 except for fatigue. Hyperglycemia was seen in 9/13 patients. There were no related serious adverse events. Among the 8 patients tested, no correlation was observed between biomarker level and response or progression free survival.Conclusion: The response rates of single-agent idelalisib (Brown JR, et al. JCO ASCO Annual Meeting Abstracts. 2013;31 abstract 7003) and buparlisib are comparable with a different toxicity profile, making buparlisib another potential therapeutic option in CLL. Better mitigation of toxicity is required to make this an acceptable therapy for patients and rational drug combinations are needed to improve the efficacy of this agent. We could not confirm the importance of P70S6K or raptor in this small clinical trial. Validating these biomarkers in a larger cohort may help to predict which CLL patients are most likely to benefit from therapy with a PI3K inhibitor. [Display omitted] DisclosuresAssouline:Bristol Myer Squibb: Speakers Bureau; Novartis Canada Inc.: Honoraria; Pfizer: Speakers Bureau; Paladin: Speakers Bureau; Janssen: Honoraria. Banerji:Roche: Research Funding; Lundbeck: Research Funding; Abbvie: Research Funding; Janssen: Research Funding; Gilead: Research Funding. Caplan:Janssen: Honoraria. Owen:Roche: Consultancy, Honoraria, Research Funding; Lundbeck: Honoraria; Gilead: Honoraria, Research Funding; Janssen: Honoraria; AbbVie: Honoraria; Celgene: Honoraria; AstraZeneca: Honoraria; Pharmacyclics LLC, an AbbVie Company: Research Funding. Robinson:Gilead: Honoraria; Roche: Honoraria; Janssen: Honoraria; Lundbeck: Honoraria; Abbvie: Honoraria. Prica:Celgene: Honoraria; Janssen: Honoraria. Peters:Abbvie: Honoraria; Roche: Honoraria; Lundbeck: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Hay:Roche: Research Funding; Abbvie: Research Funding; Kite: Research Funding; Amgen: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Novartis: Research Funding.

Glut1 expression is increased by p53 reduction to switch metabolism to glycolysis during osteoblast differentiation.

The glycolytic system is selected for ATP synthesis not only in tumor cells but also in differentiated cells. Differentiated osteoblasts also switch the dominant metabolic pathway to aerobic glycolysis. We found that primary osteoblasts increased expressions of glycolysis-related enzymes such as Glut1, hexokinase 1 and 2, lactate dehydrogenase A and pyruvate kinase M2 during their differentiation. Osteoblast differentiation decreased expression of tumor suppressor p53, which negatively regulates Glut1 expression, and enhanced phosphorylation of AKT, which is regulated by phosphoinositol-3 kinase (PI3K). An inhibitor of PI3K enhanced p53 expression and repressed Glut1 expression. Luciferase reporter assay showed that p53 negatively regulated transcriptional activity of solute carrier family 2 member 1 gene promoter region. Inhibition of glycolysis in osteoblasts reduced ATP contents more significantly than inhibition of oxidative phosphorylation by carbonyl cyanide m-chlorophenyl hydrazine. These results have indicated that osteoblasts increase Glut1 expression through the down-regulation of p53 to switch their metabolic pathway to glycolysis during differentiation.

Interleukin-6 mediates resistance to PI3K-pathway\u2013targeted therapy in lymphoma

BackgroundThe phosphoinositol 3-kinase (PI3K) pathway is associated with poor prognosis of hematologic malignancies, providing a strong rationale for the use of PI3K inhibitors in the treatment of malignant lymphoma. However, development of resistance limits the use of PI3K inhibitors in lymphoma patients.MethodsWe established copanlisib (pan-PI3K inhibitor)-resistant B-cell lymphoma and duvelisib (PI3Kδ and -γ inhibitor)-resistant T-cell lymphoma cell lines. The cytokine array and the phospho-kinase array were used to identify up-regulated proteins in the resistant cells. Cytokine expression and phospho-kinase levels were examined by ELISA and Western blot analysis, respectively. Cell proliferation capabilities were measured by using CCK-8 kit and colony formation assay. The effects of inhibitors on apoptosis were detected using an Annexin V-FITC Apoptosis Detection Kit and a flow cytometry system. The underlying mechanisms were studied by transfecting recombinant plasmids or siRNA into lymphoma cell lines. Cells were transiently transfected using the Amaxa electroporation system. We evaluated the effects of PI3K inhibitor alone and in combination with JAK inhibitor (BSK805) on lymphoma proliferation and signaling pathway activation.ResultsCytokine arrays revealed upregulation of interleukin (IL)-6 in both copanlisib- and duvelisib-resistant cell lines. Phosphorylated STAT5, AKT, p70S6K and MAPK were increased in copanlisib-resistant B-cell lymphoma cells, whereas phosphorylated STAT3 and NF-κB were increased in duvelisib-resistant T cell lymphoma cells. Conversely, depletion of IL-6 sensitized both resistant cell lines, and led to downregulation of phosphorylated STAT3 and STAT5 in copanlisib- and duvelisib-resistant cells, respectively. Moreover, combined treatment with a JAK inhibitor (BSK805) and a PI3K inhibitor circumvented the acquired resistance to PI3K inhibitors in lymphoma, and concurrent inhibition of the activated pathways produced combined effects.ConclusionsIL-6–induced STAT3 or STAT5 activation is a critical mechanism underlying PI3K inhibitor resistance in lymphoma, supporting the utility of IL-6 as an effective biomarker to predict therapeutic response to PI3K inhibitors.

