mTOR Inhibitor BEZ235 Research Articles

Three generations of mTOR kinase inhibitors in the activation of the apoptosis process in melanoma cells

Many signaling pathways are involved in the mammalian target of rapamycin (mTOR), and this serine/threonine kinase regulates the most important cellular processes such as cell proliferation, autophagy, and apoptosis. The subject of this research was the effect of protein kinase inhibitors involved in the AKT, MEK, and mTOR kinase signaling pathways on the expression of pro-survival proteins, activity of caspase-3, proliferation, and induction of apoptosis in melanoma cells. The following inhibitors were used: protein kinase inhibitors such as AKT—MK-2206, MEK—AS-703026, mTOR—everolimus and Torkinib, as well as dual PI3K and mTOR inhibitor—BEZ-235 and Omipalisib, and mTOR1/2—OSI-027 inhibitor in single-mode and their combinations with MEK1/2 kinase inhibitor AS-703026. The obtained results confirm the synergistic effect of nanomolar concentrations of mTOR inhibitors, especially the dual PI3K and mTOR inhibitors (Omipalisib, BEZ-235) in combination with the MAP kinase inhibitor (AS-703026) in the activation of caspase 3, induction of apoptosis, and inhibition of proliferation in melanoma cell lines. Our previous and current studies confirm the importance of the mTOR signal transduction pathway in the neoplastic transformation process. Melanoma is a case of a very heterogeneous neoplasm, which causes great difficulties in treating this neoplasm in an advanced stage, and the standard approach to this topic does not bring the expected results. There is a need for research on the search for new therapeutic strategies aimed at particular groups of patients.Graphical abstractEffect of three generations of mTOR kinase inhibitors on caspase-3 activity, apoptosis and proliferation in melanoma cell lines.

Treatment by PI3K/mTOR Inhibitor BEZ235 Combined with TLR-7/8 Agonist Interfere with Immune Evasion Mechanisms of WEHI-3 Mouse Leukemia Cells.

Several PI3K/Akt/mTOR pathway inhibitors and TLR agonists induce tumor cell death. However, the mechanisms of these therapeutic approaches in acute myeloid leukemia (AML) cells are still unknown. To investigate the effects of BEZ235, as a dual inhibitor of PI3K and mTOR pathways, and TLR7/8 agonist R848 on the expression and regulation of the immune inhibitory molecules in myeloid leukemia cells. WEHI-3 leukemia cells were incubated with dual PI3K and mTOR inhibitor BEZ235 and TLR7/8 agonist R848 for 48 hrs. Firstly, cell viability was assessed by MTT method. The semi-quantitative relative mRNA expression of Galectin-9 (Gal-9), PD-L1, PVR, and STAT3 was assessed according to HPRT as a housekeeping gene. Finally, the protein expression of phosphorylated STAT3 was evaluated by western blotting analysis. WEHI-3 cells showed growth inhibition following treatment with BEZ235 and R848 whose combination exerted more proliferation arrest. The mRNA expression of Gal-9, PD-L1 and PVR immune checkpoint molecules significantly reduced in treated cells with BEZ235 and R848. Combined treatment indicated more reduction compared with the single treatment. Finally, the expression and phosphorylation of STAT3 were down-regulated after a single or dual treatment with BEZ235 and R848. Our results conclude that treatment with the combination of BEZ235 and R848 interferes with immune evasion mechanisms through STAT3-signaling pathway in WEHI-3 leukemia cells.

Clusterin and Related Scoring Index as Potential Early Predictors of Response to Sorafenib in Hepatocellular Carcinoma.

