Wolfgang Hackl

Dual Inhibition of the PI3K/mTOR Pathway Increases Tumor Radiosensitivity by Normalizing Tumor Vasculature

The aberrant vascular architecture of solid tumors results in hypoxia that limits the efficacy of radiotherapy. Vascular normalization using antiangiogenic agents has been proposed as a means to improve radiation therapy by enhancing tumor oxygenation, but only short-lived effects for this strategy have been reported so far. Here, we show that NVP-BEZ235, a dual inhibitor of phosphoinositide-3-kinase (PI3K) and mTOR, can improve tumor oxygenation and vascular structure over a prolonged period that achieves the aim of effective vascular normalization. Because PI3K inhibition can radiosensitize tumor cells themselves, our experimental design explicitly distinguished effects on the blood vasculature versus tumor cells. Drug administration coincident with radiation enhanced the delay in tumor growth without changing tumor oxygenation, establishing that radiosensitization is a component of the response. However, the enhanced growth delay was substantially greater after induction of vascular normalization, meaning that this treatment enhanced the tumoral radioresponse. Importantly, changes in vascular morphology persisted throughout the entire course of the experiment. Our findings indicated that targeting the PI3K/mTOR pathway can modulate the tumor microenvironment to induce a prolonged normalization of blood vessels. The substantial therapeutic gain observed after combination of NVP-BEZ235 with irradiation has conceptual implications for cancer therapy and could be of broad translational importance.

Phase I safety, pharmacokinetic, and pharmacodynamic study of the oral phosphatidylinositol-3-kinase and mTOR inhibitor BGT226 in patients with advanced solid tumors

BACKGROUND This phase I dose-escalation study investigated the maximum tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics (PDs), and preliminary antitumor activity of BGT226, a potent, oral dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin inhibitor. PATIENTS AND METHODS Fifty-seven patients with advanced solid tumors received BGT226 2.5-125 mg/day three times weekly (TIW). Dose escalation was guided by an adaptive Bayesian logistic regression model with overdose control. Assessments included response per RECIST, [18F]-fluorodeoxyglucose uptake, and phosphorylated-S6 in skin and paired tumor samples. RESULTS Three patients (125 mg cohort) had dose-limiting toxic effects (grade 3 nausea/vomiting, diarrhea). BGT226-related adverse events included nausea (68%), diarrhea (61%), vomiting (49%), and fatigue (19%). BGT226 demonstrated rapid absorption, variable systemic exposure, and a median half-life of 6-9 h. Seventeen patients (30%) had stable disease (SD) as best response. Nine patients had SD for ≥16 weeks. Thirty patients (53%) achieved stable metabolic disease as assessed by [18F]-fluorodeoxyglucose-positron emission tomography; however, no correlation between metabolic response and tumor shrinkage according to computed tomography was observed. PD changes suggested PI3K pathway inhibition but were inconsistent. CONCLUSIONS The MTD of BGT226 was 125 mg/day TIW, and the clinically recommended dose was 100 mg/day TIW. Limited preliminary antitumor activity and inconsistent target inhibition were observed, potentially due to low systemic exposure.BACKGROUND This phase I dose-escalation study investigated the maximum tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics (PDs), and preliminary antitumor activity of BGT226, a potent, oral dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin inhibitor. PATIENTS AND METHODS Fifty-seven patients with advanced solid tumors received BGT226 2.5-125 mg/day three times weekly (TIW). Dose escalation was guided by an adaptive Bayesian logistic regression model with overdose control. Assessments included response per RECIST, [18F]-fluorodeoxyglucose uptake, and phosphorylated-S6 in skin and paired tumor samples. RESULTS Three patients (125 mg cohort) had dose-limiting toxic effects (grade 3 nausea/vomiting, diarrhea). BGT226-related adverse events included nausea (68%), diarrhea (61%), vomiting (49%), and fatigue (19%). BGT226 demonstrated rapid absorption, variable systemic exposure, and a median half-life of 6-9 h. Seventeen patients (30%) had stable disease (SD) as best response. Nine patients had SD for ≥16 weeks. Thirty patients (53%) achieved stable metabolic disease as assessed by [18F]-fluorodeoxyglucose-positron emission tomography; however, no correlation between metabolic response and tumor shrinkage according to computed tomography was observed. PD changes suggested PI3K pathway inhibition but were inconsistent. CONCLUSIONS The MTD of BGT226 was 125 mg/day TIW, and the clinically recommended dose was 100 mg/day TIW. Limited preliminary antitumor activity and inconsistent target inhibition were observed, potentially due to low systemic exposure.