PI3K activated tumors evade tumor immunity by promoting an inhibitory myeloid microenvironment

Abstract Oncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood. Here we use an in vivo screen of known cancer-associated somatic mutations in immunocompetent mouse tumor models treated with checkpoint blockade to identify oncogenes that confer immune evasion. We found that a catalytically active mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A&amp;gt;G (H1047R) confers selective growth advantage after immunotherapy. Pharmacologic PI3K inhibition resensitizes mutant tumors to immunotherapy with anti-PD-1. The tumor microenvironment (TME) in PIK3CA H1047R-expressing tumors has fewer infiltrating CD8+ T cells after immunotherapy but is enriched for immune inhibitory myeloid cells. Inhibition of myeloid infiltration in PIK3CA H1047R tumors results in increased sensitivity to PD-1 checkpoint blockade. Thus PI3K has a role in tumor immune evasion mediated by establishment of an inhibitory myeloid microenvironment in addition to its well-described, cell-intrinsic oncogenic role. Activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy. More generally, our data suggest a rationale to combine PI3K inhibition with immunotherapy of PI3KCA mutant tumors.

Optimal Management of Adverse Events From Copanlisib in the Treatment of Patients With Non-Hodgkin Lymphomas

IntroductionCopanlisib is a phosphoinositol 3-kinase (PI3K) inhibitor approved for the third-line treatment of follicular non-Hodgkin lymphoma. Although the drug is generally well-tolerated, it can be associated with several unique and potentially serious adverse effects (AEs). Two of the most common toxicities not seen with other PI3K inhibitors include hyperglycemia and hypertension, which primarily occur during infusion and resolve shortly thereafter, and likely relate to targeting the PI3K alpha isoform. Other toxicities less commonly observed with copanlisib than with other approved drugs in this class include non-infectious pneumonitis, infections, diarrhea and colitis, and hepatobiliary toxicity. Materials and MethodsA panel composed of experts in lymphoma, diabetes, and hypertension convened to develop guidance pertaining to the administration of copanlisib and the management of the AEs associated with copanlisib treatment. ResultsRecommendations were formulated pertaining to the management of AEs associated with copanlisib treatment, particularly infusion-related hyperglycemia and hypertension, noninfectious pneumonitis, infections, diarrhea, and colitis. The recommendations herein reflect the consensus of the members of this panel, all of whom contributed to these suggested approaches to patient supportive care. ConclusionThere are a number of challenges associated with the use of copanlisib. Infusion-related hypertension and hyperglycemia occur frequently, although they are transient, reversible, and rarely of clinical significance; this report provides guidance as to their management.

Abstract 706: In vivo tumor-associated mutation screen identifies PI3K activation as a mechanism of resistance to PD-1 blockade

