Adaptation to mTOR kinase inhibitors by amplification of eIF4E to maintain cap-dependent translation.

The mechanistic target of rapamycin (mTOR) protein kinase coordinates responses to nutrients and growth factors and is an anti-cancer drug target. To anticipate how cells will respond and adapt to chronic mTOR complex (mTORC)1 and mTORC2 inhibition, we have generated SW620 colon cancer cells with acquired resistance to the ATP-competitive mTOR kinase inhibitor AZD8055 (SW620:8055R). AZD8055 inhibited mTORC1 and mTORC2 signalling and caused a switch from cap-dependent to internal ribosome entry site (IRES)-dependent translation in parental SW620 cells. In contrast, SW620:8055R cells exhibited a loss of S6K signalling, an increase in expression of the eukaryotic translation initiation factor eIF4E and increased cap-dependent mRNA translation. As a result, the expression of CCND1 and MCL1, proteins encoded by eIF4E-sensitive and cap-dependent transcripts, was refractory to AZD8055 in SW620:8055R cells. RNAi-mediated knockdown of eIF4E reversed acquired resistance to AZD8055 in SW620:8055R cells; furthermore, increased expression of eIF4E was sufficient to reduce sensitivity to AZD8055 in a heterologous cell system. Finally, although the combination of MEK1/2 inhibitors with mTOR inhibitors is an attractive rational drug combination, SW620:8055R cells were actually cross-resistant to the MEK1/2 inhibitor selumetinib (AZD6244). These results exemplify the convergence of ERK1/2 and mTOR signalling at eIF4E, and the key role of eIF4E downstream of mTOR in maintaining cell proliferation. They also have important implications for therapeutic strategies based around mTOR and the MEK1/2-ERK1/2 pathway.

Impact of dual mTORC1/2 mTOR kinase inhibitor AZD8055 on acquired endocrine resistance in breast cancer in vitro.

INTRODUCTION: Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER + endocrine resistant BC cells. METHODS: In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR. RESULTS: RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 µM), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P <0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P <0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P <0.05). Co-treating with AZD8055 alongside tamoxifen (P <0.01) or oestrogen deprivation (P <0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER. CONCLUSIONS: This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance.

Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase.

INTRODUCTION: Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance. METHODS: Altered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy. RESULTS: Moderate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression. CONCLUSIONS: Moderate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.

FT-6876, a Potent and Selective Inhibitor of CBP/p300, is Active in Preclinical Models of Androgen Receptor-Positive Breast Cancer.

BACKGROUND: Patients with triple-negative breast cancer (TNBC) expressing the androgen receptor (AR) respond poorly to neoadjuvant chemotherapy, although AR antagonists have shown promising clinical activity, suggesting these tumors are AR-dependent. cAMP responsive element binding protein (CREB)-binding protein (CBP) and p300 are transcriptional co-activators for the AR, a key driver of AR+ breast and prostate cancer, and may provide a novel therapeutic target in AR+ TNBC. OBJECTIVES: The aim of this study was to determine the therapeutic potential of FT-6876, a new CBP/p300 bromodomain inhibitor, in breast cancer models with a range of AR levels in vitro and in vivo. METHODS: Effects of FT-6876 on the CBP/p300 pathway were determined by combining chromatin immunoprecipitation (ChIP) with precision run-on sequencing (PRO-seq) complemented with H3K27 acetylation (Ac) and transcriptional profiling. The antiproliferative effect of FT-6876 was also measured in vitro and in vivo. RESULTS: We describe the discovery of FT-6876, a potent and selective CBP/p300 bromodomain inhibitor. The combination of ChIP and PRO-seq confirmed the reduction in H3K27Ac at specific promoter sites concurrent with a decrease in CBP/p300 on the chromatin and a reduction in nascent RNA and enhancer RNA. This was associated with a time- and concentration-dependent reduction in H3K37Ac associated with a decrease in AR and estrogen receptor (ER) target gene expression. This led to a time-dependent growth inhibition in AR+ models, correlated with AR expression. Tumor growth inhibition was also observed in AR+ tumor models of TNBC and ER+ breast cancer subtypes with consistent pharmacokinetics and pharmacodynamics. CONCLUSION: Our findings demonstrate FT-6876 as a promising new CBP/p300 bromodomain inhibitor, with efficacy in preclinical models of AR+ breast cancer.

Olutasidenib (FT-2102) in patients with relapsed or refractory IDH1-mutant glioma: A multicenter, open-label, phase Ib/II trial.