Advanced hepatocellular carcinoma (HCC) remains a highly lethal malignancy, although several systemic therapeutic options are available, including sorafenib (SFN), which has been one of the standard treatment agents for almost a decade. As early prediction of response to SFN remains challenging, biomarkers that enable early prediction using a clinically feasible method are needed. Here, we report that the serum secretory form of clusterin (sCLU) protein and its related predictive index are potential beneficial biomarkers for early prediction of SFN response. Using high‐throughput screening and subsequent multivariate analysis in the derivation cohort, we found that changes in the concentrations of CLU, vascular cell adhesion molecule‐1 (VCAM1), and α‐fetoprotein were significantly associated with response to SFN. Furthermore, we confirmed that an increase in CLU serum level 1 month after treatment initiation was significantly associated with shorter progression‐free survival. In addition, “NR‐index,” which comprises these proteins, was evaluated as a tool for accurately predicting the efficacy of SFN and confirmed in the validation cohort. We also established SFN‐resistant HepG2 cells (HepG2‐SR) and found that sCLU significantly increased in HepG2‐SR cells compared with normal HepG2 cells, and confirmed that HepG2‐SR cells treated with SFN were resistant to apoptosis. The mechanism underlying activation of sCLU expression in acquired SFN resistance involves aberrant signaling and expression of Akt, mammalian target of rapamycin (mTOR), and a nutrient‐related transcription factor, sterol regulatory element binding protein 1c (SREBP‐1c). Furthermore, the PI3K and mTOR inhibitor BEZ235 markedly decreased sCLU expression in HepG2‐SR cells. Conclusion: These results suggest that measurement of sCLU serum levels and the sCLU‐related NR‐index are promising clinical tools for the early prediction of SFN response in HCC. Additionally, sCLU‐overexpressing HCC might be susceptible to mTOR inhibition.

Deciphering the Mechanisms of Osteoblast-Induced Resistance of Leukemic Stem Cell (LSC) in Ph+ CML: Role of PI3-Kinase, BRD4 and MYC and Development of Strategies to Overcome Osteoblast-Induced Resistance

Chronic myeloid leukemia (CML) is a stem cell neoplasms characterized by the chromosome translocation t(9;22) and the related BCR-ABL1 fusion gene. Therapy with BCR-ABL1 kinase inhibitors is highly effective in the treatment of CML and deep molecular responses are achieved in most patients. However, not all patients respond to these drugs due to resistance of leukemic stem cells (LSC). Recent data suggest that the disease-related microenvironment, known as the stem cell niche contributes to drug resistance and relapse in CML. So far, little is known about the resistance mechanisms protecting niche cells in the bone marrow of patients with CML. We have recently shown that osteoblasts are a major site of LSC-mediated resistance against BCR-ABL1-targeting drugs in CML. In the current study, we screened for drugs that are able to suppress growth and viability of osteoblasts and/or other niche-related cells and can thereby overcome drug resistance of CML LSC. Proliferation was analyzed by determining 3H-thymidine uptake in niche-related cells and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC 50: 0.05 µM; copanlisib IC 50: 0.05 µM), the osteoblastic cell line CAL-72 (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 1 µM), primary human umbilical vein endothelial cells (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 0.5 µM) and the endothelial cell line HMEC-1 (BEZ235 IC 50: 1 µM; copanlisib IC 50: 1 µM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. As determined by flow cytometry, BEZ235 and copanlisib suppressed the expression of phosphorylated (p) pAKT and pS6 in endothelial cells and osteoblasts whereas rapamycin downregulated pS6 expression but did not decrease expression of pAKT. Moreover, we found that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing growth and viability of osteoblasts and endothelial cells. Furthermore, BEZ235 and copanlisib were found to overcome osteoblast-induced resistance of K562, KU812 cells, and primary CD34 +/CD38 − CML LSC against nilotinib and ponatinib. This effect was also seen when CAL-72 cells were first exposed to BEZ235 or copanlisib and washed before co-cultures with CML cells and BCR-ABL1 inhibitors were prepared, suggesting that osteoblast inhibition was a crucial event capable of disrupting LSC resistance in these co-cultures. Of all other drugs tested, only the BRD4-targeting drug JQ1 was found to suppress CAL72-induced resistance in the CML cell lines KU812 and K562, suggesting that osteoblast-induced resistance of CML cells is also mediated by a BRD4-MYC pathway. In a next step, we examined the expression of resistance-mediating immune checkpoint molecules on CML cells (KU812, K562, LSC) and on osteoblasts by flow cytometry. We found that CML cells and CAL72 cells constitutively express PD-L1 and that interferon-gamma (IFN-G) promotes the expression of PD-L1 in all cell types tested. Moreover, we found that the BRD4 blocker JQ1 and the BRD4-degrader dBET6 suppress the IFN-G-induced upregulation of PD-L1 in CML LSC and osteoblasts. In conclusion, our data show that osteoblast-induced resistance of CML stem cells is mediated by a PI3K-dependent pathway and BRD4/MYC, and that BRD4-inhibition or BRD4-degradation counteracts osteoblast-induced resistance of CML (stem) cells against BCR-ABL1 inhibitors and PD-L1 expression on CML LSC and osteoblasts. We hypothesize that checkpoint inhibition may assist in drug-induced eradication of CML LSC and thus in the development of curative drug therapies in Ph + CML. DisclosuresHoermann: Novartis: Honoraria. Gleixner: Pfizer: Honoraria; Abbvie: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Sperr: AbbVie, BMS-Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Novartis, Pfizer, StemLine, Thermo Fisher: Honoraria, Research Funding. Valent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