Abstract Known genomic correlates of response to immunotherapy do not perfectly predict clinical outcome, supporting the existence of unknown mechanisms of resistance to tumor immunity. We hypothesize that somatic, cancer-associated mutations account for heterogeneity in spontaneous response to tumors and response to immunotherapy. We have undertaken a systematic in vivo screen to identify mechanisms of resistance to tumor immunity in order to define a comprehensive set of therapeutic targets and provide biomarkers of sensitivity to immunotherapy. Mouse tumor cell lines (MC38 colon carcinoma or B16 melanoma) were engineered to express a library of barcoded open reading frames (ORFs) mutagenized to encode known cancer-associated somatic mutations. Tumor-bearing animals were treated with anti-PD-1. A mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A&amp;gt;G, increased in representation in anti-PD-1 treated tumors but not in immunodeficient animals, suggesting that activity of the mutant allele conferred selective growth advantage in the setting of tumor immunity. This mutation encodes a constitutively active mutant catalytic domain, PIK3CA H1047R. MC38 tumors homogeneously expressing H1047R and implanted into wild-type mice failed to respond to anti-PD-1 therapy, while tumors expressing a control gene regressed. Pharmacologic PI3K inhibition resensitized tumors to treatment with anti-PD-1. PD-1-treated PIK3CA H1047R tumors had fewer infiltrating CD8+ T cells as measured by immunohistochemistry and flow cytometry. Single-cell RNA-seq of tumor-infiltrating immune cells revealed a population of myeloid cells expressing known immune inhibitory proteins that differentially enriched in PIK3CA H1047R-expressing tumors. Our data suggest that PI3K has, in addition to its well-described oncogenic role, a role in tumor immune evasion mediated by establishment of an inhibitory myeloid microenvironment. As such, activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy, and these studies provide a rationale for therapeutic combination trials of PI3K inhibition with checkpoint blockade and other myeloid-targeting immunotherapies. Citation Format: Natalie B. Collins, Brian C. Miller, Kevin Bi, Rose Al Abosy, Kathleen Yates, Yashaswi Shrestha, John Doench, Jesse Boehm, W Nicholas Haining. In vivo tumor-associated mutation screen identifies PI3K activation as a mechanism of resistance to PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 706.

Metabolic changes associated with metformin potentiates Bcl-2 inhibitor, Venetoclax, and CDK9 inhibitor, BAY1143572 and reduces viability of lymphoma cells.

Metformin exerts direct anti-tumor effects by activating AMP-activated protein kinase (AMPK), a major sensor of cellular metabolism in cancer cells. This, in turn, inhibits pro-survival mTOR signaling. Metformin has also been shown to disrupt complex 1 of the mitochondrial electron transport chain. Here, we explored the lymphoma specific anti-tumor effects of metformin using Daudi (Burkitt), SUDHL-4 (germinal center diffuse large B-cell lymphoma; GC DLBCL), Jeko-1 (Mantle-cell lymphoma; MCL) and KPUM-UH1 (double hit DLBCL) cell lines. We demonstrated that metformin as a single agent, especially at high concentrations produced significant reductions in viability and proliferation only in Daudi and SUDHL-4 cell lines with associated alterations in mitochondrial oxidative and glycolytic metabolism. As bcl-2 proteins, cyclin dependent kinases (CDK) and phosphoinositol-3- kinase (PI3K) also influence mitochondrial physiology and metabolism with clear relevance to the pathogenesis of lymphoma, we investigated the potentiating effects of metformin when combined with novel agents Venetoclax (bcl-2 inhibitor), BAY-1143572 (CDK9 inhibitor) and Idelalisib (p110δ- PI3K inhibitor). Co-treating KPUM-UH1 and SUDHL-4 cells with 10 mM of metformin resulted in 1.4 fold and 8.8 fold decreases, respectively, in IC-50 values of Venetoclax. By contrast, 3-fold and 10 fold reduction in IC-50 values of BAY-1143572 in Daudi and Jeko-1 cells respectively was seen in the presence of 10 mM of metformin. No change in IC-50 value for Idelalisib was observed across cell lines. These data suggest that although metformin is not a potent single agent, targeting cancer metabolism with similar but more effective drugs in novel combination with either bcl-2 or CDK9 inhibitors warrants further exploration.

PI3K\u03b4 contributes to ER stress-associated asthma through ER-redox disturbances: the involvement of the RIDD\u2013RIG-I\u2013NF-\u03baB axis

Hyperactivation of phosphoinositol 3-kinase (PI3K) has been suggested to be a potential mechanism for endoplasmic reticulum (ER) stress-enhanced airway hyperresponsiveness, and PI3K inhibitors have been examined as asthma therapeutics. However, the regulatory mechanism linking PI3K to ER stress and related pathological signals in asthma have not been defined. To elucidate these pathogenic pathways, we investigated the influence of a selective PI3Kδ inhibitor, IC87114, on airway inflammation in an ovalbumin/lipopolysaccharide (OVA/LPS)-induced asthma model. In OVA/LPS-induced asthmatic mice, the activity of PI3K, downstream phosphorylation of AKT and activation of nuclear factor-κB (NF-κB) were all significantly elevated; these effects were reversed by IC87114. IC87114 treatment also reduced the OVA/LPS-induced ER stress response by enhancing the intra-ER oxidative folding status through suppression of protein disulfide isomerase activity, ER-associated reactive oxygen species (ROS) accumulation and NOX4 activity. Furthermore, inositol-requiring enzyme-1α (IRE1α)-dependent degradation (RIDD) of IRE1α was reduced by IC87114, resulting in a decreased release of proinflammatory cytokines from bronchial epithelial cells. These results suggest that PI3Kδ may induce severe airway inflammation and hyperresponsiveness by activating NF-κB signaling through ER-associated ROS and RIDD–RIG-I activation. The PI3Kδ inhibitor IC87114 is a potential therapeutic agent against neutrophil-dominant asthma.