BACKGROUND: Olutasidenib (FT-2102) is a highly potent, orally bioavailable, brain-penetrant and selective inhibitor of mutant isocitrate dehydrogenase 1 (IDH1). The aim of the study was to determine the safety and clinical activity of olutasidenib in patients with relapsed/refractory gliomas harboring an IDH1R132X mutation. METHODS: This was an open-label, multicenter, nonrandomized, phase Ib/II clinical trial. Eligible patients (≥18 years) had histologically confirmed IDH1R132X-mutated glioma that relapsed or progressed on or following standard therapy and had measurable disease. Patients received olutasidenib, 150 mg orally twice daily (BID) in continuous 28-day cycles. The primary endpoints were dose-limiting toxicities (DLTs) (cycle 1) and safety in phase I and objective response rate using the Modified Response Assessment in Neuro-Oncology criteria in phase II. RESULTS: Twenty-six patients were enrolled and followed for a median 15.1 months (7.3‒19.4). No DLTs were observed in the single-agent glioma cohort and the pharmacokinetic relationship supported olutasidenib 150 mg BID as the recommended phase II dose. In the response-evaluable population, disease control rate (objective response plus stable disease) was 48%. Two (8%) patients demonstrated a best response of partial response and eight (32%) had stable disease for at least 4 months. Grade 3‒4 adverse events (≥10%) included alanine aminotransferase increased and aspartate aminotransferase increased (three [12%], each). CONCLUSIONS: Olutasidenib 150 mg BID was well tolerated in patients with relapsed/refractory gliomas harboring an IDH1R132X mutation and demonstrated preliminary evidence of clinical activity in this heavily pretreated population.

Olutasidenib alone or with azacitidine in IDH1-mutated acute myeloid leukaemia and myelodysplastic syndrome: phase 1 results of a phase 1/2 trial.

BACKGROUND: Olutasidenib (FT-2102) is a potent, selective, oral, small-molecule inhibitor of mutant isocitrate dehydrogenase 1 (IDH1). The aims for phase 1 of this phase 1/2 study were to assess the safety, pharmacokinetics, pharmacodynamics, and clinical activity of olutasidenib, as monotherapy or in combination with azacitidine, in patients with acute myeloid leukaemia or myelodysplastic syndrome, harbouring mutant IDH1. METHODS: In this phase 1/2, multicentre, open-label clinical trial, we enrolled patients aged 18 years or older with acute myeloid leukaemia or intermediate, high, or very high risk myelodysplastic syndrome harbouring mutant IDH1 at 18 study sites in the USA, Australia, France, and Spain. Other key eligibility criteria included Eastern Cooperative Oncology Group performance status 0-2 with adequate liver and renal function. The primary outcomes were dose-limiting toxicities and the maximum tolerated dose, maximum evaluated dose, and the recommended phase 2 dose of olutasidenib. Olutasidenib was administered orally in doses of 150 mg once daily, 150 mg twice per day, and 300 mg once daily. Azacitidine (75 mg/m2) was administered subcutaneously or intravenously daily for 7 days on, 21 days off. The study was ongoing at the data cutoff (Oct 2, 2019) and is registered with ClinicalTrials.gov, NCT02719574. FINDINGS: Patients were enrolled between Aug 8, 2016, and Nov 14, 2018. 78 patients received olutasidenib as monotherapy (n=32) or in combination with azacitidine (n=46). The median follow-up was 8·3 months (IQR 3·1-13·3) for monotherapy and 10·1 months (4·2-15·3) for combination therapy. 16 (50%) of 32 patients in the monotherapy group and 24 (52%) of 46 patients in the combination therapy group were women. Most patients were White (26 [81%] for monotherapy and 31 [67%] for combination therapy). No dose-limiting toxicities were reported in the dose-escalation cohorts and 150 mg twice per day was declared the recommended phase 2 dose on the basis of safety, pharmacokinetics and pharmacodynamics, and clinical activity. The most common (≥20%) grade 3-4 treatment-emergent adverse events with monotherapy were thrombocytopenia (nine [28%] of 32 patients), febrile neutropenia (seven [22%] of 32), and anaemia (seven [22%] of 32); and with combination therapy were thrombocytopenia (19 [41%] of 46), febrile neutropenia (13 [28%] of 46), neutropenia (13 [28%] of 46), and anaemia (nine [20%] of 46). 11 (34%) of 32 patients in the monotherapy group and nine (20%) of 46 patients in the combination therapy group died (most commonly from disease progression [three (9%) of 32 and four (9%) of 46]). No deaths were considered study-drug related. For patients with relapsed or refractory acute myeloid leukaemia, 41% (95% CI 21-64; nine of 22) receiving monotherapy and 46% (27-67; 12 of 26) receiving combination therapy had an overall response. For treatment-naive patients with acute myeloid leukaemia, 25% (1-81; one of four) receiving monotherapy and 77% (46-95; ten of 13) receiving combination therapy had an overall response. INTERPRETATION: Olutasidenib, with or without azacitidine, was well tolerated and showed meaningful clinical activity in patients with IDH1-mutated acute myeloid leukaemia. The results of this phase 1 study provide rationale for the continued evaluation of olutasidenib in multiple patient populations with myeloid malignancies. FUNDING: Forma Therapeutics.