NVP-BEZ235 and NVP-BGT226, dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors, enhance tumor and endothelial cell radiosensitivity

BackgroundThe phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition.Materials and methodsWe investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual γH2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well.ResultsBoth compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of γH2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor.ConclusionsThe results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy.

Multi-Level Targeting of the Phosphatidylinositol-3-Kinase Pathway in Non-Small Cell Lung Cancer Cells

Introduction We assessed expression of p85 and p110α PI3K subunits in non-small cell lung cancer (NSCLC) specimens and the association with mTOR expression, and studied effects of targeting the PI3K/AKT/mTOR pathway in NSCLC cell lines. Methods Using Automated Quantitative Analysis we quantified expression of PI3K subunits in two cohorts of 190 and 168 NSCLC specimens and correlated it with mTOR expression. We studied effects of two PI3K inhibitors, LY294002 and NVP-BKM120, alone and in combination with rapamycin in 6 NSCLC cell lines. We assessed activity of a dual PI3K/mTOR inhibitor, NVP-BEZ235 alone and with an EGFR inhibitor. Results p85 and p110α tend to be co-expressed (p<0.001); p85 expression was higher in adenocarcinomas than squamous cell carcinomas. High p85 expression was associated with advanced stage and poor survival. p110α expression correlated with mTOR (ρ = 0.276). In six NSCLC cell lines, addition of rapamycin to LY294002 or NVP-BKM120 was synergistic. Even very low rapamycin concentrations (1 nM) resulted in sensitization to PI3K inhibitors. NVP-BEZ235 was highly active in NSCLC cell lines with IC50s in the nanomolar range and resultant down-regulation of pAKT and pP70S6K. Adding Erlotinib to NVP-BEZ235 resulted in synergistic growth inhibition. Conclusions The association between PI3K expression, advanced stage and survival in NSCLC suggests that it might be a valuable drug target. Concurrent inhibition of PI3K and mTOR is synergistic in vitro, and a dual PI3K/mTOR inhibitor was highly active. Adding EGFR inhibition resulted in further growth inhibition. Targeting the PI3K/AKT/mTOR pathway at multiple levels should be tested in clinical trials for NSCLC.

Inhibition of DNA-Dependent Protein Kinase Induces Accelerated Senescence in Irradiated Human Cancer Cells

DNA-dependent protein kinase (DNA-PK) plays a pivotal role in the repair of DNA double-strand breaks (DSB) and is centrally involved in regulating cellular radiosensitivity. Here, we identify DNA-PK as a key therapeutic target for augmenting accelerated senescence in irradiated human cancer cells. We find that BEZ235, a novel inhibitor of DNA-PK and phosphoinositide 3-kinase (PI3K)/mTOR, abrogates radiation-induced DSB repair resulting in cellular radiosensitization and growth delay of irradiated tumor xenografts. Importantly, radiation enhancement by BEZ235 coincides with a prominent p53-dependent accelerated senescence phenotype characterized by positive β-galactosidase staining, G2–M cell-cycle arrest, enlarged and flattened cellular morphology, and increased p21 expression and senescence-associated cytokine secretion. Because this senescence response to BEZ235 is accompanied by unrepaired DNA DSBs, we examined whether selective targeting of DNA-PK also induces accelerated senescence in irradiated cells. Significantly, we show that specific pharmacologic inhibition of DNA-PK, but not PI3K or mTORC1, delays DSB repair leading to accelerated senescence after radiation. We additionally show that PRKDC knockdown using siRNA promotes a striking accelerated senescence phenotype in irradiated cells comparable with that of BEZ235. Thus, in the context of radiation treatment, our data indicate that inhibition of DNA-PK is sufficient for the induction of accelerated senescence. These results validate DNA-PK as an important therapeutic target in irradiated cancer cells and establish accelerated senescence as a novel mechanism of radiosensitization induced by DNA-PK blockade. Mol Cancer Res; 9(12); 1696–707. ©2011 AACR.