Abstract PR04: HER3 crosstalk with HPV16-E6E7 is a feedback resistance mechanism to PI3K-targeted therapies in head and neck cancer

Abstract Human papillomavirus (HPV) 16 plays an etiologic role in a growing subset of head and neck squamous cell carcinoma (HNSCC), where viral expression of the E6 and E7 oncoproteins is necessary for tumor growth and maintenance. Although patients with HPV(+) tumors have a more favorable prognosis, there are currently no HPV-selective therapies. Accumulating evidence indicates that HPV oncoproteins can activate the Phosphoinositol-3-Kinase (PI3K) pathway, which contributes to transformation. Furthermore, PI3K is genomically activated by PIK3CA mutation or amplification in a disproportionately high number of HPV(+) tumors as compared to HPV(-) tumors. Based on this knowledge, we investigated the efficacy of PI3K-targeted therapies in preclinical models of HPV(+) HNSCC. Our results indicate that HPV(+) preclinical models were less sensitive to the PI3K inhibitors BYL719, BKM120, and BEZ235 as compared with HPV(-) models. Sensitivity of HPV(+) cell lines to PI3K inhibitors was increased upon knockdown of the E6 and E7 oncoproteins. Reciprocally, overexpression of E6 and E7 in HPV(-) cells rendered them resistant to PI3K-targeted therapies. Proteomic analyses indicated that treatment of HPV(+) cell lines and patient-derived xenografts (PDXs) with the PI3Kα inhibitor BYL719 induced expression of the receptor tyrosine kinase HER3, as well as E6 and E7. HER3 was found to regulate the abundance of E6 and E7 in the HPV(+) models. Targeting HER3 with siRNAs or the monoclonal antibody, KTN3379, blocked the increase in E6 and E7 protein levels following BYL719 treatment, and enhanced the efficacy of PI3K inhibitors in HPV(+) cell line and PDX models. Taken together, these results suggest that crosstalk between HER3 and HPV16-E6E7 can limit the efficacy of PI3K inhibitors, and that co-targeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors. This abstract is also being presented as Poster 67. Citation Format: Toni M. Brand, Stefan Hartmann, Neil E. Bhola, Hua Li, Yan Zeng, Rachel O'Keefe, Max V. Ranall, Sourav Bandyopadhyay, Margaret Soucheray, Danielle L. Swaney, Nevan Krogan, Carolyn Kemp, Umamaheswar Duvvuri, Daniel E. Johnson, Michelle A. Ozbun, Julie E. Bauman, Jennifer R. Grandis. HER3 crosstalk with HPV16-E6E7 is a feedback resistance mechanism to PI3K-targeted therapies in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr PR04.

Impact of a novel phosphoinositol-3 kinase inhibitor in preventing mitochondrial DNA damage and damage-associated molecular pattern accumulation: Results from the Biochronicity Project.