Benefits of mTOR kinase targeting in oncology: pre-clinical evidence with AZD8055.

AZD8055 is a small-molecule inhibitor of mTOR (mammalian target of rapamycin) kinase activity. The present review highlights molecular and phenotypic differences between AZD8055 and allosteric inhibitors of mTOR such as rapamycin. Biomarkers, some of which are applicable to clinical studies, as well as biological effects such as autophagy, growth inhibition and cell death are compared between AZD8055 and rapamycin. Potential ways to develop rational combinations with mTOR kinase inhibitors are also discussed. Overall, AZD8055 may provide a better therapeutic strategy than rapamycin and analogues.

The Pharmacokinetic-Pharmacodynamic (PKPD) Relationships of AZD3229, a Novel and Selective Inhibitor of KIT, in a Range of Mouse Xenograft Models of GIST.

PURPOSE: The emergence of secondary mutations is a cause of resistance to current KIT inhibitors used in the treatment of patients with gastrointestinal stromal tumors (GIST). AZD3229 is a selective inhibitor of wild-type KIT and a wide spectrum of primary and secondary mutations seen in patients with GIST. The objective of this analysis is to establish the pharmacokinetic-pharmacodynamic (PKPD) relationship of AZD3229 in a range of mouse GIST tumor models harboring primary and secondary KIT mutations, and to benchmark AZD3229 against other KIT inhibitors. EXPERIMENTAL DESIGN: A PKPD model was developed for AZD3229 linking plasma concentrations to inhibition of phosphorylated KIT using data generated from several in vivo preclinical tumor models, and in vitro data generated in a panel of Ba/F3 cell lines. RESULTS: AZD3229 drives inhibition of phosphorylated KIT in an exposure-dependent manner, and optimal efficacy is observed when >90% inhibition of KIT phosphorylation is sustained over the dosing interval. Integrating the predicted human pharmacokinetics into the mouse PKPD model predicts that an oral twice daily human dose greater than 34 mg is required to ensure adequate coverage across the mutations investigated. Benchmarking shows that compared with standard-of-care KIT inhibitors, AZD3229 has the potential to deliver the required target coverage across a wider spectrum of primary or secondary mutations. CONCLUSIONS: We demonstrate that AZD3229 warrants clinical investigation as a new treatment for patients with GIST based on its ability to inhibit both ATP-binding and A-loop mutations of KIT at clinically relevant exposures.

AKT/mTORC2 Inhibition Activates FOXO1 Function in CLL Cells Reducing B-Cell Receptor-Mediated Survival.

PURPOSE: To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). EXPERIMENTAL DESIGN: Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. RESULTS: Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. CONCLUSIONS: Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.

Gene expression predicts differential capecitabine metabolism, impacting on both pharmacokinetics and antitumour activity.

Capecitabine is converted into 5'-deoxy-5-fluorocytidine (5'DFCR), 5'-deoxy-5-fluorouridine (5'DFUR) and 5-fluorouracil (5-FU) by CES1 and 2, CDD, and TP, in both liver and tumour. 5-FU is catabolised by DPD. Gene expression analysis of these enzymes was undertaken in fresh human hepatocytes, mouse liver, colorectal cancer cell lines and xenografts. Cell lines with low CDD expression (<1.5) had 5'DFCR/5'DFUR cytotoxicity ratios>2 and cell lines with TP/DPD<0.6 had 5'DFUR IC50>50 microM (SRB assay). A pharmacokinetic/pharmacodynamic study in nude mice bearing HCT 116 xenografts and treated with capecitabine by oral gavage assessed pharmacokinetic, gene expression and antitumour activity. Low liver CDD correlated with high 5'DFCR plasma concentrations in mice. CDD expression was approximately 100-fold higher in fresh human hepatocytes than mouse liver, explaining the higher plasma 5'DFUR concentrations reported previously in humans. Tumour 5-FU concentration correlated with TP/DPD and with tumour response. These studies identify the potential utility of gene expression analysis and drug monitoring in tumour in patients.