Characterization and targeting of phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) in renal cell cancer.

BackgroundPI3K and mTOR are key components of signal transduction pathways critical for cell survival. Numerous PI3K inhibitors have entered clinical trials, while mTOR is the target of approved drugs for metastatic renal cell carcinoma (RCC). We characterized expression of p85 and p110α PI3K subunits and mTOR in RCC specimens and assessed pharmacologic co-targeting of these molecules in vitro.MethodsWe employed tissue microarrays containing 330 nephrectomy cases using a novel immunofluorescence-based method of Automated Quantitative Analysis (AQUA) of in situ protein expression. In RCC cell lines we assessed synergism between PI3K and mTOR inhibitors and activity of NVP-BEZ235, which co-targets PI3K and mTOR.Resultsp85 expression was associated with high stage and grade (P < 0.0001 for both). High p85 and high mTOR expression were strongly associated with decreased survival, and high p85 was independently prognostic on multi-variable analysis. Strong co-expression of both PI3K subunits and mTOR was found in the human specimens. The PI3K inhibitor LY294002 and rapamycin were highly synergistic in all six RCC cell lines studied. Similar synergism was seen with all rapamycin concentrations used. NVP-BEZ235 inhibited RCC cell growth in vitro with IC50s in the low ηM range and resultant PARP cleavage.ConclusionsHigh PI3K and mTOR expression in RCC defines populations with decreased survival, suggesting that they are good drug targets in RCC. These targets tend to be co-expressed, and co-targeting these molecules is synergistic. NVP-BEZ235 is active in RCC cells in vitro; suggesting that concurrent PI3K and mTOR targeting in RCC warrants further investigation.

Serum epidermal growth factor receptor/HER-2 predicts poor survival in patients with metastatic breast cancer.

Epidermal growth factor receptor (EGFR, HER‐1, and erbB1) is overexpressed in primary breast cancer and had been identified as a poor prognostic factor.

Abstract P6-15-07: A Dose-Escalation Study with a Special Drug Delivery System (SDS) of BEZ235, a Novel Dual PI3K/mTOR Inhibitor, in Patients with Metastatic/Advanced Solid Tumors

Background: The PI3K pathway plays a major role in cancer cell growth and survival and is the most frequently altered pathway in cancer. BEZ235 is a potent and highly specific oral PI3K/mTOR inhibitor. Administered as hard-gelatine capsule, BEZ235 was well tolerated with a favorable safety profile. Available data showed that BEZ235 led to PRs (1 patient (pt) with NSCLC [PTEN null ], 1 pt with ER+ breast cancer [mutation unknown]) and prolonged disease stabilization (4 to >24 months) in pts with PI3K pathway dysregulated tumors (N=9/51), 5/9 with ER+, 1/9 with HER2+ breast cancer. Here, we present data from patients (pts) receiving treatment with a special drug delivery system of BEZ235 (SDS) with improved PK properties (Cao SABCS 2010). Results: 22 pts have been treated with BEZ235 SDS capsule at 3 dose levels (qd): 400 mg (5); 800 mg (6), 1000 mg (11). Median age was 56 years. The most common tumor types among pts enrolled were: breast cancer (4), CRC (4) and NSCLC (3). 2/22 pts were treated for >3 cycles, 16/22 pts progressed within the first three cycles of treatment. The maximum tolerated dose (MTD) is 1000 mg/d. Dose-limiting toxicities (DLTs) included: 2 Grade (G)3 fatigue (800 mg and 1000 mg) and 1 G3 skin rash (at 1000 mg). The most common treatment-related AEs included: fatigue, diarrhea, nausea, vomiting (G1-3). Preliminary PK data showed a slow absorption rate, C max at ∼6 h post-dose (range 2-8 h). The elimination halflife is ∼6 h across doses and visits. Steady state was reached after 8 days of treatment; the mean (SD) exposures at steady state (AUC, rss ) for the dose of 400, 800, and 1000 mg/d were 8864 (12210), 19060 (19860), 46010 (36150) ng*h/mL, respectively. The exposure across dose levels (AUC 0- 24, SS ) was above the exposure required for tumor stasis in PI3K pathway dysregulated tumors, estimated based on CT measurements in patients(Bottino SABCS 2010). Currently, the efficacy of BEZ235 SDS, alone and in combination with trastuzumab, in patients with PIK3CA mutated breast cancer is investigated. As of today 1 clinical PR was observed with BEZ235+trastuzumab in a patient with breast cancer (HER2+, PIK3CA mutated, trastuzumab refractory) with brain and peripheral metastases. Conclusions: BEZ235 SDS treatment is well tolerated. The MTD is 1000 mg/d. Available PD and efficacy data show that BEZ235 is active in breast cancer (especially PI3K pathway dysregulated tumors). Phase II studies in breast cancer combining BEZ235 SDS with other agents will start soon. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-07.