A phase I study of a dual PI3-kinase/mTOR inhibitor BEZ235 in adult patients with relapsed or refractory acute leukemia

BackgroundCombined inhibition of phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) complexes may be an efficient treatment for acute leukemia. The primary objective of this phase I single center open label study was to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of the dual pan-class I PI3K and mTOR inhibitor BEZ235 in patients with advanced leukemia.MethodsHerein patients > 18 years of age who had relapsed or showed refractory leukemia were treated with BEZ235 (orally at 300–400 mg BID (cohort − 1/1)) to assess safety, tolerability, preliminary efficacy and pharmacokinetic (PK). Adverse events data and serious adverse events were analyzed and haematological and clinical biochemistry toxicities were assessed from laboratory test parameters. Response was assessed for the first time at the end of cycle 1 (day 29) and after every subsequent cycle. Pharmacokinetic and pharmacodynamic analyses of BEZ235 were also included (BEZ235 plasma levels, phosphorylation of AKT, S6 and 4EBP1). On statistics this trial is a multiple ascending dose study in which a following variant of the 3 + 3 rule (“Rolling Six”), a minimum of 6 and a maximum of 12 patients was recruited for the dose escalation and another 5 were planned for the expansion phase.ResultsTwenty-four patients with ALL (n = 11) or AML (n = 12) or CML-BP (n = 1) were enrolled. All patients had failed one (n = 5) or more lines of therapy (n = 5) and 14 patients were in refractory / refractory relapse. No formal MTD was defined, stomatitis and gastrointestinal toxicity at 400 mg BID dose was considered incompatible with prolonged treatment. The RP2D of BEZ235 was defined as 300 mg BID. Four of 24 patients showed clinical benefit. Twenty-two of 24 patients discontinued because of progression, (median time to progression 27 days (4d-112d). There was no association between PK parameters and efficacy or tolerability.ConclusionsCombined inhibition of PI3K and mTOR inhibits a clinically meaningful driver pathway in a small subset of patients with ALL, with no benefit in patients with AML.Trial registrationClinicalTrials.gov, identifier NCT01756118. retrospectively registered 19th December 2012, https://clinicaltrials.gov/ct2/show/NCT01756118.

Inhibition of esophageal cancer growth through the suppression of PI3K/AKT/mTOR signaling pathway.

BackgroundThe phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is implicated in several cancers. AKT allosteric inhibitor MK2206 and dual PI3K and mTOR inhibitor BEZ235 are promising drug candidates with potential anti-tumor effects. PurposeIn this study, we aimed to detect the activation of PI3K/AKT/mTOR pathway and assess the efficacy of MK2206 and BEZ235 in inhibiting esophageal cancer growth.Materials and methodsWe used three different systems including carcinogen-induced animal model, human esophageal squamous cell carcinoma (SCC) cell lines, and xenograft mouse model. ResultsOur data indicated that components of the PI3K/AKT/mTOR pathway were overexpressed and activated in esophageal SCC. MK2206 and BEZ235 inhibited cell proliferation, enhanced apoptosis, and induced cell-cycle arrest through downstream effectors SKP2, MCL-1, and cyclin D1 in esophageal SCC cells. MK2206 and BEZ235 also inhibited tumor growth in xenograft mice through the inhibition of AKT phosphorylation. MK2206/BEZ235 combination showed greater anti-tumor effect than MK2206 or BEZ235 alone. The enhanced efficacy of the combination was associated with the inhibition of phosphorylation ATK on both Thr308 and Ser473. ConclusionThe combination of MK2206 and BEZ235 exhibits potent antitumor effects and may have important clinical applications for esophageal SCC treatment.

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment.