Despite improvements in the management of severely injured patients, development of multiple organ dysfunction syndrome (MODS) remains a morbid complication of traumatic shock. One of the key attributes of MODS is a profound bioenergetics crisis, for which the mediators and mechanisms are poorly understood. We hypothesized that metabolic uncoupling using an experimental phosphoinositol-3 kinase (PI3-K) inhibitor, LY294002 (LY), may prevent mitochondrial abnormalities that lead to the generation of mitochondrial DNA (mtDNA) damage and the release of mtDNA damage-associated molecular patterns (DAMPs). Sixteen swine were studied using LY, a nonselective PI3-K inhibitor. Animals were assigned to trauma only (TO, n = 3), LY drug only (LYO, n = 3), and experimental (n = 10), trauma + drug (LY + T) groups. Both trauma groups underwent laparotomy, 35% hemorrhage, severe ischemia-reperfusion injury, and protocolized resuscitation. A battery of hemodynamic, laboratory, histological, and bioenergetics parameters were monitored. Mitochondrial DNA damage was determined in lung, liver, and kidney using Southern blot analyses, whereas plasma mtDNA DAMP analysis used polymerase chain reaction amplification of a 200-bp sequence of the mtDNA D-loop region. Relative to control animals, H + I/R (hemorrhage and ischemia/reperfusion) produced severe, time-dependent decrements in hepatic, renal, cardiovascular, and pulmonary function accompanied by severe acidosis and lactate accumulation indicative of bioenergetics insufficiency. The H-I/R animals displayed prominent oxidative mtDNA damage in all organs studied, with the most prominent damage in the liver. Mitochondrial DNA damage was accompanied by accumulation of mtDNA DAMPs in plasma. Pretreatment of H + I/R animals with LY resulted in profound metabolic suppression, with approximately 50% decreases in O2 consumption and CO2 production. In addition, it prevented organ and bioenergetics dysfunction and was associated with a significant decrease in plasma mtDNA DAMPs to the levels of control animals. These findings show that H + I/R injury in anesthetized swine is accompanied by MODS and by significant mitochondrial bioenergetics dysfunction, including oxidative mtDNA damage and accumulation in plasma of mtDNA DAMPs. Suppression of these changes with the PI3-K inhibitor LY indicates that pharmacologically induced metabolic uncoupling may comprise a new pharmacologic strategy to prevent mtDNA damage and DAMP release and prevent or treat trauma-related MODS. Therapeutic study, level III.

METABOLIC CHANGES ASSOCIATED WITH METFORMIN POTENTIATES ANTI‐BCL‐2 INHIBITOR, ABT‐199, AND CDK9 INHIBITOR, BAY‐1143572 AND REDUCES VIABILITY OF LYMPHOMA CELLS

Introduction: Metformin exerts anti-tumor effects in part by activating AMP-activated protein kinase (AMPK) with resultant reduction in pro-survival mTOR signaling. Here, we test the hypothesis that metformin induces cell death by altering mitochondrial metabolism in lymphoma cell lines. As Bcl-2 proteins, cyclin-dependent kinases (CDK) and Phosphoinositol-3-kinase (PI3K) also influence mitochondrial physiology and metabolism, we also explored the additive effects of metformin with novel agents including the Bcl-2 inhibitor (ABT-199), the CDK9 inhibitor (BAY-1143572) and the p110δ-selective PI3K inhibitor (CAL-101). Methods: Daudi (Burkitt), SUDHL-4 (GC DLBCL), Jeko-1 (MCL) and KPUM-UH1 (double hit DLBCL) cells were plated in 96-well plates and treated with varying doses of metformin (0–5000 μM). Hoechst 33342 DNA-binding dye or MTS assays were used to measure proliferation and cell viability, respectively. Perturbations in oxidative and glycolytic metabolism were monitored via Seahorse XF Energy Phenotype kits. Lastly, efficacy of combination therapy was evaluated in cells co-treated with metformin and ABT-199 or BAY-1143572 or CAL-101. Results: Daudi and SUDHL-4 cells showed 80% and 50% reduction in viability, respectively, with metformin at levels greater than 1 mM at day 7, while KPUM-UH1 and Jeko-1 cells were unaffected. The Seahorse analyses showed reduction in oxidative phosphorylation, in cells treated with 1mM metformin with a corresponding increase in glycolysis. Co-treating KPUM-UH1 and SUDHL-4 cells with 10 mM metformin resulted in 1.4-fold and 8.8-fold decreases, respectively, in IC50 values of ABT-199 at day 3 (Table 1). Jeko-1 and Daudi cells were resistant to ABT-199. By contrast, 3-fold and 10 fold reduction in IC50 values of BAY-1143572 in Daudi and Jeko-1, respectively, was seen in the presence of 10 mM Metformin, and SUDHL-4 or KPUM-UH1 cells were unaffected. No change in IC50 value for CAL-101 was observed. Conclusions: Metformin induces significant changes in cellular metabolism leading to decreased proliferation and viability in lymphoma cell lines. The combination of metformin and the anti-Bcl-2 agent ABT-199 achieves an additive effect in DLBCL (including double hit) cell lines, whereas the combination with CDK9 inhibitor BAY-1143572 provides more selective additive anti-tumor effect in Burkitt and MCL cell lines. These data support the idea of targeting cancer metabolism with metformin in novel combinatorial therapeutic strategies. Table: IC-50 concentrations for novel agents ±10 mM metformin Keywords: “double-hit” lymphomas; ABT-199; B-cell lymphoma.