Simultaneous determination of decitabine and vorinostat (Suberoylanalide hydroxamic acid, SAHA) by liquid chromatography tandem mass spectrometry for clinical studies.

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by tandem mass spectrometry is described for the simultaneous quantitative determination of decitabine (5-aza-2'-deoxycytidine) and vorinostat (Suberoylanalide hydroxamic acid, SAHA) in human plasma. The method involves a simple acetonitrile precipitation step and centrifugation followed by injection of the supernatant onto a C18 150mmx2.1mm I.D., 3microm HPLC column at 36 degrees C. Separation of decitabine, SAHA and their respective internal standards was achieved with a gradient elution and detection was via the mass spectrometer operated in selected reaction monitoring mode. The method was within the defined validation parameters for linearity, repeatability, reproducibility and stability. The limit of detection was determined as 1.0 and 0.125ngml(-1) and lower limits of quantitation were 10 and 1ngml(-1) for decitabine and SAHA, respectively. Effects of sample preparation on stability were also evaluated in human plasma. For clinical sample handling tetrahydrouridine, an inhibitor of cytidine deaminase was found to help prevent decitabine degradation. The method is currently being used in clinical pharmacokinetic studies for the evaluation of decitabine and SAHA combination therapies.

Time-dependent cytotoxicity induced by SJG-136 (NSC 694501): influence of the rate of interstrand cross-link formation on DNA damage signaling.

SJG-136 is a new pyrrolobenzodiazepine dimer inducing time-dependent cytotoxicity. HCT 116 cells were exposed to 50 nmol/L of SJG-136 for 1 hour or 1 nmol/L of SJG-136 for 24 hours to achieve similar levels of interstrand cross-links (ICL). The short exposure led to a rapid formation of ICLs (1 hour), early H2AX foci formation (4 hours), prominent S phase arrest, and greater phosphorylation of Nbs1 (on serine 343) and Chk1 (on serine 317) than a 24-hour exposure. The prolonged exposure at low concentrations of SJG-136 induced a gradual formation of ICLs (up to 24 hours) which was associated with a limited S phase arrest and delayed Nbs1 phosphorylation. Prolonged exposure was also associated with a reduced phosphorylation of p53 on serines 15 and 20, a limited and delayed phosphorylation on serine 392, and a less prominent increase in p21 levels. These data suggest that the 24-hour exposure to a low concentration of SJG-136 led to delayed and reduced DNA damage signaling compared with a higher concentration of SJG-136 for 1 hour, resulting in greater cytotoxicity and contributing to the time-dependent cytotoxic effect of SJG-136.

A pharmacokinetic-pharmacodynamic model predicting tumour growth inhibition after intermittent administration with the mTOR kinase inhibitor AZD8055.

BACKGROUND AND PURPOSE: AZD8055 is a potent orally available mTOR kinase inhibitor with in vitro and in vivo antitumour activity against a range of tumour types. Preclinical studies showed that AZD8055 induced a dose-dependent pharmacodynamic effect in xenograft models in vivo, but a lack of understanding of the relative contributions of the maximum inhibition of the biomarkers and the duration of inhibition to the antitumour effect, limited the rational design of experiments to optimize the dose and schedules of treatment. EXPERIMENTAL APPROACH: In this study, a mathematical modelling approach was developed to relate pharmacodynamics and antitumour activity using preclinical data generated in mice bearing U87-MG xenografts. KEY RESULTS: Refinement and validation of the model was carried out in a panel of additional human tumour xenograft models with different growth rates and different sensitivity to AZD8055 (from partial growth inhibition to regression). Finally, the model was applied to accurately predict the efficacy of high, intermittent dosing schedules of AZD8055. CONCLUSIONS AND IMPLICATIONS: Overall, this new model linking pharmacokinetics, pharmacodynamic biomarkers and efficacy across several tumour xenografts with different sensitivity to AZD8055 was able to identify the optimal dose and route of administration to maximize the antitumour efficacy in preclinical models and its potential for translation into man.

Efficacy and toxicity of a virus-directed enzyme prodrug therapy purging method: preclinical assessment and application to bone marrow samples from neuroblastoma patients.