Targeting PI3K/mTOR signaling exerts potent antitumor activity in pheochromocytoma in vivo

Pheochromocytomas (PCCs) are mostly benign tumors, amenable to complete surgical resection. However, 10-17% of cases can become malignant, and once metastasized, there is no curative treatment for this disease. Given the need to identify the effective therapeutic approaches for PCC, we evaluated the antitumor potential of the dual-PI3K/mTOR inhibitor BEZ235 against these tumors. We employed an in vivo model of endogenous PCCs (MENX mutant rats), which closely recapitulate the human tumors. Mutant rats with PCCs were treated with 2 doses of BEZ235 (20 and 30 mg/kg), or with placebo, for 2 weeks. Treatment with BEZ235 induced cytostatic and cytotoxic effects on rat PCCs, which could be appreciated by both staining the tumors ex vivo with appropriate markers and non-invasively by functional imaging (diffusion-weighted magnetic resonance imaging) in vivo Transcriptomic analyses of tumors from rats treated with BEZ235 or placebo-identified potential mediators of therapy response were performed. Slc6a2, encoding the norepinephrine transporter (NET), was downregulated in a dose-dependent manner by BEZ235 in rat PCCs. Moreover, BEZ235 reduced Slc6a2/NET expression in PCC cell lines (MPC) also. Studies of a BEZ235-resistant derivative of the MPC cell line confirmed that the reduction of NET expression associates with the response to the drug. Reduction of NET expression after BEZ235 treatment in vivo could be monitored by positron emission tomography (PET) using a tracer targeting NET. Altogether, here we demonstrate the efficacy of BEZ235 against PCC in vivo, and show that functional imaging can be employed to monitor the response of PCC to PI3K/mTOR inhibition therapy.

PI3K/Akt/mTOR Pathway Inhibition in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Introduction: Burkitt lymphoma (BL) is the most common form of B-cell non-Hodgkin lymphoma (B-NHL) in children. Despite significant improvements in survival with de novo disease, treatment of relapsed or refractory BL remains a significant hurdle with survival in only about 20% of patients. Novel therapeutic approaches are necessary to improve outcomes in this group of childhood B-NHL patients with the worst prognosis. Recent literature has identified a high rate of recurrent mutations that result in activation of the PI3K/Akt pathway in BL and have implicated activation of PI3K/Akt in coordination with Myc in BL lymphomagenesis. Our laboratory has developed rituximab and chemotherapy resistant cell line models and subsequently found that these cell lines exhibit increased activation of Akt. We hypothesized that increased activation of Akt may be contributing to chemoresistance and that targeting the PI3K/Akt/mTOR pathway may increase chemoresponsiveness. To that end, we have investigated the effect of inhibiting the PI3K/Akt/mTOR pathway with either the PI3K-delta inhibitor idelalisib or the pan-PI3K/mTOR inhibitor BEZ-235 in cell line models of BL.Methods: The in vitro effect of idelalisib or BEZ-235 was investigated in BL cell lines including Raji, Raji 2R and Raji 4RH (rituximab-chemotherapy resistant), Raji 7R and Raji 8RH (rituximab resistant), Ramos and Daudi. Cell viability following inhibitor exposure was assessed by Alamar blu and cell-titer glo assays. The effect of inhibitor exposure on cell cycle progression was determined by flow cytometry using propidium iodide staining. Inhibition of Akt activation following inhibitor exposure was determined using phospho-flow cytometry. The activity of cytotoxic chemotherapeutic agents following inhibition by idelalisib or BEZ-235 was assessed using Alamar blu and cell titer glo assays.Results: In vitro exposure of BL cell lines to idelalisib in concentrations from 0.1-100µM for 24, 48 or 72 hours resulted in a dose and time-dependent decrease in viable cells in all cell lines tested with IC50 concentrations of 60-300uM. Pre-treatment with the pan-caspase inhibitor QVD resulted in a small reversal in the decrease in cell viability suggesting only a minimal portion of the activity was caspase dependent. When induction of apoptosis was measured using annexin V-propidium iodide staining, little induction of apoptosis was observed with single agent idelalisib at concentrations up to 100uM. Determination of cell cycle progression following exposure to idelalisib at 1, 10, 50 or 100 uM for 24, 48 or 72 hours indicated a time and dose dependent cell cycle arrest in all cell lines. In chemotherapy-sensitive cell lines the arrest was primarily noted in G1, while the chemotherapy-resistant Raji 2R and Raji 4RH cell lines exhibited arrest primarily in G2/M. A significant reduction in cell viability following chemotherapy exposure for 48 hours was noted in chemotherapy resistant Raji 2R cells following pre-treatment for 48 hours with idelalisib 10uM compared to non-idelalisib exposed cells (doxorubicin 10uM 55% vs 77%, p<0.001; vincristine 0.05uM, 48% vs 61%, P<0.001). At higher idelalisib pre-treatment concentrations (50uM) additional synergistic activity was observed in Raji 2R cells (cisplatin 48% vs 61%, p<0.001; dexamethasone 67% vs 87%, p<0.01). To further assess the effect of dual inhibition of PI3K and mTOR, cell lines were exposed to the dual inhibitor BEZ-235. BEZ-235 exhibited a more potent decrease in cell viability compared to idelalisib with activity at nM concentrations. Unlike idelalisib, exposure to BEZ-235 resulted in significant induction of apoptosis by Annexin V-propidium iodide staining. BEZ-235 also exhibited synergistic activity in combination with chemotherapy in all cell lines. At equivalent dosing, BEZ-235 exposure resulted in a more significant decrease in Akt phosphorylation compared to idelalisib as determined by flow cytometry for p-Akt at Ser and Thr phosphorylation sites.Conclusions: Chemotherapy sensitive and resistant BL cell line models are susceptible to inhibition of the PI3K/Akt/mTOR pathway. Targeted inhibition of this pathway leads to a decrease in AKT activation, decrease in cell viability, cell cycle arrest and an increase in sensitivity to cytotoxic chemotherapeutic agents. Broader inhibition of both PI3K and mTOR is more effective than more targeted inhibition of PI3K-delta alone. DisclosuresNo relevant conflicts of interest to declare.