PIK3CA Mutations Can Be Acquired during Tumor Progression in Breast Cancer.

Background: Activation of the PI3K pathway by somatic mutations in the PI3KCA oncogene plays an important role in breast cancer. Mutations in PI3KCA have been described in approximately 30 % of breast carcinomas. Dysregulation of the PI3K pathway may have a significant impact on primary or acquired resistance towards endocrine and anti HER2 targeted therapy. The objective of this study was to assess changes in mutations in PI3KCA between the primary tumor and corresponding later metastatic lesions.Material: Formalin-fixed paraffin-embedded tumor blocks were available from 104 paired samples from primary tumors and corresponding distant metastases. In 99 patients paired PI3KCA mutation results were obtained.Methods: HE69(24 Suppl):Abstract nr 6023.

Association of Somatic Driver Alterations With Prognosis in Postmenopausal, Hormone Receptor–Positive, HER2-Negative Early Breast Cancer: A Secondary Analysis of the BIG 1-98 Randomized Clinical Trial

Importance A range of somatic driver alterations has been described in estrogen receptor–positive, HER2-negative (ER+/HER2−) early breast cancer (BC); however, the clinical relevance is unknown. Objective To investigate associations of driver alterations with prognosis and the role of PIK3CA mutations in prediction of benefit associated with endocrine therapy in postmenopausal patients with ER+/HER2− early BC treated with tamoxifen or letrozole. Design, Setting, and Participants The Breast International Group (BIG) 1-98 trial randomized 8010 postmenopausal patients with hormone receptor–positive, operable, invasive BC to monotherapy with letrozole, tamoxifen, or a sequential strategy for 5 years. Driver alterations were characterized using next-generation sequencing in primary tumors from a subset of 764 patients from 7329 eligible patients with ER+/HER2− BC, with 841 distant recurrences after a median of 8.1 years of follow-up. To correct for the oversampling of distant recurrences, weighted analysis methods were used. This analysis was conducted from April 4, 2016, to November 30, 2016. Main Outcomes and Measures The prevalence of driver alterations, associations with clinicopathologic factors, distant recurrence-free interval, and treatment interactions were analyzed. Multivariable analyses were performed to adjust for clinicopathologic factors. Results Of 764 samples, 538 (70.4%), including 140 distant recurrence events, were successfully sequenced. Nineteen driver alterations were observed with 5% or greater frequency, with a mean of 4 alterations (range, 0-15) per tumor. PIK3CA mutations were the most common (49%) and were significantly associated with reduction in the risk for distant recurrence (hazard ratio [HR], 0.57; 95% CI, 0.38-0.85; P = .006). TP53 mutations (HR, 1.92; 95% CI, 1.21-3.04; P = .006), amplifications on 11q13 (HR, 2.14; 95% CI, 1.36-3.37; P = .001) and 8p11 (HR, 3.02; 95% CI, 1.88-4.84; P < .001), and increasing number of driver alterations (HR per additional alteration, 1.18; 95% CI, 1.11-1.25; P < .001) were associated with significantly greater risk. Amplifications on 11q13 and 8p11 remained significant predictors in multivariable analysis, but not PIK3CA and TP53 mutations. Patients with tumors harboring kinase or helical domain PIK3CA mutations derived significantly greater benefit from letrozole over tamoxifen than patients whose tumors did not (P interaction = .002). Conclusions and Relevance In ER+/HER2− postmenopausal, early-stage BC, amplifications on 11q13 and 8p11 were significantly associated with increased risk for distant recurrence and PIK3CA mutations were predictive of greater magnitude of benefit from letrozole. With these findings, DNA-based classification may aid adjuvant treatment decision making in this setting. Trial Registration ClinicalTrials.gov Identifier: NCT00004205