Autologous stem cell transplantation is used to rescue cancer patients from myelosuppression caused by high-dose chemotherapy. However, autologous grafts often contain tumor cells that can contribute directly to relapse. Current purging methods are useful when fewer than 1% tumor cells contaminate the bone marrow, and patients with tumor burdens of >1% are considered ineligible for chemotherapy that necessitates stem cell rescue. Using neuroblastoma (NB) as a model system, we developed a method that is effective even with tumor burdens of 10-25%. Mixtures of NB-1691 NB cells and CD34(+) hematopoietic cells purged by this method showed no evidence of viable tumor cells as assessed by clonogenic assays or reverse transcription-PCR for the NB cell markers tyrosine hydroxylase and N-MYC. The efficacy and lack of toxicity of the method were verified using in vivo mouse models. Severe combined immunodeficient mice that received purged cell preparations containing 10% NB-1691 cells survived without evidence of disease for the observation period (>1 year), whereas mice that received unpurged cells developed disseminated disease requiring euthanasia 73-96 days after injection of cells. No evidence of toxicity to the mice was detected by numerous laboratory values for bone marrow, liver, and kidney function, and no difference was seen in the ability of purged cell mixtures versus unmanipulated CD34(+) cells to reconstitute the marrow of non-obese diabetic severe combined immunodeficient mice. In a pilot study, marrow was obtained from eight patients who had >/=1% metastatic tumor burden. All eight samples were purged to the level of detection by reverse transcription-PCR (samples from seven patients) or clonogenic potential (sample from one patient), whichever assay was used. The described adenovirus/rabbit carboxylesterase/CPT-11 (irinotecan, 7-ethyl-10[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) virus-directed enzyme prodrug method may be useful for patients whose tumor burdens exceed 1% at the time of stem cell harvest. Assessment of purging efficacy with additional samples from NB patients is ongoing.

Simultaneous determination of capecitabine and its metabolites by HPLC and mass spectrometry for preclinical and clinical studies.

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of capecitabine, its intermediate metabolites (DFCR, DFUR) and the active metabolite 5-fluorouracil in mouse plasma, liver and human xenograft tumours. The method was also cross-validated in human plasma and human tumour for clinical application. The method has greater sensitivity than previously published methods with an equivalent accuracy and precision. It uses less biological material (plasma, tissue) and should therefore be applicable to biopsies in patients treated with capecitabine.

AZD2014, an Inhibitor of mTORC1 and mTORC2, Is Highly Effective in ER+ Breast Cancer When Administered Using Intermittent or Continuous Schedules.

mTOR is an atypical serine threonine kinase involved in regulating major cellular functions, such as nutrients sensing, growth, and proliferation. mTOR is part of the multiprotein complexes mTORC1 and mTORC2, which have been shown to play critical yet functionally distinct roles in the regulation of cellular processes. Current clinical mTOR inhibitors only inhibit the mTORC1 complex and are derivatives of the macrolide rapamycin (rapalogs). Encouraging effects have been observed with rapalogs in estrogen receptor-positive (ER(+)) breast cancer patients in combination with endocrine therapy, such as aromatase inhibitors. AZD2014 is a small-molecule ATP competitive inhibitor of mTOR that inhibits both mTORC1 and mTORC2 complexes and has a greater inhibitory function against mTORC1 than the clinically approved rapalogs. Here, we demonstrate that AZD2014 has broad antiproliferative effects across multiple cell lines, including ER(+) breast models with acquired resistance to hormonal therapy and cell lines with acquired resistance to rapalogs. In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials.

Examining changes in [18 F]FDG and [18 F]FLT uptake in U87-MG glioma xenografts as early response biomarkers to treatment with the dual mTOR1/2 inhibitor AZD8055.

PURPOSE: The mTOR kinase inhibitor AZD8055 inhibits both mTORC1 and mTORC2 leading to disruption of glucose metabolism and proliferation pathways. This study assessed the impact of single and multiple doses of AZD8055 on the uptake of the glucose metabolism marker 2-deoxy-2-[(18) F]fluoro-D-glucose ([(18) F]FDG) and the proliferation marker 3'-deoxy-3'-[(18) F]fluorothymidine ([(18) F]FLT) in U87-MG glioma xenografts. PROCEDURES: Mice bearing U87-MG tumours received either vehicle or AZD8055 (20 mg/kg) once daily p.o. Mice were imaged with either [(18) F]FDG or [(18) F]FLT PET to assess treatment response. Comparisons were made between in vivo imaging and ex vivo histopathology data. RESULTS: Tumour uptake of [(18) F]FDG was reduced by 33 % 1 h after a single dose of AZD8055 and by 49 % following 4 days of dosing. These changes coincided with suppression of the mTOR pathway biomarkers pS6 and pAKT. In contrast, the effect of AZD8055 on [(18) F]FLT uptake was inconsistent. CONCLUSIONS: The very rapid change in [(18) F]FDG uptake following acute AZD8055 treatment suggests that this could be used as an early mechanistic biomarker of metabolic changes resulting from mTOR inhibition. The utility of [(18) F]FLT for measuring the anti-proliferative effect of AZD8055 remains unclear.