The effect of the dual PI3K and mTOR inhibitor BEZ235 on tumour growth and osteolytic bone disease in multiple myeloma.

The plasma cell malignancy multiple myeloma (MM) is unique among haematological malignancies in its capacity to cause osteoclast-mediated skeletal destruction. The PI3K/Akt/mTOR pathway mediates proliferation, survival and drug resistance in MM plasma cells and is also involved in regulating the formation and activity of bone-forming osteoblasts and bone-resorbing osteoclasts. NVP-BEZ235 is a dual pan class I PI3K and mTOR inhibitor that is currently undergoing clinical evaluation in several tumour settings. In this study, we examined the anti-tumorigenic effects of BEZ235 in an immunocompetent mouse model of MM and assessed the effects of BEZ235 on osteoblast and osteoclast formation and function. BEZ235 treatment (50mg/kg) resulted in a significant decrease in serum paraprotein and tumour burden, and μCT analysis of the proximal tibia revealed a significant reduction in the number of osteolytic bone lesions in BEZ235-treated animals. Levels of the serum osteoblast marker P1NP were significantly higher in BEZ235-treated animals, while levels of the osteoclast marker TRAcP5 were reduced. In vitro, BEZ235 decreased MM plasma cell proliferation, osteoclast formation and function and promoted osteoblast formation and function. These findings suggest that, in addition to its anti-tumour properties, BEZ235 could be useful in treating osteolytic bone disease in MM patients.

Preclinical evaluation of the mTOR–PI3K inhibitor BEZ235 in nasopharyngeal cancer models

The dual PI3K–mTOR inhibitor BEZ235 was evaluated in preclinical models of nasopharyngeal carcinoma (NPC). The IC50 value of BEZ235 for growth was in the nanomolar range in vitro, induce G1 cycle arrest and apoptosis, and inhibited AKT and mTOR signaling in most NPC cell lines. No synergistic effect was observed when BEZ235 was combined with chemotherapy. BEZ235 increased MAPK activation in vitro but not in vivo. A daily schedule was more effective than a weekly schedule on tumor growth and inhibition of downstream mTOR signaling in vivo. The activity of BEZ235 maybe independent of the PIK3CA amplification and mutation status.

Dual PI3K/AKT/mTOR inhibitor BEZ235 synergistically enhances the activity of JAK2 inhibitor against cultured and primary human myeloproliferative neoplasm cells.