Abstract A16: Defining molecular mechanisms of resistance to tumor immunity

Abstract Recent success of immune checkpoint blockade solidifies the importance of the immune system in the defense against cancer. The clinical impact of the immune response is, however, very heterogeneous, with some patients achieving dramatic responses while others fail to respond. Known genomic correlates of response to immunotherapy are not perfectly predictive of clinical outcome, supporting the existence of unknown mechanisms of resistance to tumor immunity. I hypothesize somatic acquired mutations of individual tumors may account for heterogeneity in the spontaneous response to tumors and response to immunotherapy. I have undertaken a systematic in vivo screen to identify mechanisms of resistance to tumor immunity in order to discover new mechanisms of immune resistance, define a comprehensive set of therapeutic targets and provide biomarkers of sensitivity to immunotherapeutic strategies. Mouse tumor cell lines (MC38 colon carcinoma or B16 melanoma) were engineered to express a library of barcoded open reading frames (ORFs) mutagenized to encode known cancer-associated somatic mutations from the Pan Cancer analysis within The Cancer Genome Atlas (TCGA). These cell lines form tumors when implanted subcutaneously in immunocompetent animals. Tumor-bearing animals were then subjected to immunotherapy with either therapeutic vaccination or checkpoint blockade with anti-PD-1. Barcode relative representation was measured by next generation sequencing at the time of tumor implantation and at the time of tumor harvest post-immunotherapy. Barcoded mutant ORFs that confer immune resistance increased in representation under immune pressure in comparison to untreated or immunodeficient animals. A mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A&amp;gt;G, consistently increased in representation in both B16 and MC38 immunotherapy-treated tumors. This mutation encodes a constitutively active mutant catalytic domain of PI3K, PIK3CA H1047R. MC38 tumors homogenously expressing PIK3CA H1047R and implanted into wild type mice failed to respond to anti-PD-1 therapy, while tumors expressing a control gene or non-scoring mutation in PI3K regressed after treatment with anti-PD-1. Pharmacologic PI3K inhibition resensitized tumors to treatment with anti-PD-1. PD-1-treated PIK3CA H1047R tumors had fewer infiltrating CD8+ T cells as measured by immunohistochemistry and flow cytometry of tumor infiltrating lymphocytes. I conclude that PI3K has, in addition to its well-described oncogenic role, a role in tumor immune evasion. As such, activation of PI3K may be useful as a predictor of resistance to immunotherapy. Importantly, these findings also provide a rationale for therapeutic combination trials of immune checkpoint blockade and PI3K inhibition. Citation Format: Natalie B. Collins, Robert Manguso, Hans Pope, W. Nicholas Haining. Defining molecular mechanisms of resistance to tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A16.

Abstract 372: Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. Taselisib (GDC 0032) is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Previously published data demonstrated that taselisib has increased activity against PIK3CA mutant cancer cell lines (Ndubaku CO et al, J Med Chem, 2013). To determine if there are additional predictive biomarkers outside of PIK3CA mutations for sensitivity to taselisib, we profiled over 550 cell lines, encompassing 13 of the main tissue types, to increasing concentrations of taselisib. As expected, a small percentage of all tumor types responded to taselisib and correlated strongly with PIK3CA mutations. Intriguingly, the majority of head and neck squamous cell cancer (HNSCC) cell lines showed an IC50 concentration of less than 0.5uM, suggesting that HNSCC cell lines may be particularly susceptible to PI3K inhibition. Interestingly, the majority of HNSCC cell lines displayed an activated PI3K pathway as evidenced by phosphorylation of key proteins, such as AKT, S6 and PRAS40 implicating that downstream phospho-protein analysis of the PI3K pathway may not predict for sensitivity to taselisib. Mutational profiling identified that only 3 out of the tested 31 HNSCC cell lines harbored a mutation within PIK3CA, suggesting that additional genotypes may explain the sensitivity to taselisib. Additional molecular analysis assessing both gene expression and copy number levels of genes relevant to the PI3K pathway found that many of the taselisib sensitive cell lines had an activated ERBB signaling node through low level amplification of ERBB receptors (e.g. EGFR, FGFR1) or via a NRG1:ERBB3 autocrine mechanism. As many ERBB receptors heterodimerize with ERBB3, which contains p85 binding sites and potentially activates the PI3K pathway, we next tested whether taselisib would enhance the potency of ERBB receptor inhibitors in biomarker-defined subset of HNSCC cell lines. Combination screens were performed in cell lines with amplified EGFR, ERBB2, and NRG1:ERBB3 autocrine signaling with taselisib + tarceva or lapatinib. We found that combination effects assessed using the BLISS excess method showed a synergistic interaction. These results suggest that taselisib may have therapeutic potential for the treatment of HNSCC. Citation Format: Heidi M. Savage, Carol O’Brien, Heather Moore, Mark R. Lackner, Robert Yauch, Jeffrey Settleman, Timothy R. Wilson. Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines. [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 372.