Delineating the mTOR kinase pathway using a dual TORC1/2 inhibitor, AZD8055, in multiple myeloma.

Despite promising preclinical results with mTOR kinase inhibitors in multiple myeloma, resistance to these drugs may arise via feedback activation loops. This concern is especially true for insulin-like growth factor 1 receptor (IGF1R), because IGF1R signaling is downregulated by multiple AKT and mTOR feedback mechanisms. We have tested this hypothesis in multiple myeloma using the novel selective mTOR kinase inhibitor AZD8055. We evaluated p-mTOR S(2481) as the readout for mTORC2/Akt activity in multiple myeloma cells in the context of mTOR inhibition via AZD8055 or rapamycin. We next validated AZD8055 inhibition of mTORC1 and mTORC2 functions in multiple myeloma cells alone or in culture with bone marrow stroma cells and growth factors. Unlike rapamycin, AZD8055 resulted in apoptosis of multiple myeloma cells. AZD8055 treatment, however, induced upregulation of IGF1R phosphorylation in p-Akt S(473)-expressing multiple myeloma cell lines. Furthermore, exposure of AZD8055-treated cells to IGF1 induced p-Akt S(473) and rescued multiple myeloma cells from apoptosis despite mTOR kinase inhibition and TORC2/Akt blockage. The addition of blocking IGF1R antibody resulted in reversing this effect and increased AZD8055-induced apoptosis. Our study suggests that combination treatment with AZD8055 and IGF1R-blocking agents is a promising strategy in multiple myeloma with potential IGF1R/Akt signaling-mediated survival.

Enhanced apoptosis and tumor growth suppression elicited by combination of MEK (selumetinib) and mTOR kinase inhibitors (AZD8055).

The mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT signaling pathways interact at multiple nodes in cancer, including at mTOR complexes, suggesting an increased likelihood of redundancy and innate resistance to any therapeutic effects of single pathway inhibition. In this study, we investigated the therapeutic effects of combining the MAPK extracellular signal-regulated kinase (MEK)1/2 inhibitor selumetinib (AZD6244) with the dual mTORC1 and mTORC2 inhibitor (AZD8055). Concurrent dosing in nude mouse xenograft models of human lung adenocarcinoma (non-small cell lung cancers) and colorectal carcinoma was well tolerated and produced increased antitumor efficacy relative to the respective monotherapies. Pharmacodynamic analysis documented reciprocal pathway inhibition associated with increased apoptosis and Bim expression in tumor tissue from the combination group, where key genes such as DUSP6 that are under MEK functional control were also modulated. Our work offers a strong rationale to combine selumetinib and AZD8055 in clinical trials as an attractive therapeutic strategy.

Optimization of the antitumor activity of sequence-specific pyrrolobenzodiazepine derivatives based on their affinity for ABC transporters.

Pyrrolobenzodiazepine (PBD) derivatives are highly potent sequence-specific DNA cross-linking agents. The present study aimed to identify key physicochemical properties influencing the interaction of a series of PBDs (four dimers and 12 monomers) with the three major human ATP-binding cassette (ABC) transporters (P-gp, ABCG2, and MRP1). Isogenic cell lines expressing P-gp and ABCG2, cell lines with acquired resistance to cytotoxic agents due to the high expression of ABC transporters, and specific inhibitors against P-gp, ABCG2, and MRP1 were used. P-gp and ABCG2 decreased the permeability of the PBD dimers across cell membranes and their interaction with DNA, reducing DNA damage and the overall cytotoxic effect. PBD monomer SG-2823 formed a conjugate with glutathione and interacted with MRP1, reducing its cytotoxic effect in A549 cells. Structure-activity relationship revealed that the interaction of PBDs with the transporters could be predicted considering the molecular weight, the lipophilicity, the number of (N + O) atoms and aromatic rings, the polar surface area, the hydrogen bonding energy, and electrophilic centers. A rational design of novel PBDs with increased potency and reduced interaction with the ABC transporters is proposed.