Hemopoietic progenitor cells (HPC) from myeloproliferative neoplasms (MPN) such as myelofibrosis commonly express mutant JAK2-V617F or other mutations that are associated with increased activities of JAK-STAT5/3, RAS/RAF/MAPK, and PI3K/AKT/mTOR pathways. This confers proliferative and survival advantage on the MPN HPCs. Treatment with JAK tyrosine kinase inhibitor (TKI), for example, TG101209, TG101348 (SAR302503), or INCB018424 (ruxolitinib), inhibits mutant JAK2-mediated signaling. Although effective in reducing constitutional symptoms and splenomegaly, treatment with JAK-TKI does not ameliorate myelofibrosis or significantly improve survival of patients with advanced myelofibrosis. Here, we show that treatment with the dual phosphoinositide-3-kinase (PI3K)/AKT and mTOR inhibitor BEZ235 attenuated PI3K/AKT and mTOR signaling, as well as induced cell-cycle growth arrest and apoptosis of the cultured human JAK2-V617F-expressing HEL92.1.7 (HEL), UKE1 cells, and primary CD34+ myelofibrosis (MF)-MPN cells. Treatment with BEZ235 also induced significant apoptosis of the JAK2-TKI resistant HEL/TGR cells that were selected for resistance against JAK-TKI. Cotreatment with BEZ235 and JAK2-TKI (TG101209 and SAR302503) synergistically induced lethal activity against the cultured and primary CD34+ MPN cells while relatively sparing the normal CD34+ HPCs. These findings create a compelling rationale to determine the in vivo activity of dual PI3K/mTOR inhibitors in combination with JAK inhibitors against myelofibrosis HPCs.

Abstract P6-10-05: SU2C Phase 1b Trial of Dual PI3K/ mTOR Inhibitor BEZ235 with Letrozole in ER+/HER2− Metastatic Breast Cancer (MBC)

Abstract Background: Mutations in PIK3CA, the gene encoding the p110alpha subunit of PI3K, have been associated with antiestrogen resistance in ER+ BC. In general, antiestrogen-resistant cancers retain ER and responsiveness to estradiol, suggesting that treatment of ER+/PI3K mutant BC should include PI3K inhibitors plus endocrine therapy. Methods: We are conducting a phase Ib open-label trial of letrozole (2.5 mg/d) with the dual PI3K/ mTOR inhibitor BEZ235 (400 mg BID, sachet formulation) in post-menopausal patients with ER+/HER2 negative MBC. Upon toxicity, BEZ235 was reduced to 400 mg in AM/ 200 mg in PM (dose level 1), and 200 mg BID (dose level 2), in a standard 3+3 de-escalation design. Treatment continues until unacceptable toxicity or disease progression. Tumor assessments are performed every 2 months. All tumor biopsies are being analyzed for PI3K pathway alterations. Results: To date, 9 patients have been accrued; 6 on dose level 1 (400 mg BID) and 3 on dose level 1 (400 mg in AM/ 200 mg in PM). Of note, the latter 3 patients have been on treatment for less than 2 weeks at the time of abstract submission. Median age was 55 years (range 40–65); all patients had disease progression on previous endocrine therapy, and 100% had bone metastases and 30% had visceral metastases. Preliminary toxicities for dose level 1 cohort (400 mg BID) are summarized in the table below. The DLT was grade 3 mucositis, which happened less than 3 weeks of study initiation, in a total of 3 out of 6 patients. All grade 3 mucositis cases improved after BEZ235 interruption for 7 days and subsequent dose reduction. These 3 events triggered activation of the dose level 1 cohort (400 mg AM/ 200 mg PM). The first 3 patients on dose level 1 cohort (2 of which have PIK3CA mutated tumors) had their tumor assessment scans at 8 weeks post treatment initiation; all patients were found to have stable disease. Discussion: The combination of letrozole/BEZ235 is still being evaluated in patients with ER+/HER2MBC. Safety, tolerability and preliminary antitumor activity data will be updated at the meeting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-05.

Abstract B55: Glioblastoma xenografts obtained from patient derived cancer stem cells preserve the heterogeneous response to targeted therapy