Abstract LB-B10: Chemical Proteomic Analysis of BTK and PI3K Using Chloroalkane-Derivatized Small Molecule Inhibitors

Abstract Chemical proteomics is a rapidly evolving technology that allows for drug target identification for small molecule inhibitors. Here we present an approach utilizing a chloroalkane (CA) moiety capture handle, which can be chemically attached to small molecules and used to pull-down protein binding partners using the HaloTag technology. CA-modified compounds are cell permeable and have minimal impact on potency, allowing for phenotypic assays of the derivatized compound to be recapitulated. Here we present a study looking at the targets of two compounds - a Brutons Tyrosine Kinase (BTK) small molecule inhibitor and a Phospho-inositol 3 Kinase (PI3K) small molecule inhibitor. BTK is a non-receptor tyrosine kinase expressed in most hematopoietic cells except T cells, it is essential for B cell receptor (BCR) signaling in B cells and as such is the therapeutic focus in many autoimmune diseases, including rheumatoid arthritis (RA) and lupus. Upon binding to growth factor receptor, PI3K is activated which initiates a signaling cascade involving AKT and mTOR that promotes cell cycle progression and cell proliferation. Mutations of the catalytic subunit of PI3KA are observed in several types of cancers. Inhibition of PI3K is therefore an important therapeutic focus. Taselisib, a selective inhibitor of PIK3CA, and G02599124, an inhibitor of BTK, were derivitized with the CA linker. Phenotypic screens compared to the parental compound showed comparable IC50 values. Both CA-compounds showed good binding to HaloTag and were cell permeable in a U2OS stable cell line. Control and test experiments were performed with BTK-CA compound using Jurkat (T cells) and Ramos (B cells) cell lines. Taselib-CA was studied after 1h and 8h treatment in the HCC1954 cell line. Cells were lysed and the CA compounds, together with the bound targets, were rapidly captured onto magnetic resin coated with HaloTag. Unmodified compound was used to competitively elute interacting proteins. Eluted proteins were processed using SDS-PAGE and in-gel digestion. Peptides were analyzed using nanoscale LC-MS/MS coupled with an Orbitrap Velos Pro mass spectrometer. Using this approach we were able to specifically isolate BTK when compared to control experiments in T cells. In the Taselib-CA experiment, we were able to specifically isolate the PIK3CA catalytic subunit and PIK3R1 and PIK3R2 regulatory subunits. Observation of the wild type and mutant forms of PI3KCA will be discussed. Citation Format: Victoria Pham, James Crawford, Steve Saben, Michael Ford, Richard Jones, Danette Daniels, Thomas Kirkland, Marjeta Urh, Jennie Lill. Chemical Proteomic Analysis of BTK and PI3K Using Chloroalkane-Derivatized Small Molecule Inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B10.

Protein kinase Cδ mediates trimethyltin-induced neurotoxicity in mice in vivo via inhibition of glutathione defense mechanism.

We investigated whether protein kinase C (PKC) is involved in trimethyltin (TMT)-induced neurotoxicity. TMT treatment (2.8 mg/kg, i.p.) significantly increased PKCδ expression out of PKC isozymes (i.e., α, βI, βII, δ, and ς) in the hippocampus of wild-type (WT) mice. Consistently, treatment with TMT resulted in significant increases in cleaved PKCδ expression. Genetic or pharmacological inhibition (PKCδ knockout or rottlerin) was less susceptible to TMT-induced seizures than WT mice. TMT treatment increased glutathione oxidation, lipid peroxidation, protein oxidation, and levels of reactive oxygen species. These effects were more pronounced in the WT mice than in PKCδ knockout mice. In addition, the ability of TMT to induce nuclear translocation of Nrf2, Nrf2 DNA-binding activity, and upregulation of γ-glutamylcysteine ligase was significantly increased in the PKCδ knockout mice and rottlerin (10 or 20 mg/kg, p.o. × 6)-treated WT mice. Furthermore, neuronal degeneration (as shown by nuclear chromatin clumping and TUNEL staining) in WT mice was most pronounced 2 days after TMT. At the same time, TMT-induced inhibition of phosphoinositol 3-kinase (PI3K)/Akt signaling was evident, thereby decreasing phospho-Bad, expression of Bcl-xL and Bcl-2, and the interaction between phospho-Bad and 14-3-3 protein, and increasing Bax expression and caspase-3 cleavage were observed. Rottlerin or PKCδ knockout significantly protected these changes in anti- and pro-apoptotic factors. Importantly, treatment of the PI3K inhibitor LY294002 (0.8 or 1.6 µg, i.c.v.) 4 h before TMT counteracted protective effects (i.e., Nrf-2-dependent glutathione induction and pro-survival phenomenon) of rottlerin. Therefore, our results suggest that down-regulation of PKCδ and up-regulations of Nrf2-dependent glutathione defense mechanism and PI3K/Akt signaling are critical for attenuating TMT neurotoxicity.