Abstract Molecular and signaling pathway heterogeneity contributes to the poor prognosis and high recurrence rate after therapy prevalent for glioblastoma (GBM) patients. Our goal was to test response to pharmacological agents targeting signaling pathways deregulated in glioblastomas: mTOR, RAF/MEK/ERK, VEGFR-2/PDGFRbeta, in preclinical models that use cancer stem cells to preserve the molecular signatures and heterogeneity characteristic of GBMs. Response to the DNA alkylating agent temozolomide (TMZ), used in the standard of care of GBMs, was assessed and correlated with the 06-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, a known predictive biomarker. The efficacy of targeting the base-excision DNA repair protein poly (ADP-ribose) polymerase (PARP) in potentiating TMZ therapy was tested. Neurosphere cultures enriched in cancer stem cells were obtained from four GBM tumors resected at our institution, three from first surgery and one from a post-treatment recurrence. Neurosphere cells were orthotopically implanted in nude mice. TMZ (Schering-Plough), the PI3K/mT0R dual inhibitor BEZ-235 (Novartis), the PARP inhibitor ABT-888 (Abbott Laboratories), and the multi-kinase inhibitor Sorafenib Tosylate (BAY43-9006, Nexavar), which blocks RAF kinase and the VEGFR-2/PDGFRbeta signaling cascade, were tested as part of the Ivy Genomics-Based Medicine Consortium. For the chemoprofiling studies, mice were implanted with each GBM neurosphere line, and divided into treatment cohorts. Drugs were administered 5 days/week intragastrically, for 4 weeks (BEZ-235, Sorafenib and control), or 2 cycles of 1 week each (TMZ and TMZ/ABT-888). Animals were sacrificed when moribund and brain tissue was harvested for molecular profiling and immunohistochemistry. Kaplan-Meier survival curves were compared by log-rank test to evaluate response to treatment. TMZ monotherapy significantly increased survival of xenografts derived from treatment naïve tumors with methylated MGMT promoter. The PARP inhibitor in combination to TMZ did not further improve survival for this cohort. TMZ treatment had no significant effect in survival for the xenografts obtained from the glioblastoma with unmethylated MGMT promoter, but the PARP inhibitor significantly potentiated TMZ for this line. As monotherapy, both the multi-kinase inhibitor Sorafenib and the pan-PI3K/mTOR inhibitor BEZ-235 were effective for one of the four lines. Xenografts derived from the recurrent, previously treated tumor, did not exhibited a significant response to the regimens tested. Improved design of pre-clinical studies using patient derived cancer stem cells will accelerate the pace of translational research, allowing for biomarker identification and testing of targeted agents currently under clinical investigation in animal models that preserve the molecular signatures and heterogeneity of GBMs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B55.

Gastric Cancer Growth Control by BEZ235In VivoDoes Not Correlate with PI3K/mTOR Target Inhibition but with [18F]FLT Uptake

In this study, we tested the antitumor activity of the dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor BEZ235 against gastric cancer in vitro and in vivo. Gastric cancer cell lines (N87, MKN45, and MKN28) were incubated with BEZ235 and assessed for cell viability, cell cycle, and PI3K/mTOR target inhibition. In vivo, athymic nude mice were inoculated with N87, MKN28, or MKN45 cells and treated daily with BEZ235. 3'-Deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) uptake was measured via small animal positron emission tomography (PET). In vitro, BEZ235 dose dependently decreased the cell viability of gastric cancer cell lines. The antiproliferative activity of BEZ235 was linked to a G(1) cell-cycle arrest. In vivo, BEZ235 treatment resulted in PI3K/mTOR target inhibition as shown by dephosphorylation of AKT and S6 protein in all xenograft models. However, BEZ235 treatment only inhibited tumor growth of N87 xenografts, whereas no antitumor effect was observed in the MKN28 and MKN45 xenograft models. Sensitivity to BEZ235 in vivo correlated with downregulation of the proliferation marker thymidine kinase 1. Accordingly, [(18)F]FLT uptake was only significantly reduced in the BEZ235-sensitive N87 xenograft model as measured by PET. In conclusion, in vivo sensitivity of gastric cancer xenografts to BEZ235 did not correlate with in vitro antiproliferative activity or in vivo PI3K/mTOR target inhibition by BEZ235. In contrast, [(18)F]FLT uptake was linked to BEZ235 in vivo sensitivity. Noninvasive [(18)F]FLT PET imaging might qualify as a novel marker for optimizing future clinical testing of dual PI3K/mTOR inhibitors.

BEZ235 impairs gastric cancer growth by inhibition of PI3K/mTOR in vitro and in vivo

Background Gastric cancer at advanced stage of disease is a major health problem and the prognosis with the current therapeutic treatment strategies remains poor. PI3K/mTOR pathway mutations, especially PTEN, PI3K3C and AKT mutations and pS6 overexpression, are found frequently in gastric cancer patients and are linked with poor outcome. Thus, we evaluated the dual PI3K and mTOR inhibitor BEZ235 against gastric cancer in vitro and in vivo.