Intrinsic resistance triggered under acid loading within normal esophageal epithelial cells: NHE1- and ROS-mediated survival.

The transition to a pathological phenotype such as Barrett's esophagus occurs via induction of resistance upon repeated contact with gastric refluxate in esophagus. This study examined the molecular changes within normal esophageal epithelial cells (EECs) under short-term acid loading and the role of these changes in defensive resistance against acidic cytotoxicity. After primary cultured EECs were exposed to pH 4-acidified medium (AM4), cell viability was determined by the MTT assay. Reactive oxygen species (ROS) and NAD(P)H oxidase (NOX) activity were measured. Activation of the mitogen-activated protein kinases (MAPKs) MEK/ERK1/2, p38 and JNK; phosphoinositol-3-kinase (PI3K)/Akt, and nuclear factor-kappa B (NF-κB) were detected by Western blot analysis or immunofluorescence staining. AM4 incubation induced intracellular ROS generation accompanied by increase in NOX activity, which was further increased by Na(+) /H(+) exchange-1 (NHE1)-dependent inhibition but was prevented by inhibition of NOX or mitochondria complex I. AM4 also induced phosphorylation of MEK/ERK1/2, p38 MAPK, PI3K/Akt, and nuclear translocation of NF-κB, and all these effects, except for p38 MAPK phosphorylation, were abolished by inhibition of ROS. ROS-dependent PI3K/Akt activation, which mediates NF-κB nuclear translocation, was inhibited by protein tyrosine kinase (PTK) inhibitors and NHE1-specific inhibitor. All inhibitors of NHE, ROS, PTK, PI3K, or NF-κB further decreased AM4-induced cell viability. Acid loading in the presence of NHE1-dependent protection induced ROS generation by activating NOX and mitochondria complex I, which stimulated PTK/PI3K/Akt/NF-κB-dependent survival in EEC. Our data indicate that normal EEC initially respond to acid loading through intrinsic survival activation.

Inhibition of PI3K signaling triggered apoptotic potential of curcumin which is hindered by Bcl-2 through activation of autophagy in MCF-7 cells

Curcumin is a natural anti-cancer agent derived from turmeric (Curcuma longa). Curcumin triggers intrinsic apoptotic cell death by activating mitochondrial permeabilization due to the altered expression of pro- and anti-apoptotic Bcl-2 family members. Phosphoinositol-3-kinase (PI3K) and Akt, key molecular players in the survival mechanism, have been shown to be associated with the Bcl-2 signaling cascade; therefore, evaluating the therapeutic efficiency of drugs that target both survival and the apoptosis mechanism has gained importance in cancer therapy. We found that Bcl-2 overexpression is a limiting factor for curcumin-induced apoptosis in highly metastatic MCF-7 breast cancer cells. Forced overexpression of Bcl-2 also blocked curcumin-induced autophagy in MCF-7 cells, through its inhibitory interactions with Beclin-1. Pre-treatment of PI3K inhibitor LY294002 enhanced curcumin-induced cell death, apoptosis, and autophagy via modulating the expression of Bcl-2 family members and autophagosome formation in MCF-7 breast cancer cells. Atg7 silencing further increased apoptotic potential of curcumin in the presence or absence of LY294002 in wt and Bcl-2+ MCF-7 cells. The findings of this study support the hypothesis that blocking the PI3K/Akt pathway may further increased curcumin-induced apoptosis and overcome forced Bcl-2 expression level mediated autophagic responses against curcumin treatment in MCF-7 cells.